Realmente existe una vacuna contra la leishmaniasis ???

Data-Medicos 
Dermagic/Express No. 5-(118) 
28 Febrero 2.003 / 28 February 2.003 
 

Cutaneous Leishmaniasis Pentamidine e itraconazole.
                                               (hot link)

EDITORIAL ESPANOL
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Hola amigos de la red, DERMAGIC de nuevo con ustedes, despues de vivir una situacion dificil en Venezuela durante los meses de DICIEMBRE Y ENERO, la cual retardo la presente publicacion sobre LEISHMANIASIS Y VACUNAS

Puedo comentarles mi experiencia con un caso de Leishmanisis cutanea que tuve la oportunidad de ver en mi consulta, el paciente un joven de 16 años quien referi al Hospital Central de Maracay, alli se le comenzo a practicar inmunoterapia (BCG mas formas amastigostas de Leishmania), el paciente no respondio al tratamiento y tuvo que ser tratado con el clasico tratamiento de 3 ciclos de 20 ampollas de glucantime via intramuscular.

Este evento me llevo a realizar la presente revision, para evaluar las diferentes metodologias existentes para elaborar una vacuna contra esta enfermedad.

Se ha dicho mucho sobre la LEISHMANIASIS, enfermedad parasitaria con tres variantes, CUTANEA, CUTANEO MUCOSA, Y VISCERAL (KALA-AZAR). En el mundo se producen aproximadamente 15 millones casos nuevos al año con unas 500.000 mil muertes anuales entre niños y adultos.

Numerosos paises alrededor del mundo se han dedicado a la dura tarea de fabricar una VACUNA EFICAZ contra la enfermedad, entre ellos Venezuela, encontre muchas referencias BIBLIOGRAFICAS, de muchos paises, diferentes metodos y diferentes estudios TODOS ellos con relativo exito para encontrar una VACUNA tanto para la LEISHMANIASIS CUTANEA, MUCOSA y VISCERAL. La mayoria de estos metodos son muy parecidos en su escencia; combinacion de formas amastigotas de diferentes cepas de Leishmania mas BCG (bacilo de calmette Guerin), en forma de inmunoterapia, y tambien vacunas en base a ADN y antigenos de Leishmania.

El unico trabajo o estudio que en esencia se diferencia a los "clasicos" ya mencionados es el del Dr Valenzuela y Dr Ribeiro de la Universidad de Bethesda USA año 2.001 Agosto 6, quienes logran aislar una proteina de la saliva de de la mosca de arena (sand fly) flebotomo papatasi quien es el encargado de transmitir y diseminar la Leishmaniasis mayor en el Medio Oriente. Con esta proteina, que denominaron SP15 construyeron una vacuna en base a ADN que que utilizaron para inmunizar ratones. Cuando los ratones fueron inyectados posteriormente con parasitos de Leishmania mayor mezclados con la saliva de la mosca, la infeccion fue marcadamente menor comparada con la infeccion en ratones no vacunados. Las Lesiones en los ratones vacunados fueron mucho menores y desaparecieron en seis (6) semanas. Los ratones no vacunados desarrollaron grandes ulceras y no eliminaron el parasito.

Otro aspecto a resaltar es la DONACION DE NUESTRO querido amigo BILL GATES Y SU ESPOSA Melinda de 15. millones de dolares a traves de una fundacion, para el desarrollo de una vacuna contra la LEISHMANIASIS a la compañia CORIXA, quien en base a nuevos antigenos realizo en el año 2.001 los primeros estudios de campo en BRAZIL.

Grandes esfuerzos de todos estos investigadores, los FELICITO a todos por su colaboracion a la salud de NUESTRA bella poblacion HUMANA. Grandes esfuerzos que espero sean recompensados pronto con la salida al MERCADO de una VACUNA contra esta enfermedad, pues segun lo revisado TODAVIA no EXISTE UNA VACUNA COMO TAL disponible comercialmente contra la LEISHMANIASIS

Los hechos en esta apocaliptica revision con mas de 100 referencias bibliograficas

Saludos a Todos

Dr Jose Lapenta R.
 

ENGLISH EDITORIAL
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Hello friends of the net, DERMAGIC again with you, after living a difficult situation in Venezuela during the months of DECEMBER AND JANUARY, which I stop the present publication LEISHMANIASIS AND VACCINES.

I can comment them my experience with a case of cutaneous Leishmanisis that I had the opportunity to see in my office, the patient a 16 year-old boy who I referred to the Central Hospital of Maracay, there he was begun to practice immunotherapy (BCG -Calmette-Guerin- plus amastigotes forms of Leishmania), the patient didn't respond to the treatment and he had to be treated with the classic treatment of 3 cycles of 20 glucantime amps (chemotherapy) via IM.

This event takes myself to carry out the present revision, to evaluate the different existent methodologies to elaborate a vaccine against this illness.

A lot of things about LEISHMANIASIS has been said, parasitic illness with three variants, CUTANEOUS, MUCOUS, AND VISCERAL (KALA-AZAR). In the world they take place 15 millions new cases approximately a year with some 500.000 thousand annual deaths between children and adults.

Numerous countries around the world have been devoted to the hard task of manufacturing an EFFECTIVE VACCINE against the illness, among them Venezuela, I found many BIBLIOGRAPHICAL references, of many countries, different methods and different studies ALL them with relative success to find a VACCINE for the CUTANEOUS, MUCOUS and VISCERAL LEISHMANIASIS. Most of these methods are very similar in their essence: combination of amastigotes different strains of Leishmania plus BCG (calmette Guerin bacillus), in immunotherapy form, and also VACCINES based on DNA and antigens of Leishmania.

The only work or study that differs to those in essence "classic" already mentioned it is that of the Dr Valenzuela and Dr Ribeiro of the University of Bethesda USA year 2.001 August 6 who are able to isolate a protein of the saliva of of the fly of sand (sand fly) phlebotomus papatasi who is the one in charge of to transmit and to disseminate the Leishmaniasis major in the Middle East. With this protein that SP15 denominated they built a vaccine based on DNA that used to immunize mice. When the mice were injected later on with parasites of Leishmania adult mixed with the saliva of the fly, the infection was markedly minor compared with the infection in not vaccinated mice. The Lesions in the vaccinated mice were much smaller and they disappeared in six (6) weeks. The not vaccinated mice developed big ulcers and they didn't eliminate the parasite.

Another aspect to stand out is that OUR dear friend BILL GATES AND ITS WIFE Melinda they made a DONATION of 15 millions of dollars through a foundation, for the development of a vaccine against the LEISHMANIASIS to the company CORIXA who I carry out in the year 2.001 the first trail field studies in BRAZIL, based on novel antigens.

All these investigators' big efforts, I CONGRATULATE them to all for their collaboration to OUR beautiful HUMAN population's health. Big efforts that I wait are rewarded soon with the release to the MARKET of a VACCINE against this illness, because according to that revised A VACCINE it doesn't STILL EXIST commercially available one against the LEISHMANIASIS

The facts in this apocalyptic revision with but of 100 bibliographical references

Greetings to All

Dr José Lapenta R.
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REFERENCIAS BIBLIOGRAFICAS /BIBLIOGRAPHICAL REFERENCES
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1.) Vaccination against Leishmania infections.
2.) Leishmania transmission-blocking vaccines: a review.
3.) Vaccination against cutaneous leishmaniasis: current status.
4.) Leishmaniasis: current status of vaccine development.
5.) Protective immunity against cutaneous leishmaniasis achieved by partly purified vaccine in a volunteer.
6.) Vaccines against leishmaniasis.
7.) Mechanisms of acquired immunity in leishmaniasis.
8.) Experimental leishmaniasis in humans: review.
9.) Heterologous protection in murine cutaneous leishmaniasis.
10.) IgE antibody against surface antigens of Leishmania promastigotes in American cutaneous leishmaniasis.
11.) A comparison of the molecular biology of trypanosomes and leishmaniae, and its impact on the development of methods for the diagnosis and vaccination of leishmaniasis and Chagas disease.
12.) Dendritic cell-based vaccination strategies: induction of protective immunity against leishmaniasis.
13.) Effective immunization against cutaneous leishmaniasis with defined membrane antigens reconstituted into liposomes.
14.) [An immunologic method of producing antigens for the treatment and prevention of leishmaniasis]
15.) Development of vaccines against leishmaniasis.
16.) Further trials of a vaccine against American cutaneous leishmaniasis.
17.) Vaccination against cutaneous leishmaniasis in mice using nonpathogenic cloned promastigotes of Leishmania major and importance of route of injection.
18.) Leishmaniasis.
19.) Leishmania (Leishmania) amazonensis-induced cutaneous leishmaniasis in the primate Cebus apella: a model for vaccine trials.
20.) [Experimental skin leishmaniasis: II--course of the infection in the Cebus apella primate (Cebidae) caused by Leishmania (V.) braziliensis and L. (L.) amazonensis]
21.) Leishmania (Leishmania) major-infected rhesus macaques (Macaca mulatta) develop varying levels of resistance against homologous re-infections.
22.) Protection of C57BL/10 mice by vaccination with association of purified proteins from Leishmania (Leishmania) amazonensis.
23.) Experimental cutaneous leishmaniasis. IV. The humoral response of Cebus apella (Primates: Cebidae) to infections of Leishmania (Leishmania) amazonensis, L. (Viannia) lainsoni and L. (V.) braziliensis using the direct agglutination test.
24.) Vaccination of C57BL/10 mice against cutaneous Leishmaniasis using killed promastigotes of different strains and species of Leishmania.
25.) [Experimental cutaneous leishmaniasis: I--on the susceptibility of the primate Cebus apella (Cebidae) to the infection caused by Leishmania (Viannia) lainsoni Silveira, Shaw and Ishikawa, 1987]
26.) [Experimental cutaneous leishmaniasis. III. Histopathological aspects of the developmental behavior of the cutaneous lesion induced in Cebus apella (Primates: Cebidae) by Leishmania (Viannia) lainsoni, L. (V.) braziliensis and L. (Leishmania) amazonensis]
27.) Immune responses induced by a Leishmania (Leishmania) amazonensis recombinant antigen in mice and lymphocytes from vaccinated subjects.
27A.) Vervet monkeys vaccinated with killed Leishmania major parasites and interleukin-12 develop a type 1 immune response but are not protected against challenge infection.
28.) Protective immunity using recombinant human IL-12 and alum as adjuvants in a primate model of cutaneous leishmaniasis.
29.) Leishmaniasis in Brazil: XII. Observations on cross-immunity in monkeys and man infected with Leishmania mexicana mexicana, L. m. amazonensis, L. braziliensis braziliensis, L. b. guyanensis and L. b. panamensis.
30.) Leishmania pifanoi amastigote antigens protect mice against cutaneous leishmaniasis.
31.) Specific immunization of mice against Leishmania mexicana amazonensis using solubilized promastigotes.
32.) Membrane glycoprotein M-2 protects against Leishmania amazonensis infection.
33.) The role of BCG in human immune responses induced by multiple injections of autoclaved Leishmania major as a candidate vaccine against leishmaniasis.
34.) Randomised vaccine trial of single dose of killed Leishmania major plus BCG against anthroponotic cutaneous leishmaniasis in Bam, Iran.
35.) A randomised, double-blind, controlled trial of a killed L. major vaccine plus BCG against zoonotic cutaneous leishmaniasis in Iran.
36.) Immunogenicity and safety of autoclaved Leishmania major plus BCG vaccine in healthy Sudanese volunteers.
37.) Immune responses in vaccinated dogs with autoclaved Leishmania major promastigotes.
38.) Study of the safety, immunogenicity and efficacy of attenuated and killed Leishmania (Leishmania) major vaccines in a rhesus monkey (Macaca mulatta) model of the human disease.
39.) Safety and immunogenicity of a killed Leishmania (L.) amazonensis vaccine against cutaneous leishmaniasis in Colombia: a randomized controlled trial.
40.) Immune response in healthy volunteers vaccinated with killed leishmanial promastigotes plus BCG. I: Skin-test reactivity, T-cell proliferation and interferon-gamma production.
41.) The effect of repeated leishmanin skin testing on the immune responses to Leishmania antigen in healthy volunteers.
42.) Evaluation of the stability and immunogenicity of autoclaved and nonautoclaved preparations of a vaccine against American tegumentary leishmaniasis.
43.) A randomized double-blind placebo-controlled trial to evaluate the immunogenicity of a candidate vaccine against American tegumentary leishmaniasis.
44.) BCG expressing LCR1 of Leishmania chagasi induces protective immunity in susceptible mice.
45.) Immune response in healthy volunteers vaccinated with BCG plus killed leishmanial promastigotes: antibody responses to mycobacterial and leishmanial antigens.
46.) Immunotherapy with live BCG plus heat killed Leishmania induces a T helper 1-like response in American cutaneous leishmaniasis patients.
47.) Vaccination of mice with a combination of BCG and killed Leishmania promastigotes reduces acute Trypanosoma cruzi infection by promoting an IFN-gamma response.
48.) Immunization of BALB/c mice with mIFN-gamma-secreting Mycobacterium bovis BCG provides early protection against Leishmania major infection.
49.) Short report: evaluation of the potency and stability of a candidate vaccine against American cutaneous leishmaniasis.
50.) The immunology of susceptibility and resistance to Leishmania major in mice.
51.) Optimization of DNA vaccination against cutaneous leishmaniasis.
52.) Canine leishmaniasis: epidemiological risk and the experimental model.
53.) Protective efficacy of a tandemly linked, multi-subunit recombinant leishmanial vaccine (Leish-111f) formulated in MPL adjuvant.
54.) Molecular biological applications in the diagnosis and control of leishmaniasis and parasite identification.
55.) Immunochemotherapy in American cutaneous leishmaniasis: immunological aspects before and after treatment.
56.) T cell responses to crude and defined leishmanial antigens in patients from the lower Amazon region of Brazil infected with different species of Leishmania of the subgenera Leishmania and Viannia.
57.) Cell-mediated immunity in localized cutaneous leishmaniasis patients before and after treatment with immunotherapy or chemotherapy.
58.) Immunotherapy as a treatment of American cutaneous leishmaniasis: preliminary studies in Brazil.
59.) T-cell responsiveness of American cutaneous leishmaniasis patients to purified Leishmania pifanoi amastigote antigens and Leishmania braziliensis promastigote antigens: immunologic patterns associated with cure.
60.) Dichotomy of the T cell response to Leishmania antigens in patients suffering from cutaneous leishmaniasis; absence or scarcity of Th1 activity is associated with severe infections.
61.) Chemotherapy for cutaneous leishmaniasis: a controlled trial using killed Leishmania (Leishmania) amazonensis vaccine plus antimonial.
62.) Immunotherapy versus chemotherapy in localised cutaneous leishmaniasis.
63.)Immunotherapy of localized, intermediate, and diffuse forms of American cutaneous leishmaniasis.
64.) Characterization of the immune response in subjects with self-healing cutaneous leishmaniasis.
65.) Antigen provoking gamma interferon production in response to Mycobacterium bovis BCG and functional difference in T-cell responses to this antigen between viable and killed BCG-immunized mice.
66.) Autoclaved Leishmania major vaccine for prevention of visceral leishmaniasis: a randomised, double-blind, BCG-controlled trial in Sudan.
67.) Leishmania donovani p36(LACK) DNA vaccine is highly immunogenic but not protective against experimental visceral leishmaniasis.
68.) Vaccination of Balb/c mice against experimental visceral leishmaniasis with the GP36 glycoprotein antigen of Leishmania donovani.
69.) Vaccination of langur monkeys (Presbytis entellus) against Leishmania donovani with autoclaved L. major plus BCG.
70.) Successful vaccination against Leishmania donovani infection in Indian langur using alum-precipitated autoclaved Leishmania major with BCG.
71.) [Protective effects of leishmanial antigens against Leishmania infantum infection in Lagurus lagurus]
72.) Vaccination with DNA encoding ORFF antigen confers protective immunity in mice infected with Leishmania donovani.
73.) Intradermal infection model for pathogenesis and vaccine studies of murine visceral leishmaniasis.
74.) Saponins, IL12 and BCG adjuvant in the FML-vaccine formulation against murine visceral leishmaniasis.
75.) COMPONENT OF FLY SALIVA MAKES PROMISING LEISHMANIASIS VACCINE
76.) Effort to Target Clinical Development of Corixa's Leishmaniasis Vaccine
77.) Report on the fourth TDR/IDRI meeting on second generation vaccine against Leishmaniasis. Merida, Yucatan, Mexico, May 1-3, 2001.
78.) Drug development: LEISHMANIASIS
79.) Vaccines for cutaneous leishmaniasis
80.) Vaccine development: LEISHMANIASIS
81.) Vaccine from fly spit
82.)Toward a defined anti-Leishmania vaccine targeting vector antigens: characterization of a protective salivary protein.
83.) Other studies and Bibliographical references
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1.) Vaccination against Leishmania infections.
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Curr Drug Targets Immune Endocr Metabol Disord 2002 Oct;2(3):201-26

Mauel J.

Institute of Biochemistry, Ch. des Boveresses 155, CH-1066 Epalinges, Switzerland. [email protected]

Leishmaniasis, that affects millions of people worldwide, is an infectious disease caused by the protozoan parasite Leishmania. Incidence of the condition appears to be increasing in several parts of the world. Of the three main presentations of the disease, i.e. cutaneous, mucocutaneous and visceral, only the first one tends to heal spontaneously, while the other two are considered fatal if left to run their natural course. Recovery from leishmaniasis, whether spontaneous or drug-induced, is usually accompanied by solid immunity against reinfection, which provides a rationale for attempting to design vaccines against the disease. This review presents an outline of the main immunological features of Leishmania infections and of the mechanisms thought to operate in recovery from the disease. It describes various experimental approaches to vaccination in man and animal models, including the use of virulent and avirulent organisms, of dead parasites and extracts thereof, and of purified parasite proteins. Assays using novel technologies, such as the direct injection of DNAs encoding parasite proteins, or the inoculation of viral or bacterial vectors expressing such molecules, as well as recent experiments aimed at inducing an immune response against saliva of the insect vector, are also reviewed. Observations made during the course of these studies have reinforced the notion that vaccination against leishmaniasis is indeed feasible. However, in spite of intensive efforts by many groups and many reports of success in man and in animal models, a consensus is yet to emerge as to what constitutes the best approach to vaccination against leishmaniasis.

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2.) Leishmania transmission-blocking vaccines: a review.
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East Afr Med J 1999 Feb;76(2):93-6

Tonui WK.

Centre for Biotechnology Research and Development, Kenya Medical Research Institute, Nairobi.
OBJECTIVE: To review information on transmission-blocking vaccine against Leishmania infections. DATA SOURCES: Peer-reviewed journals and accepted manuscripts in press. STUDY SELECTION: Laboratory studies based on raising of immune sera against Leishmania-derived antigens using the BALB/c and hamster murine models. Testing of the immune sera for their potential to limit the transmission of cutaneous leishmaniasis in its natural vector Phlebotomus duboscqi. DATA SYNTHESIS: Leishmania major-derived recombinant 63 kilodalton glycoprotein (rgp63), lipophosphoglycan (LPG), whole parasite, flagella and nuclear fractions; monoclonal antibodies and sandfly gut antigens have all been tested for their candidacy as Leishmania-transmission blocking vaccines. CONCLUSION: Liposphoglycan proved to be a promising candidate for Leishmania transmission-blocking vaccine studies.

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3.) Vaccination against cutaneous leishmaniasis: current status.
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Am J Clin Dermatol 2002;3(8):557-70

Melby PC.

Department of Veterans Affairs Medical Center, Medical Service, South Texas Veterans Health Care System, San Antonio, TX 78229, USA. [email protected]

The different cutaneous leishmaniases are distinct in their etiology, epidemiology, transmission, and geographical distribution. In most instances cutaneous leishmaniasis is limited to one or a few skin ulcers that develop at the site where the parasites were deposited during the bite of the sandfly vector. Lesions typically heal spontaneously after several months but some lesions can be large and follow a chronic, more severe course. Protective immunity is usually acquired following cutaneous infection with Leishmania spp., so prevention of disease through prophylactic immunization appears to be feasible. Since vaccination with live, virulent parasites is associated with an unacceptable rate of adverse events, attention has turned to the use of killed or attenuated parasite vaccines and defined subunit vaccines. Whole parasite vaccines have the advantage of delivering multiple antigenic epitopes that may be necessary for initiation of a broad-based immune response. Persistent or repeated immune-stimulation by parasite antigens and/or sustained expression of interleukin-12 appear to be critical elements in the development of durable immunity. A number of purified or recombinant antigens, when co-administered with a vaccine adjuvant, appear promising as vaccine candidates against cutaneous leishmaniasis. The sustained expression of recombinant Leishmania antigens by vaccination with DNA is an attractive approach because it mimics the persistent antigenic stimulation of subclinical infection. Effective vaccine-induced immunity must generate an antigen-specific memory T cell population that, upon exposure to the infecting parasite, rapidly produces a type 1 effector T cell response that leads to interferon-gamma-mediated activation of infected macrophages to kill the intracellular parasites. This parasite-directed recall response must be prompt and of sufficient magnitude to overcome the subversive effect that the intracellular infection has on macrophage effector function. It is unlikely that vaccination against cutaneous leishmaniasis would induce sterile immunity, but a small number of parasites are likely to persist subclinically.

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4.) Leishmaniasis: current status of vaccine development.
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Clin Microbiol Rev 2001 Apr;14(2):229-43

Handman E.

Infection and Immunity Division, The Walter and Eliza Hall Institute of Medical Research, The Royal Melbourne Hospital, Parkville 3050, Australia. [email protected]

Leishmaniae are obligatory intracellular protozoa in mononuclear phagocytes. They cause a spectrum of diseases, ranging in severity from spontaneously healing skin lesions to fatal visceral disease. Worldwide, there are 2 million new cases each year and 1/10 of the world's population is at risk of infection. To date, there are no vaccines against leishmaniasis and control measures rely on chemotherapy to alleviate disease and on vector control to reduce transmission. However, a major vaccine development program aimed initially at cutaneous leishmaniasis is under way. Studies in animal models and humans are evaluating the potential of genetically modified live attenuated vaccines, as well as a variety of recombinant antigens or the DNA encoding them. The program also focuses on new adjuvants, including cytokines, and delivery systems to target the T helper type 1 immune responses required for the elimination of this intracellular organism. The availability, in the near future, of the DNA sequences of the human and Leishmania genomes will extend the vaccine program. New vaccine candidates such as parasite virulence factors will be identified. Host susceptibility genes will be mapped to allow the vaccine to be targeted to the population most in need of protection.


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5.) Protective immunity against cutaneous leishmaniasis achieved by partly purified vaccine in a volunteer.
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Lancet 1986 Jun 28;1(8496):1490


Monjour L, Monjour E, Vouldoukis I, Ogunkolade BW, Frommel D.
Publication Types:
Letter
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6.) Vaccines against leishmaniasis.
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Ann Trop Med Parasitol 1995 Dec;89 Suppl 1:83-8

Modabber F.

UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, World Health Organization, Geneva, Switzerland.

Unlike some other parasites, Leishmania can be grown in cell-free media with ease. This simple cultivation and the use of killed parasites as skin-test antigens (leishmanin) for diagnosis in humans during the past several decades have prompted scientists to try using the killed parasites, with or without adjuvant, as vaccines or for immunotherapy. In addition, different recombinant molecules, either parasite fractions or genetically engineered organisms (i.e. Leishmania made avirulent by removing specific genes, or bacteria carrying and expressing leishmanial genes), are being investigated as potential future vaccines against leishmaniasis. The 'first-generation' vaccines, composed of killed parasites with or without adjuvant, have been derived using an empirical approach. The 'second-generation' vaccines have been genetically constructed, using a more rational approach. At present, the first-generation vaccines are at various stages of Phase I (safety), II (reactivity) or III (efficacy) trials in humans. Results are expected in 1-2 years. The second-generation vaccines are, however, only in a preclinical state and are not expected to reach clinical trials for at least 3 years. The Special Programme for Research and Training in Tropical Diseases (TDR) is actively involved in most clinical trials of the first-generation vaccines and supports many of the second-generation candidates. In the present article, the advantages and disadvantages of each approach to vaccine development are discussed and the progress being made is briefly reviewed.

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7.) Mechanisms of acquired immunity in leishmaniasis.
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Philos Trans R Soc Lond B Biol Sci 1984 Nov 13;307(1131):87-98

Howard JG, Liew FY.

Self-curing cutaneous leishmaniasis depends on T cell-mediated immune activation of infected macrophages. Failure of immune control in inbred mouse models of metastasizing mucocutaneous and visceralizing forms of the disease involves, respectively, insusceptibility of the parasite and the generation of T cells that suppress a potentially curative response. Prophylactic immunization in man has so far been restricted to cutaneous leishmaniasis and based on inducing infection under controlled conditions with virulent Leishmania tropica major promastigotes. The feasibility of immunization against visceral leishmaniasis merits reconsideration. BALB/c mice are genetically vulnerable to L. tropica major, which produces a fatal visceralizing type of disease involving specific suppression of cell-mediated immunity. Potent and lasting protection can be induced by repeated intravenous immunization with irradiated promastigotes. The efficacy of this 'vaccine' is relatively heat-stable (1 h at 56 degrees C). Immunity is not attributable to antibody but to the generation of Lyt-1+2- T cells which, although possessing helper and macrophage-activating functions, do not express classical delayed-type hypersensitivity. The immunological features of this system and its relevance to the possibility of protection against human Leishmania donovani infection are considered.

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8.) Experimental leishmaniasis in humans: review.
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Rev Infect Dis 1991 Sep-Oct;13(5):1009-17

Melby PC.

Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland.

Experimental infection of humans with Leishmania parasites has contributed significantly to the understanding of the etiology, transmission, and pathogenesis of leishmaniasis and the immunity associated with it. Leishmania organisms recovered from human and animal tissue, insect vectors, and in vitro cultures have all produced cutaneous or visceral leishmaniasis in human subjects who were voluntarily inoculated with them. Volunteers bitten by infected Phlebotomine sandflies also developed cutaneous or visceral disease. In these experiments, it appeared that the parasite must undergo certain developmental changes within the sandfly for it to become infective and that the parasites in sandflies were far more efficient in causing full-blown infection than were cultured Leishmania organisms. The clinical manifestations of these experimental infections did not differ from infections that were acquired naturally. Natural or experimental infections appeared to confer resistance to subsequent leishmanial infection. This immunity was best documented to be a species-specific phenomenon; however, a small number of studies have demonstrated cross protection between some Leishmania species. In this review article, data from human experimental infections are summarized and discussed in light of recent advances in the field.

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9.) Heterologous protection in murine cutaneous leishmaniasis.
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Immunol Cell Biol 1987 Oct;65 Pt 5:387-92

Mitchell GF, Handman E.

Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia.

Mice immunized with a glycolipid antigen (GL) of Leishmania major plus adjuvant are relatively resistant to subsequent infection with this protozoan parasite. The GL is affinity purified on the monoclonal antibody WIC-79.3 which is L. major-specific and does not react with L. donovani. When another monoclonal, WIC-108.3, which cross-reacts with several Leishmania species, is used to affinity purify GL from L. donovani, the eluted material can partially protect genetically resistant mice against L. major. Thus, GL cross-reactions may in part underlie the known protective effects of crude L. donovani antigens against L. major infection. Experiments with live parasites of the L. major isolate LRC-L119, that is non-pathogenic in mice, that does not survive long in macrophages in vitro, and that has not been shown to contain any WIC-79.3 reactive GL, indicated that this isolate will very effectively protect mice against subsequent infection. This raises the possibility that GL is only one of at least two different classes of vaccinating antigen capable of protectively immunizing mice in this cutaneous leishmaniasis model.

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10.) IgE antibody against surface antigens of Leishmania promastigotes in American cutaneous leishmaniasis.
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Parasite Immunol 1986 Mar;8(2):109-16

Lynch NR, Malave C, Turner KJ, Infante B.

As IgE-mediated immune mechanisms participate in the host defence against some types of parasites, we evaluated sera from American cutaneous leishmaniasis (ACL) patients for the presence of this antibody against Leishmania. Using monoclonal antibodies against human IgE and an immunoperoxidase staining technique, 48% of the patients sera tested were found to contain IgE antibody that bound strongly to Leishmania promastigotes. A much lower proportion of sera from non-symptomatic subjects from either endemic or non-endemic areas of the disease contained significant levels of anti-Leishmania IgE antibody (6.5% and 0% respectively). The results indicated that the IgE antibody bound predominantly to surface components of the promastigotes, and reactivity against the intracellular amastigote form of the parasite was rarely detected. Somewhat unexpectedly, in a small proportion of the sera, the IgE antibody showed apparent specificity for L. mexicana or L. braziliensis. This study demonstrates that ACL patients can develop anti-Leishmania IgE antibody responses, that seem to be directed preferentially against surface antigens of promastigotes, and that can be strain specific. This raises the question as to the possible contribution of this antibody to the immune defence mechanisms against the parasite.

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11.) A comparison of the molecular biology of trypanosomes and leishmaniae, and its impact on the development of methods for the diagnosis and vaccination of leishmaniasis and Chagas disease.
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Biol Res 1993;26(1-2):219-24

O'Daly JA.

Centro de Microbiologia y Biologia Celular, Instituto Venezolano de Investigaciones Cientificas (IVIC), Caracas.

Our main interest have focused on Chagas disease and Leishmaniasis, working in the areas of: 1--The molecular biology of Trypanosomes and Leishmaniae, and 2--The immunology of Chagas disease, cutaneous leishmaniasis and visceral leishmaniasis. In this article we summarize the work realized in the last 20 years in the Immunobiology Laboratory at the IVIC with special emphasis in the development of a vaccine against leishmaniasis that is being currently used in a field trial in human beings of the endemic area of Guatire, Miranda State, Venezuela.

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12.) Dendritic cell-based vaccination strategies: induction of protective immunity against leishmaniasis.
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Immunobiology 2001 Dec;204(5):659-66

Moll H, Berberich C.

Institute for Molecular Biology of Infectious Diseases, University of Wurzburg, Germany. [email protected]

The clinical symptoms caused by infections with Leishmania parasites range from self-healing cutaneous to uncontrolled visceral disease and depend not only on the parasite species but also on the type of the host's immune response. Infection of genetically susceptible mice with Leishmania major results in the development of disease-promoting T helper cells of type 2 (Th2). On the other hand, healing of lesions is dependent on the induction of Th1 cells producing interferon-gamma (IFN-gamma). The presence of interleukin 12 (IL-12) is known to be crucial for the differentiation of Th1 cells. Whereas IL-12 release and the T cell stimulatory functions of macrophages are down-regulated by L. major infection, dendritic cells (DC) exposed to L. major readily produce IL-12 and are highly potent antigen-presenting cells. Moreover, DC pulsed ex vivo with L. major antigen induce protection in otherwise susceptible mice against subsequent challenges with the parasites. The protection is long-lasting and correlates with a shift of the cytokine expression pattern towards a Th1 response. Thus, DC serve as immunomodulators in vivo and can be used as an effective adjuvant for vaccination against experimental leishmaniasis. Studies on the ability of DC to induce protective immunity to leishmaniasis may have important implications for the development of novel strategies for prophylactic and therapeutic immunizations against microbial pathogens.

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13.) Effective immunization against cutaneous leishmaniasis with defined membrane antigens reconstituted into liposomes.
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J Immunol 1988 Feb 15;140(4):1274-9 Related Articles, Links

Erratum in:
J Immunol 1988 Apr 15;140(8):2858

Russell DG, Alexander J.

Max-Planck-Institut fur Biologie, Tubingen, Federal Republic of Germany.

The abundant Leishmania promastigote surface Ag gp63 and Leishmania promastigote lipophosphoglycan were reconstituted into liposomes and used as a vaccine against the agent of New World cutaneous leishmaniasis, Leishmania mexicana. The Ag were inoculated s.c., i.p., and i.v. into CBA/ca and BALB/c mice. Even at low Ag dosages, 8 to 10 micrograms/mouse, the Ag induced appreciable levels of protection. In CBA/ca mice complete protection was obtained by s.c. inoculation of antigen-containing liposomes. Protection could be transferred with T cells to naive mice. Interestingly, the Ag-containing liposomes did not cause the disease exacerbation observed in previous vaccine studies with crude parasite extracts.

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14.) [An immunologic method of producing antigens for the treatment and prevention of leishmaniasis]
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C R Acad Sci III 1989;308(5):129-34

[Article in French]

Vouldoukis I, Monjour L.

Laboratoire de Parasitologie experimentale et I.N.S.E.R.M. U n. 313, Faculte de Medecine Pitie-Salpetriere, Paris.

Antigens isolated from lysates of L. infantum promastigotes by electroelution from polyacrylamide gels and by gel filtration, have already been proven to induce anti-Leishmania protective immunity in BALB/c mice when injected subcutaneously. At the present time, five preparations including either Leishmania antigens or anti-idiotypic reagents as vaccines can be considered as candidates for immunoprophylaxis in natural hosts of Leishmania parasites. We present a new method for producing Leishmania antigenic preparations which is of considerable interest, since it can be proposed for immunotherapy and prevention of other parasitic, bacterial, viral and perhaps, retroviral diseases.

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15.) Development of vaccines against leishmaniasis.
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Scand J Infect Dis Suppl 1990;76:72-8

Modabber F.

UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR), Geneva, Switzerland.

A vaccine against leishmaniasis is the only practical means to control this disease in many epidemiological situations. Two approaches have been adopted: pragmatic and systematic. The pragmatic approach involves trial of crude leishmanial components in animals and then in humans if they meet safety requirements. The systematic approach requires identification of the protective immunogen(s), appropriate carrier and adjuvant, and determination of the immune responses and modes of presentation of the immunogens to achieve the desired effect. Progress have been made with both approaches. Killed Leishmania promastigotes have been used in Brazil for high risk individuals with encouraging results. Impressive results have also been observed with killed Leishmania plus BCG for immunotherapy of cutaneous leishmaniasis in Venezuela. With the systematic approach, recent research has identified some protective immunogens, cloned protective murine T-cells, developed primate models resembling the human disease, cloned and expressed genes of some potential immunogens, identified some features of the protective immune response, determined modes of presentation of immunogen to produce a protective response, and been able to protect mice (even/Balb/c) against L. major infection. The difficult part that remains is the implementation of a vaccine or any control measure in the poor communities where they are needed and where the lack of required infrastructure does not allow adequate coverage.

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16.) Further trials of a vaccine against American cutaneous leishmaniasis.
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Trans R Soc Trop Med Hyg 1986;80(6):1001 Related Articles, Links

Mayrink W, Antunes CM, Da Costa CA, Melo MN, Dias M, Michalick MS, Magalhaes PA, De Oliveira Lima A, Williams P.

Publication Types:
Clinical Trial
Letter
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17.) Vaccination against cutaneous leishmaniasis in mice using nonpathogenic cloned promastigotes of Leishmania major and importance of route of injection.
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Aust J Exp Biol Med Sci 1984 Apr;62 ( Pt 2):145-53

Mitchell GF, Handman E, Spithill TW.

In vaccination studies, mice have been injected by different routes with living promastigotes of nonpathogenic leishmania followed by cutaneous challenge with pathogenic promastigotes. Parasites used for vaccination have been promastigotes of the cloned parasite lines A12 and A52 derived from Leishmania major isolate L137, or long-term cultured promastigotes of the leishmaniasis recidiva isolate, L32 (L.t.tropica). None of these protozoa causes lesions after cutaneous injection to mice. Disease in previously injected mice has been monitored after cutaneous challenge with promastigotes of a virulent cloned line, V121, derived from isolate L137. Mice used were C57BL/6 (genetically resistant), BALB/c and BALB/c.H-2b (genetically susceptible) and BALB/c.H-2k (also genetically susceptible but sometimes less so than BALB/c). C57BL/6 mice were almost completely resistant to subsequent cutaneous disease when challenged after intraperitoneal injection of viable nonpathogenic cloned promastigotes. In contrast, BALB/c, BALB/c.H-2b and BALB/c.H-2k mice challenged after intravenous or intraperitoneal injection were only protected partially against cutaneous leishmaniasis. These vaccinated mice generally showed persistent low grade cutaneous disease for many months after challenge. High doses of viable L32 promastigotes injected intraperitoneally were also able to induce a degree of resistance to subsequent cutaneous leishmaniasis. Using any protocol, subcutaneous injections have been totally without protective effects as have been killed promastigotes injected by any route to mice. Subcutaneous injections appear to be ineffective rather than counterproductive in that mice injected by both the intravenous and subcutaneous routes with nonpathogenic living cloned promastigotes resemble mice injected by the intravenous route in their disease status following challenge.

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18.) Leishmaniasis.
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Infect Dis Clin North Am 1993 Sep;7(3):527-46 Related Articles, Links

Evans TG.

Department of Medicine, University of Utah, Salt Lake City.

Leishmanial infections include three major clinical syndromes: visceral, cutaneous, and mucosal leishmaniasis. Visceral leishmaniasis, usually due to Leishmania donovani, has received increasing attention in the United States because of the growing number of cases seen in AIDS patients and the occurrence of viscerotropic L. tropica disease among Persian Gulf war participants. Cutaneous leishmaniasis is a relatively benign disease caused by L. Mexicana and L. (Viannia) species in the New World, and L. major, L. tropica, and L. aethiopica in the Old World. Many of these cutaneous lesions are self-healing, and treatment recommendations vary and continue to undergo evolution and study. Mucosal disease, caused by Leishmania (V.) braziliensis, is difficult to diagnose as well as to treat. A practical approach to the clinical presentation, diagnostic measures, and some treatment options of these syndromes is presented in relation to specific case studies.


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19.) Leishmania (Leishmania) amazonensis-induced cutaneous leishmaniasis in the primate Cebus apella: a model for vaccine trials.
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Int J Parasitol 2002 Dec 19;32(14):1755-64

Garcez LM, Goto H, Ramos PK, Brigido Mdo C, Gomes PA, Souza RA, De Luca PM, Mendonca SC, Muniz JA, Shaw JJ.

Secao de Parasitologia, Programa de Imunologia, Instituto Evandro Chagas, Fundacao Nacional de Saude, Ministerio da Saude-BR 316, Km 7 S/N, CEP 67.030-070, Ananindeua, PA, Brazil. [email protected]

A primate model of leishmaniasis was developed with the objective of future vaccine testing. Lesion development and immunological parameters were studied upon primary and secondary infections. Seven Cebus apella were injected subcutaneously with 2 x 10(6) Leishmania (Leishmania) amazonensis promastigotes. Erythematous nodules appeared 19-29 days p.i., which disappeared 100 days p.i. Four months later, six of the monkeys were challenged with the same inoculum; three of them developed erythematous nodules after 7 days p.i., with ulcer formation in two of these subjects. The lesions were short-lived and all were cured 40 days post challenge. Anti-Leishmania IgG antibodies were detected and they increased after the challenge infection. Leishmania antigen-induced lymphoproliferation was found 1 month post-primary infection, which coincided with IFN-gamma production and lesion development. It decreased to control levels afterwards, but at the time of the challenge dose, it was significantly above the initial level. After the challenge infection, it first increased then decreased sharply at 40 days post-challenge, coinciding with the healing of the lesion. It increased again to a higher level at 60 days post-challenge. Leishmania (Leishmania) amazonensis-infection in C. apella did not induce complete protection against a secondary infection with a homologous parasite although specific antibody production and lymphoproliferation with IFN-gamma production were observed. This fact indicates that vaccine has to be better than infection in the induction of protective immunity, and raises a question on in vitro parameters that should be considered as a counterpart of expected protection induced by vaccine candidate. In addition, we conclude that this is a useful primate model for the evaluation of candidate vaccines.

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20.) [Experimental skin leishmaniasis: II--course of the infection in the Cebus apella primate (Cebidae) caused by Leishmania (V.) braziliensis and L. (L.) amazonensis]
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Rev Soc Bras Med Trop 1990 Jan-Mar;23(1):5-12

[Article in Portuguese]

Silveira FT, Lainson R, Shaw JJ, Garcez LM, Souza AA, Braga RR, Ishikawa EA.

Instituto Evandro Chagas Fundacao SESP, Brasil.

As a means of assessing the usefulness of the monkey Cebus apella as an experimental model for the study of cutaneous leishmaniasis, 5 of these animals were inoculated intradermally at 8 sites along the tail with 3 X 10(6) promastigotes of L. (V.) braziliensis, while a further 5 monkeys received similar inoculations with 3 X 10(6) promastigotes of L. (L.) amazonensis. Following the inoculations, weekly examinations and monthly biopsies showed evolution of resulting skin lesions to be as follows: a) L. (V.) braziliensis: lesions were first visible 15-20 days post-inoculation (p.i), and at 30 days they were clearly of an erythematous-papular nature, which assumed a nodular form at 60 days; after 3 months a spontaneous ulceration of these lesions was noted and, at 4 months, the initiation of healing. In one animal total healing was apparent 5 months p.i; in two others at 6 months, in another monkey after 7 months, and in the last animal at 10 months p.i. Amastigotes were demonstrated in smears from the lesions of all monkeys up to 90 days p.i; up to 120 days in two animals, and at 180 days p.i. in the monkey which showed resolution of the lesions after 10 months. b) L. (L.) amazonensis lesions were first apparent after 20 days p.i; at 30 days they were of an erythematous-papular nature, developing into nodules at 60 days. From the third month of infection onwards, however, the lesions diminished rapidly and, at 90 days p.i. amastigotes were no longer detectable in the skin.(ABSTRACT TRUNCATED AT 250 WORDS)

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21.) Leishmania (Leishmania) major-infected rhesus macaques (Macaca mulatta) develop varying levels of resistance against homologous re-infections.
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Mem Inst Oswaldo Cruz 2001 Aug;96(6):795-804

Amaral VF, Teva A, Porrozzi R, Silva AJ, Pereira MS, Oliveira-Neto MP, Grimaldi G Jr.

Departamento de Imunologia, Instituto Oswaldo Cruz-Fiocruz, 21045-900 Rio de Janeiro, RJ, Brasil.

Seven rhesus macaques were infected intradermally with 10(7) promastigotes of Leishmania (Leishmania) major. All monkeys developed a localized, ulcerative, self-healing nodular skin lesion at the site of inoculation of the parasite. Non-specific chronic inflammation and/or tuberculoid-type granulomatous reaction were the main histopathological manifestations of the disease. Serum Leishmania-specific antibodies (IgG and IgG1) were detected by ELISA in all infected animals; immunoblot analyses indicated that numerous antigens were recognized. A very high degree of variability was observed in the parasite-specific cell-mediated immune responses [as detected by measuring delayed-type hypersensitivity (DTH) reaction, in vitro lymphocyte proliferation, and gamma interferon (IFN-gamma) production] for individuals over time post challenge. From all the recovered monkeys (which showed resolution of the lesions after 11 weeks of infection), 57.2% (4/7) and 28.6% (2/7) animals remained susceptible to secondary and tertiary infections, respectively, but the disease severity was altered (i.e. lesion size was smaller and healed faster than in the primary infection). The remaining monkeys exhibited complete resistance (i.e. no lesion) to each rechallenge. Despite the inability to consistently detect correlates of cell-mediated immunity to Leishmania or correlation between resistance to challenge and DTH, lymphocyte transformation or IFN-gamma production, partial or complete acquired resistance was conferred by experimental infection. This primate model should be useful for measuring vaccine effectiveness against the human disease.

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22.) Protection of C57BL/10 mice by vaccination with association of purified proteins from Leishmania (Leishmania) amazonensis.
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Rev Inst Med Trop Sao Paulo 1999 Jul-Aug;41(4):243-8

Mora AM, Mayrink W, Costa RT, Costa CA, Genaro O, Nascimento E.

Department of Parasitology, Institute of Biological Sciences, Federal University of Minas Gerais, Brazil.

In the past few years, induction of protective immunity to cutaneous leishmaniasis has been attempted by many researchers using a variety of antigenic preparations, such as living promastigotes or promastigote extracts, partially purified, or defined proteins. In this study, eleven proteins from Leishmania (Leishmania) amazonensis (LLa) with estimated molecular mass ranging from 97 to 13.5kDa were isolated by polyacrylamide gel electrophoresis and electro-elution. The proteins were associated as vaccine in different preparations with gp63 and BCG (Bacilli Calmette-Guerin). The antigenicity of these vaccines was measured by their ability to induce the production of IFN-gamma by lymphocyte from subjects vaccinated with Leishvacinregister mark or target. The immunogenicity was evaluated in vaccinated mice. C57BL/10 mice were vaccinated with three doses of each vaccine consisting of 30 microg of each protein at 15 days interval. One hundred microg of live BCG was only used in the first dose. Seven days after the last dose, they received a first challenge infection with 10(5) infective promastigotes and four months later, a second challenge was done. Two months after the second challenge, 42.86% of protection was obtained in the group of mice vaccinated with association of proteins of gp63+46+22kDa, gp63+13.5+25+42kDa, gp63+46+42kDa, gp63+66kDa, and gp63+97kDa; 57. 14% of protection was demonstrated with gp63+46+97+13.5kDa, gp63+46+97kDa, gp63+46+33kDa, and 71.43% protection for gp63 plus all proteins. The vaccine of gp63+46+40kDa that did not protect the mice, despite the good specific stimulation of lymphocytes (LSI = 7. 60) and 10.77UI/ml of IFN-gamma production. When crude extract of L. (L.) amazonensis was used with BCG a 57.14% of protection was found after the first challenge and 28.57% after the second, the same result was observed for gp63. The data obtained with the vaccines can suggest that the future vaccine probably have to contain, except the 40kDa, a cocktail of proteins that would protect mice against cutaneous leishmaniasis.

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23.) Experimental cutaneous leishmaniasis. IV. The humoral response of Cebus apella (Primates: Cebidae) to infections of Leishmania (Leishmania) amazonensis, L. (Viannia) lainsoni and L. (V.) braziliensis using the direct agglutination test.
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Acta Trop 1997 Oct 14;68(1):65-76

Garcez LM, Silveira FT, el Harith A, Lainson R, Shaw JJ.

Leishmaniasis Program, Evandro Chagas Institute, Belem, Para, Brazil.

The direct agglutination test (DAT) was used to evaluate the serological response of 150 serum samples taken from 15 captive-bred capuchin monkeys Cebus apella. These animals had been experimentally infected with either L. (Leishmania) amazonensis, L. (Viannia) lainsoni or L. (V.) braziliensis. Monkeys infected with L. (L.) amazonensis or L. (V.) lainsoni were challenged with the homologous parasite one month after their spontaneous cure. DAT antigens were prepared from L. (L.) donovani, L. (L.) amazonensis and L. (V.) braziliensis. Antigens were difficult to standardise and it was impossible to produce an L. (V.) lainsoni antigen as parasites remained aggregated even after trypsinization. The DAT detected significant humoral responses in all the infected monkeys. Titres were higher when homologous antigens were used, especially in secondary responses. This suggests that homologous antigen should be used to detect antibodies in human cutaneous leishmaniasis.

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24.) Vaccination of C57BL/10 mice against cutaneous Leishmaniasis using killed promastigotes of different strains and species of Leishmania.
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Rev Soc Bras Med Trop 2002 Mar-Apr;35(2):125-32

Mayrink W, Santos GC, Toledo Vde P, Guimaraes TM, Machado-Coelho GL, Genaro O, da Costa CA.

Departamento de Parasitologia, Instituto de Ciencias Biologicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG.

Antigenic extracts from five Leishmania stocks were used to vaccinate C57BL/10 mice. The Leishvacin(R) and PH8 monovalent vaccine yielded the highest IFN-gamma levels in the supernatants of spleen cell culture from vaccinated animals. Each single strain immunized group showed evidence of protective immunity six months after the challenge with promastigotes of Leishmania (Leishmania) amazonensis. No differences were detected between the vaccinated groups. It can be concluded that vaccines composed of single Leishmania stocks can provide protection to C57BL/10 mice against L. (L.) amazonensis infection.

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25.) [Experimental cutaneous leishmaniasis: I--on the susceptibility of the primate Cebus apella (Cebidae) to the infection caused by Leishmania (Viannia) lainsoni Silveira, Shaw and Ishikawa, 1987]
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Rev Soc Bras Med Trop 1989 Jul-Sep;22(3):125-30

[Article in Portuguese]

Silveira FT, Lainson R, Shaw JJ, Garcez LM, Souza AA, Braga RR, Ishikawa EA.

The susceptibility of the monkey Cebus apella (Cebidae) to experimental infection by Leishmania (Viannia) lainsoni has been investigated. For this purpose, five young monkeys, 2 males and 3 females, were intradermally, inoculated, in eight different places along the dorsal surface of the tail with 3 x 10(6) promastigotes of the parasite (MHOM/BR/81/M6426, Benevides, Para), from stationary phase culture in Difco B45 medium. After inoculations, infection in the monkeys was indicated by the presence of amastigotes in the skin lesions produced in these animals at the points of inoculation, confirming the susceptibility of the monkey Cebus apella to experimental infection by Leishmania lainsoni, with an infection period of four months. This represents a suitable period for testing antileishmanial drugs or studying the pathogenesis of the disease caused by this parasite.

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26.) [Experimental cutaneous leishmaniasis. III. Histopathological aspects of the developmental behavior of the cutaneous lesion induced in Cebus apella (Primates: Cebidae) by Leishmania (Viannia) lainsoni, L. (V.) braziliensis and L. (Leishmania) amazonensis] ====================================================
Rev Inst Med Trop Sao Paulo 1990 Nov-Dec;32(6):387-94

[Article in Portuguese]

Silveira FT, Moraes MA, Lainson R, Shaw JJ.

Secao de Parasitologia, Instituto Evandro Chagas (Fundacao SESP), Belem, Para, Brasil.

We have studied the histopathological aspects related to the evolution of cutaneous lesions experimentally produced in the monkey Cebus apella (Primates: Cebidae) by Leishmania (V.) lainsoni, L. (V.) braziliensis and L. (L.) amazonensis. Microscopical examination of a series of biopsies obtained from these animals showed the kinetics of the cutaneous lesions regarding three species of Leishmania inoculated, as follows: 1) an initial non-specific chronic inflammatory infiltrate; 2) macrophagic nodules; 3) necrosis of parasitized phagocytic cells; 4) epitheliode granuloma; 5) absorption of the necrotic area (sometimes forming "foreign-body granuloma"); 6) a non-specific residual inflammatory infiltration; and 7) cicatrization. These pathological processes are, of course, responsible for both development and resolution of the leishmaniotic lesion. We also discuss some immunopathological mechanisms probably related with the sequential events, and that could be also responsible for the different clinical aspects found in man.

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27.) Immune responses induced by a Leishmania (Leishmania) amazonensis recombinant antigen in mice and lymphocytes from vaccinated subjects.
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Rev Inst Med Trop Sao Paulo 1997 Mar-Apr;39(2):71-8

Fernandes AP, Herrera EC, Mayrink W, Gazzinelli RT, Liu WY, de Costa CA, Tavares CA, Melo MN, Michalick MS, Gentz R, Nascimento E.

Departamento de Parasitologia, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.

In the search for Leishmania recombinant antigens that can be used as a vaccine against American Cutaneous Leishmaniasis, we identified a Leishmania (Leishmania) amazonensis recombinant protein of 33 kD (Larp33) which is recognized by antibodies and peripheral blood leukocytes (PBL) from subjects vaccinated with Leishvacin, Larp33 was expressed in Escherichia coli after cloning of a 2.2 kb Sau3 digested genomic fragment of L. (L.) amazonensis into the pDS56-6 His vector. Immunoblotting analysis indicated that Larp33 corresponds to an approximately 40-kD native protein expressed in promastigotes of L. (L.) amazonensis and L. (Viannia) braziliensis. Northern blots of total RNA also demonstrated that the gene coding for this protein is expressed in promastigotes of the major lineages of Leishmania causing American Cutaneous Leishmaniasis. Larp33 induced partial protection in susceptible mouse strains (BALB/c and C57BL/10) against L. (L.) amazonensis after vaccination using Bacille Calmette-Guerin (BCG) as adjuvant. In vitro stimulation of splenocytes from BALB/c protected mice with Larp33 elicited the secretion of IL-2 and IFN-gamma, suggesting that a Th1 cell-mediated protective response is associated with the resistance observed in these mice. As revealed by its immunogenic and antigenic properties, this novel recombinant antigen is a suitable candidate to compose a vaccine against cutaneous leishmaniasis.

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27.) Vervet monkeys vaccinated with killed Leishmania major parasites and interleukin-12 develop a type 1 immune response but are not protected against challenge infection.
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Infect Immun 2001 Jan;69(1):245-51

Gicheru MM, Olobo JO, Anjili CO, Orago AS, Modabber F, Scott P.

Institute of Primate Research, Nairobi, Kenya.

Leishmania major is a protozoan parasite that causes chronic cutaneous lesions that often leave disfiguring scars. Infections in mice have demonstrated that leishmanial vaccines that include interleukin-12 (IL-12) as an adjuvant are able to induce protective immunity. In this study, we assessed the safety, immunopotency, and adjuvant potential of two doses of IL-12 when used with a killed L. major vaccine in vervet monkeys. The induction of cell-mediated immunity following vaccination was determined by measuring delayed-type hypersensitivity, in vitro lymphocyte proliferation, and gamma interferon (IFN-gamma) production. Protection was assessed by challenging the animals with L. major parasites and monitoring the course of infection. At low doses of IL-12 (10 microg), a small increase in the parameters of cell-mediated immunity was observed, relative to those in animals that received antigen without IL-12. However, none of these animals were protected against a challenge infection. At higher doses of IL-12 (30 microg), a substantial increase in Leishmania-specific immune responses was observed, and monkeys immunized with antigen and IL-12 exhibited an IFN-gamma response that was as great as that in animals that had resolved a primary infection and were immune. Nevertheless, despite the presence of correlates of protection, the disease course was only slightly altered, and protection was low compared to that in self-cured monkeys. These data suggest that protection against leishmaniasis may require more than the activation of Leishmania-specific IFN-gamma-producing T cells, which has important implications for designing a vaccine against leishmaniasis.


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28.) Protective immunity using recombinant human IL-12 and alum as adjuvants in a primate model of cutaneous leishmaniasis.
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J Immunol 1999 Oct 15;163(8):4481-8

Kenney RT, Sacks DL, Sypek JP, Vilela L, Gam AA, Evans-Davis K.

Laboratory of Parasitic Biology, Division of Veterinary Medicine, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20852, USA. [email protected]

Protection from cutaneous leishmaniasis, a chronic ulcerating skin lesion affecting millions, has been achieved historically using live virulent preparations of the parasite. Killed or recombinant Ags that could be safer as vaccines generally require an adjuvant for induction of a strong Th1 response in murine models. Murine rIL-12 as an adjuvant with soluble Leishmania Ag has been shown to protect susceptible mice. We used 48 rhesus macaques to assess the safety, immunogenicity, and efficacy of a vaccine combining heat-killed Leishmania amazonensis with human rIL-12 (rhIL-12) and alum (aluminum hydroxide gel) as adjuvants. The single s.c. vaccination was found to be safe and immunogenic, although a small transient s.c. nodule developed at the site. Groups receiving rhIL-12 had an augmented in vitro Ag-specific IFN-gamma response after vaccination, as well as increased production of IgG. No increase in IL-4 or IL-10 was found in cell culture supernatants from either control or experimental groups. Delayed hypersensitivity reactions were not predictive of protection. Intradermal forehead challenge infection with 107 metacyclic L. amazonensis promastigotes at 4 wk demonstrated protective immunity in all 12 monkeys receiving 2 microgram rhIL-12 with alum and Ag. Partial efficacy was seen with lower doses of rhIL-12 and in groups lacking either adjuvant. Thus, a single dose vaccine with killed Ag using rhIL-12 and alum as adjuvants was safe and fully effective in this primate model of cutaneous leishmaniasis. This study extends the murine data to primates, and provides a basis for further human trials.

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29.) Leishmaniasis in Brazil: XII. Observations on cross-immunity in monkeys and man infected with Leishmania mexicana mexicana, L. m. amazonensis, L. braziliensis braziliensis, L. b. guyanensis and L. b. panamensis.
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J Trop Med Hyg 1977 Feb;80(2):29-35

Lainson R, Shaw JJ.

Cross-immunity trials in monkeys (Cebus apella apella) and observations on experimental and natural infections in man confirm the separate identity of L. mexicana mexicana, L. m. amazonensis, L. b. braziliensis, L. b. guyanensis and L. b. panamensis. Neither L. m. mexicana nor L. m. amazonensis infections gave protection against subsequent challenge with parasites of the L. braziliensis complex; but recovery from infection with subspecies of L. braziliensis in most cases gave firm resistance to infection with the mexicana parasites. The failure of certain New World leishmanias to immunize against each other has an important bearing on taxonomy, future attempts to prepare vaccines against Leishmania, epidemiology and diagnosis.

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30.) Leishmania pifanoi amastigote antigens protect mice against cutaneous leishmaniasis.
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Infect Immun 1995 Sep;63(9):3559-66

Soong L, Duboise SM, Kima P, McMahon-Pratt D.

Department of Epidemiology and Public Health, Yale University School of Medicine, New Haven, Connecticut 06520-8034, USA.

In the search for a leishmaniasis vaccine, extensive studies have been carried out with promastigote (insect stage) molecules. Information in this regard on amastigote (mammalian host stage) molecules is limited. To investigate host immune responses to Leishmania amastigote antigens, we purified three stage-specific antigens (A2, P4, and P8) from in vitro-cultivated amastigotes of Leishmania pifanoi by using immunoaffinity chromatography. We found that with Corynebacterium parvum as an adjuvant, three intraperitoneal injections of 5 micrograms of P4 or P8 antigen provided partial to complete protection of BALB/c mice challenged with 10(5) to 10(7) L. pifanoi promastigotes. These immunized mice developed significantly smaller or no lesions and exhibited a 39- to 1.6 x 10(5)-fold reduction of lesion parasite burden after 15 to 20 weeks of infection. In addition, P8 immunization resulted in complete protection against L. amazonensis infection of CBA/J mice and partial protection of BALB/c mice, suggesting that this antigen provided cross-species protection of mice with different H-2 haplotypes. At different stages during infection, vaccinated mice exhibited profound proliferative responses to parasite antigens and increased levels of gamma interferon production, suggesting that a Th1 cell-mediated immune response is associated with the resistance in these mice. Taken together, the data in this report indicate the vaccine potential of amastigote-derived antigens.

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31.) Specific immunization of mice against Leishmania mexicana amazonensis using solubilized promastigotes.
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Clin Exp Immunol 1987 Jan;67(1):11-9

Barral-Netto M, Reed SG, Sadigursky M, Sonnenfeld G.

Successful immunization of highly susceptible BALB/c mice against progressive infection by Leishmania mexicana amazonensis, using whole solubilized promastigotes was achieved. The best immunization schedule consisted of three weekly injections of 5 X 10(7) parasite equivalents. Intravenous was superior to intraperitoneal or subcutaneous immunization. Protection persisted for up to 2 months after immunization, and beneficial effects could be observed in long-term follow-up (24 weeks after infection). Immunized mice exhibited marked reduction in primary lesion size, as well as reduction of the number of parasites in the spleen, and developed less metastases. High titres of specific anti-L. m. amazonensis IgG antibodies resulted from immunization, but titres did not correlate with protection. Groups with widely differing pre-infection antibody titres were equally protected, and similar antibody titres resulted in different levels of protection. Immunization alone did not induce significant serum interferon-gamma levels and specific delayed-type hypersensitivity (DTH) reactions, but resulted in the persistence of positive (DTH) reactions after infection, at a time when infected control animals had suppressed responses. Resistance to leishmaniasis appears to depend on cell mediated immune mechanisms, and the possibility of immunization with a solubilized antigen without adjuvant is intriguing and opens new perspectives in this area.

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32.) Membrane glycoprotein M-2 protects against Leishmania amazonensis infection.
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Infect Immun 1988 Dec;56(12):3272-9

Champsi J, McMahon-Pratt D.

Department of Epidemiology, Yale University School of Medicine, New Haven, Connecticut 06510.

Previous passive antibody transfer experiments have indicated that immunity to a 46-kilodalton membrane glycoprotein (M-2) of Leishmania amazonensis may protect against infection with this parasite. In the studies described in this paper, we investigated the ability of the purified M-2 molecule to elicit a protective immune response in conjunction with Freund incomplete and complete adjuvants, saponin, and Corynebacterium parvum. Both relatively susceptible (BALB/c and CBA) and resistant (C57BL/6) strains of mice were examined. C. parvum appeared to be the most effective adjuvant in the three mouse strains tested. The level of protection varied with the mouse strain, although all animals received identical preparations of antigen and adjuvant. Immunization of CBA mice with the M-2 glycoprotein and C. parvum resulted in complete protection against a challenge infection of 10(4) and 10(6) late log-phase promastigotes of L. amazonensis. In the BALB/c strain, complete protection was observed in some of the immunized animals (28 to 50%); in the rest of the mice the onset of infection was significantly delayed. Protective immunity for C57BL/6 mice was observed only at the low infecting dose (10(4) L. amazonensis organisms). The level of protection observed is reflected by increased antibody response (immunoglobulins G1 and G2) developed to the M-2 molecule. The relationship of pure T-cell (nonantibody) immunity to this protection remains to be elucidated.

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33.) The role of BCG in human immune responses induced by multiple injections of autoclaved Leishmania major as a candidate vaccine against leishmaniasis.
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Vaccine 2002 Dec 13;21(3-4):174-80 Related Articles, Links

Alimohammadian MH, Khamesipour A, Darabi H, Firooz A, Malekzadeh S, Bahonar A, Dowlati Y, Modabber F.

Department of Immunology, Pasteur Institute of Iran, Tehran, Iran

To determine if BCG was required in booster injections for autoclaved Leishmania major (ALM) vaccine, 75 volunteers with no response to leishmanin were injected double-blind and randomly with either ALM+BCG or BCG alone for the first injection and boosted either with ALM+BCG, ALM or BCG alone for the second and third. Addition of BCG to the boosters significantly increased the frequency and the magnitude of leishmanin skin tests (LSTs); however, there was no difference in proliferative and IFN-gamma responses (a month and a year later). Three injections of BCG produced no observable adverse reaction; hence BCG could be used in booster injections to increase the protective potential of this candidate vaccine.

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34.) Randomised vaccine trial of single dose of killed Leishmania major plus BCG against anthroponotic cutaneous leishmaniasis in Bam, Iran.
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Lancet 1998 May 23;351(9115):1540-3

Sharifi I, FeKri AR, Aflatonian MR, Khamesipour A, Nadim A, Mousavi MR, Momeni AZ, Dowlati Y, Godal T, Zicker F, Smith PG, Modabber F.

Kerman University of Medical Sciences, Medical School, Iran.

BACKGROUND: A vaccine consisting of a single dose of whole-cell autoclave-killed Leishmania major (ALM) mixed with BCG was assessed in comparison with BCG alone against anthroponotic (human to human transmission) cutaneous leishmaniasis in a randomised double-blind trial in Bam, Iran. METHODS: 3637 schoolchildren, aged 6-15 years, with no history of cutaneous leishmaniasis and no response to a leishmanin skin test, were randomly assigned to receive 1 mg ALM mixed with BCG (n = 1839), or BCG alone (n = 1798). Safety of the vaccine and the incidence of confirmed cases of cutaneous leishmaniasis were followed up for 2 years. FINDINGS: Side-effects were those usually associated with BCG vaccination, but tended to persist longer in the ALM + BCG group. After exclusion of four cases occurring within 80 days of vaccination (one in the ALM + BCG group and three in the BCG group), the 2-year incidence of cutaneous leishmaniasis did not differ significantly between vaccine and BCG groups: 2.8% vs 3.3%, respectively (total cases 112). A sex-stratified analysis showed that in boys the vaccine conferred a protective efficacy of 18% and 78% for the first and second years, respectively--a crude 2-year overall protection of 55% (95% CI 19-75%, p < 0.01). In the first 9 months after vaccination, there was a non-significant excess of cases in the ALM + BCG group (25 vs 16), whereas the incidence of cutaneous leishmaniasis thereafter was significantly reduced in the ALM + BCG group (27 vs 44, p < 0.05). INTERPRETATION: A single dose of ALM + BCG was safe and more immunogenic than BCG alone, as measured by leishmanin skin test. The exact reason for the apparent protective effect of the vaccine in boys is unknown, and may be a chance finding. However, since boys are more exposed to the infection, which is indicated by higher disease prevalence in boys in this study population, the preferential protective effect in boys may have resulted from a greater booster effect produced by repeated exposure to infected sandflies. Booster injections or alternative adjuvants should be tried to improve the potential efficacy of this vaccine.

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35.) A randomised, double-blind, controlled trial of a killed L. major vaccine plus BCG against zoonotic cutaneous leishmaniasis in Iran.
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Vaccine 1999 Feb 5;17(5):466-72

Momeni AZ, Jalayer T, Emamjomeh M, Khamesipour A, Zicker F, Ghassemi RL, Dowlati Y, Sharifi I, Aminjavaheri M, Shafiei A, Alimohammadian MH, Hashemi-Fesharki R, Nasseri K, Godal T, Smith PG, Modabber F.

Isfahan University of Medical Sciences, Iran.

Safety and efficacy of killed (autoclaved) L. major promastigotes, ALM, mixed with BCG against zoonotic cutaneous leishmaniasis was tested in healthy volunteers (n = 2453) in a randomized double blind trial vs. BCG as control. Side-effects were similar in both groups but tended to be slightly more frequent and prolonged in the ALM + BCG group. Leishmanin skin test conversion (induration > or =5 mm) was significantly greater in the ALM + BCG than in the BCG group (36.2% vs. 7.9% on day-80 and 33% vs. 19%, after 1 year, respectively). Cumulative incidence rates for 2 years, were similar in both groups (18.0% vs. 18.5%). However, LST responders on day 80 (> or =5 mm) had a significantly lower incidence (35%) of CL during the first year than non-responders. A single dose of ALM + BCG is not sufficiently immunogenic to provide a measurable response when compared to BCG alone. A single dose of this vaccine has been shown to be safe with no evidence of an exacerbating response following natural infection; hence, multiple doses or other adjuvants should be considered to increase its immunogenicity.

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36.) Immunogenicity and safety of autoclaved Leishmania major plus BCG vaccine in healthy Sudanese volunteers.
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Vaccine 2001 Feb 28;19(15-16):2100-6

Satti IN, Osman HY, Daifalla NS, Younis SA, Khalil EA, Zijlstra EE, El Hassan AM, Ghalib HW.

Institute of Endemic Diseases, University of Khartoum, P. O. Box 102, Khartoum, Sudan. [email protected]

In a longitudinal study in the epidemiology of Leishmania donovani infection in an endemic focus in eastern Sudan, we observed that previous exposure or infection with Leishmania major appeared to protect against visceral leishmaniasis caused by L. donovani. We therefore conducted a study to test the safety and immunogenicity of a vaccine consisting of autoclaved L. major (ALM) plus BCG in inducing protection in vaccinated individuals. Leishmanin-negative healthy Sudanese volunteers were enrolled in the study and were divided into three groups: group (A) received ALM+BCG, group (B) received BCG alone, and group (C) received the vaccine diluent. The subjects were examined for their clinical and immunological responses before intervention, following intervention and 6-8 weeks after vaccination. Vaccinated subjects (group A) developed localized reactions at the sites of vaccine inoculation that ulcerated and healed within 4-6 weeks; 61.6% of them converted to leishmanin reactive following vaccination. Only one subject in group (C) became leishmanin-positive. A total 76.9% of the vaccinated volunteers in group (A) produced significant levels of interferon-gamma in response to L. major antigen. The vaccine produced significant cellular immune responses that may protect against natural challenge. None of the groups had systemic reactions and all the reactions observed in the vaccinated group were comparable with the BCG-vaccinated group.

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37.) Immune responses in vaccinated dogs with autoclaved Leishmania major promastigotes.
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Vet Res 1999 Sep-Oct;30(5):441-9

Lasri S, Sahibi H, Sadak A, Jaffe CL, Rhalem A.

Departement de parasitologie et maladies parasitaires, Institut agronomique et veterinaire Hassan-II, Rabat, Morocco.

A comparative study was undertaken on the immunogen power of autoclaved Leishmania major promastigotes (ALM) vaccines given simultaneously with either BCG or saponin against canine leishmaniasis. The humoral immune response was assessed by ELISA and western blotting. The cellular immune response was evaluated by the lymphocyte transformation test. Dogs vaccinated simultaneously with ALM and saponin showed high antibody titres to crude L. infantum antigens after the first vaccine booster and reacted with several antigens, with molecular weights from 26 to 108 kDa by western blotting. However, the lymphocyte proliferation of these dogs to the crude L. infantum antigen was not significantly different from the control group. In contrast, in dogs vaccinated simultaneously with ALM and BCG, the antibody titres to crude antigen were low. Their sera reacted with the same proteins recognised by sera from dogs vaccinated simultaneously with ALM and saponin by western blotting. However, the 85-kDa protein was only identified by sera taken from dogs vaccinated simultaneously with ALM and BCG. These latter exhibited specific lymphocyte proliferation to the L. infantum antigen. This cell proliferation was observed for approximately 9 months after the first dose of the vaccine. This study indicates that a combination of ALM as the vaccine and BCG as the adjuvant, in the dog model, was successful in inducing a cell immune response, which is implicated in protection of dogs against a Leishmania infection.

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38.) Study of the safety, immunogenicity and efficacy of attenuated and killed Leishmania (Leishmania) major vaccines in a rhesus monkey (Macaca mulatta) model of the human disease.
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Mem Inst Oswaldo Cruz 2002 Oct;97(7):1041-8

Amaral VF, Teva A, Oliveira-Neto MP, Silva AJ, Pereira MS, Cupolillo E, Porrozzi R, Coutinho SG, Pirmez C, Beverley SM, Grimaldi G Jr.

Departamento de Imunologia, Instituto Oswaldo Cruz-Fiocruz, Rio de Janeiro, Brasil.

We have compared the efficacy of two Leishmania (Leishmania) major vaccines, one genetically attenuated (DHFR-TS deficient organisms), the other inactivated [autoclaved promastigotes (ALM) with bacillus Calmete-Guerin (BCG)], in protecting rhesus macaques (Macaca mulatta) against infection with virulent L. (L.) major. Positive antigen-specific recall proliferative response was observed in vaccinees (79% in attenuated parasite-vaccinated monkeys, versus 75% in ALM-plus-BCG-vaccinated animals), although none of these animals exhibited either augmented in vitro gamma interferon (IFN-gamma) production or positive delayed-type hypersensitivity (DTH) response to the leishmanin skin test prior to the challenge. Following challenge, there were significant differences in blastogenic responses (p < 0.05) between attenuated-vaccinated monkeys and naive controls. In both vaccinated groups very low levels of antibody were found before challenge, which increased after infective challenge. Protective immunity did not follow vaccination, in that monkeys exhibited skin lesion at the site of challenge in all the groups. The most striking result was the lack of pathogenicity of the attenuated parasite, which persisted in infected animals for up to three months, but were incapable of causing disease under the conditions employed. We concluded that both vaccine protocols used in this study are safe in primates, but require further improvement for vaccine application.




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39.) Safety and immunogenicity of a killed Leishmania (L.) amazonensis vaccine against cutaneous leishmaniasis in Colombia: a randomized controlled trial.
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Trans R Soc Trop Med Hyg 2000 Nov-Dec;94(6):698-703

Velez ID, del Pilar Agudelo S, Arbelaez MP, Gilchrist K, Robledo SM, Puerta JA, Zicker F, Berman J, Modabber F.

Programme for the Study and Control of Tropical Diseases (PECET), Universidad de Antioquia, Apartado Aereo 1226, Medellin, Colombia. [email protected]

The safety and immunogenicity of an intramuscular (i.m.) and intradermal (ID) formulation of autoclaved Leishmania (Leishmania) amazonensis vaccine was evaluated in 296 volunteers in a randomized, placebo-controlled, double-blind trial in Colombia. There were 4 vaccination groups: i.m. vaccine, i.m. placebo, ID vaccine, and ID placebo. The ID formulations were mixed with BCG as adjuvant at the time of injection. For each group, 3 vaccinations were given with a 20-day interval between injections, and adverse events were monitored at 20 min, and at 2, 7 and 21 days after each injection. BCG-induced adverse reactions resulted in cancellation of the third vaccine administration in the ID groups. Antibody titres did not differ significantly between the groups. Montenegro skin-test conversion was achieved by 86.4% and 90% of the i.m. vaccine group and by 25% and 5% of the i.m. placebo group 80 days and 1 year after vaccination, respectively. A significant increase in mean Leishmania-antigen lymphocyte proliferation indexes was observed after i.m. vaccine immunization, but not after i.m. placebo immunization, 80 days and 1 year after vaccination. Significant levels of IFN gamma but not IL-10 were observed 1 year after vaccination in the i.m. vaccine group compared to the i.m. placebo group. The good safety profile and evidence of Th1 immune reactions due to i.m. vaccination in this phase-I/II study suggest that a population-based phase-III efficacy trial of the i.m. vaccine should be initiated.

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40.) Immune response in healthy volunteers vaccinated with killed leishmanial promastigotes plus BCG. I: Skin-test reactivity, T-cell proliferation and interferon-gamma production.
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Vaccine 1994 Aug;12(11):1041-51

Castes M, Blackwell J, Trujillo D, Formica S, Cabrera M, Zorrilla G, Rodas A, Castellanos PL, Convit J.

Instituto de Biomedicina, Facultad de Medicina, Universidad Central de Venezuela, Caracas.

This study reports the results of a vaccine trial established to study the cellular immune responses in vivo (skin-test reactivity) and in vitro (T-cell proliferation and interferon-gamma production) to both leishmanial and mycobacterial antigens following vaccination of healthy volunteers from a leishmaniasis-endemic area with killed leishmanial promastigotes, with or without BCG (Bacille Calmette-Guerin). Skin tests were performed using purified protein derivative of tuberculin (PPD) and leishmanial antigen in 692 volunteers, and 208 doubly negative subjects (< or = 7 mm induration) were selected to participate in the trial. The study subjects were divided into four vaccine groups: (A) killed promastigotes plus BCG, (B) BCG alone, (C) killed promastigotes alone, and (D) placebo. Three vaccine doses were administered at 6-10-week intervals. The skin-test responses to PPD and leishmanial antigen were reassessed at 4-6- and 12-18-month follow-ups. The results of this trial demonstrated that the combined vaccine, i.e. killed promastigotes of Leishmania plus BCG, results in the stimulation of an immune response to both leishmania and mycobacterial antigens in a high percentage of vaccines (> 85%), manifested either by skin-test conversion, lymphocyte proliferation and/or interferon-gamma production. This was evident after the first dose of vaccine for lymphocyte proliferation and interferon-gamma production and was maintained for a year after the three doses of vaccine. Group B (which received BCG alone), responded as well as group A to PPD but not as well to leishmanial antigen. The reverse was true for group C which received promastigotes alone. Group A attained a 38% leishmanin skin-test conversion at the 4-6-month follow-up, which was associated with double PPD/leishmanial antigen responder status. In contrast, a 35% skin-test conversion was found at the 12-18-month follow-up in group C (promastigotes alone), but this was not associated with responses to PPD. A significant percentage of conversion was observed in the placebo group at the 12-18-month follow-up, both to PPD (58%) and leishmanial (21%) antigens, which suggests either environmental exposure to mycobacterial or leishmanial antigens during the vaccine trial or, more probably, a response to the repeated leishmanial skin tests. Further studies are required to determine whether the presence of proliferative and/or interferon-gamma responses in the absence of a skin-test response are sufficient indicators of potential vaccine success.

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41.) The effect of repeated leishmanin skin testing on the immune responses to Leishmania antigen in healthy volunteers.
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Trans R Soc Trop Med Hyg 2002 Sep-Oct;96(5):565-7

Satti I, el Hassan A, Khalil el TA, Akuffo H.

Microbiology and Tumour Biology Centre, Karolinska Institute, Box 280, S-171 77 Stockholm, Sweden.

The leishmanin skin test (LST) is used in immunogenicity studies. The effect of multiple LSTs on immune responses was assessed. None of the volunteers converted to LST positive. IFN-gamma and IL-10 levels remained unchanged. Repetition of LST does not modulate the in vivo or in vitro immune responses to Leishmania antigen.

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42.) Evaluation of the stability and immunogenicity of autoclaved and nonautoclaved preparations of a vaccine against American tegumentary leishmaniasis.
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Vaccine 1999 Mar 5;17(9-10):1179-85

De Luca PM, Mayrink W, Alves CR, Coutinho SG, Oliveira MP, Bertho AL, Toledo VP, Costa CA, Genaro O, Mendonca SC.

Departamento de Protozoologia, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro-RJ, Brasil.

This study was designed to evaluate the immunogenicity of autoclaved and nonautoclaved preparations of a vaccine composed of whole antigens from killed promastigotes of Leishmania amazonensis. Leishmanin skin-test (LST)-negative volunteers were immunized with either autoclaved or nonautoclaved vaccine preparations (32 and 36 subjects, respectively) that had been maintained at 4 degrees C for one year before the onset of this trial. Immunological tests were performed two days before and 40 days after vaccination. The LST conversion rates induced by the autoclaved and nonautoclaved vaccines were significantly different: 59% and 83%, respectively. Leishmania antigen-stimulated proliferative responses of peripheral blood mononuclear cells (PBMC) were significantly higher after vaccination than before vaccination in both groups. The CD8+ subset was predominant over the CD4+ subset among the leishmania-reactive cells after vaccination in both groups. The production of IFN-gamma by the leishmania antigen-stimulated PBMC was significantly higher after vaccination than before vaccination in the group receiving the nonautoclaved vaccine but not in the autoclaved vaccine group. IL-2 was found both before and after vaccination with no differences between its levels in these time points in either group. IL-4 was not detected for either group during the study period.

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43.) A randomized double-blind placebo-controlled trial to evaluate the immunogenicity of a candidate vaccine against American tegumentary leishmaniasis.
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Acta Trop 2001 Dec 21;80(3):251-60

De Luca PM, Mayrink W, Pinto JA, Coutinho SG, Santiago MA, Toledo VP, Costa CA, Genaro O, Reis AB, Mendonca SC.

Departamento de Imunologia, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil 4365-Manguinhos, CP 926, CEP 21045-900, Rio de Janeiro RJ, Brazil.

This study was aimed at evaluating the immunogenicity of a vaccine composed of killed Leishmania amazonensis promastigotes using several different protocols in a randomized, double-blind and controlled trial design in order to select one of them for further efficacy trials. One hundred and fourteen leishmanin skin test (LST)-negative healthy volunteers were allocated into eight groups that received either two or three deep intramuscular injections of vaccine at doses of 180, 360 and 540 microg or similar injections of placebo. Cell-mediated immune responses were evaluated before and after vaccination by means of LST as well as proliferative responses and cytokine production in Leishmania antigen-stimulated peripheral blood mononuclear cell cultures. The majority of the subjects who actually received vaccine converted to positive LST (89.5%). On the other hand, none of the subjects who received placebo converted to positive LST. Proliferative responses and production of interferon-gamma and interleukin-2 were significantly higher after vaccination than before vaccination in all groups, including those that received placebo. The dose of 360 microg provided the highest LST conversion rate (100%), as well as the greatest increase in interferon-gamma and interleukin-2 production after vaccination.

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44.) BCG expressing LCR1 of Leishmania chagasi induces protective immunity in susceptible mice.
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Exp Parasitol 2000 Jan;94(1):33-41

Streit JA, Recker TJ, Donelson JE, Wilson ME.

Department of Internal Medicine, 300D EMRB, University of Iowa, Iowa City 52242, USA.

Cellular immune responses are required for protective immunity against Leishmania chagasi. Immunization strategies using live intracellular bacteria (e.g., bacille-Calmette Guerin strain of Mycobacterium bovis) expressing recombinant antigens can induce cellular immune responses to these antigens. Previous studies demonstrated that the L. chagasi antigen LCR1 stimulates IFN-gamma production from T cells of infected BALB/c mice, and immunization with recombinant LCR1 partially protects against L. chagasi infection. To determine whether live bacteria could enhance the immunization potential of LCR1, we engineered BCG expressing LCR1 (BCG-LCR1). Subcutaneous immunization with BCG-LCR1, but not with BCG containing plasmid only (BCG-pMV261), elicited better protective immunity against L. chagasi infection than LCR1 protein alone. BCG-LCR1 administered intraperitoneally did not protect. Splenocytes from mice immunized s.c. with either BCG-LCR1 or BCG-pMV261 and then infected with L. chagasi promastigotes had increased antigen-induced IFN-gamma and reduced IL-10 production compared to splenocytes of control mice. We propose that BCG-LCR1 promotes a Th1-type protective immune response, and it may be a useful component of a Leishmania vaccine. Copyright 2000 Academic Press.

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45.) Immune response in healthy volunteers vaccinated with BCG plus killed leishmanial promastigotes: antibody responses to mycobacterial and leishmanial antigens.
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Vaccine 1994 Nov;12(15):1402-12 Related Articles, Links

Sharples CE, Shaw MA, Castes M, Convit J, Blackwell JM.

University of Cambridge Clinical School, Department of Medicine, Addenbrooke's Hospital, UK.

Antibody (IgG) responses to mycobacterial (BCG; PPD; Mycobacterium leprae soluble antigen, MLSA) and leishmanial (Leishmania mexicana LV4) antigens were measured in 208 initially PPD and leishmanin skin-test negative volunteers divided into four vaccine groups as follows: 68 received BCG plus killed promastigotes (group A), 47 received BCG alone (group B), 47 received killed promastigotes alone (group C), and 46 formed the diluent control (placebo, group D). Three vaccine doses were administered at 8-12 week intervals. Vaccinees were bled immediately prior to each vaccination, and again at 3- and 12-month follow-up. Skin tests were performed prevaccination, and again at the 3- and 12-month follow-up. Anti-BCG, anti-PPD and anti-MLSA IgG levels increased significantly in groups A and B receiving BCG. The presence of leishmanial antigen (with BCG) in the inoculum suppressed the IgG response to Mycobacterium tuberculosis/Mycobacterium bovis-related (PPD and BCG), but not M. leprae-related (MLSA)-related, antigens. A small but significant increase (relative to prevaccination level) in response to MLSA, but not to BCG or PPD was observed in the non-BCG-vaccinated groups. The background level of response to mycobacterial and leishmanial antigens was higher in the Venezuelan vaccinees than in non-endemic (British) volunteers. Responses to leishmanial antigen were not enhanced in the two vaccine groups receiving killed promastigotes (with/without BCG) compared with the BCG alone and placebo groups. Instead, all vaccine groups showed a pattern of response consistent with either (i) a response to the skin-test antigen or, more likely, (ii) seasonal endemic exposure to leishmanial antigen. Interestingly, this endemic response to leishmanial antigen was enhanced in the vaccine groups receiving BCG, despite the fact that group B received no leishmanial antigen in the vaccine inoculum. When prevaccination IgG levels (mean + 3 standard deviations) were used to determine a negative cut-off, a low percentage (< 38%) of vaccinees converted to responder status for either anti-mycobacterial or anti-leishmanial responses, and those who did would be classified as 'low-responder' status compared with titres observed in severe forms of disease. Hence, although there was evidence for a background endemic response to both leishmanial and mycobacterial antigens, there was no evidence that vaccination per se led to a potentially disease exacerbatory level of TH2-associated antibody response especially with respect to the anti-leishmanial response.(ABSTRACT TRUNCATED AT 400 WORDS)

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46.) Immunotherapy with live BCG plus heat killed Leishmania induces a T helper 1-like response in American cutaneous leishmaniasis patients.
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Parasite Immunol 2000 Feb;22(2):73-9

Cabrera M, Blackwell JM, Castes M, Trujillo D, Convit J, Shaw MA.

Instituto de Biomedicina, Facultad de Medicina, Universidad Central de Venezuela, Apdo 4043 (Carmelitas), Caracas 1010-A Venezuela.

Previous work has shown that American cutaneous leishmaniasis (ACL) patients treated with viable BCG plus heat killed promastigotes of Leishmania amazonensis show the same rate of cure as patients receiving conventional chemotherapy. The treatment is safe and economical, but the immunological correlates of cure have not been examined. In the present study, T cell responses have been analysed in 43 ACL patients, including patient groups sampled before and after therapy, and in 10 endemic controls. Lymphocyte proliferation, interferon (IFN)-gamma and interleukin (IL)-5 responses to crude antigen (L. amazonensis, MEL; Mycobacterium tuberculosis PPD; M. bovis BCG) stimulation, and serum IL-5 levels, were analysed. In endemic volunteers, proliferative responses to BCG were high and IFN-gamma responses low. In contrast, localized cutaneous (LCL) and mucocutaneous (MCL) patients showed low proliferative and high IFN-gamma responses to BCG. Treatment enhanced the IFN-gamma response and further decreased the proliferative response to BCG, especially in MCL patients. LCL and MCL patients showed an increase in proliferative and IFN-gamma responses to MEL with treatment, but the response was not exaggerated in MCL patients, either before or after treatment, compared to LCL patients. IL-5 production was low in T cell assays, and > 62% of untreated patients had very low serum IL-5 levels. There were no significant changes in serum IL-5 with treatment. Overall results show enhanced antigen-specific IFN-gamma responses to the two components of the immunotherapy, live M. bovis BCG and heat killed L. amazonensis, which is consistent with a shift in balance of T cell response towards a T helper 1 response and clinical cure mediated by IFN-gamma.

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47.) Vaccination of mice with a combination of BCG and killed Leishmania promastigotes reduces acute Trypanosoma cruzi infection by promoting an IFN-gamma response.
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Vaccine 1999 Feb 26;17(7-8):957-64

Araujo Z, El Bouhdidi A, Heremans H, Van Marck E, Castes M, Carlier Y.

Catedra de Inmunologia, Escuela de Medicina Jose Maria Vargas, Facultad de Medicina, Universidad Central de Venezuela, Caracas. [email protected]

The combination of BCG with killed Leishmania promastigotes, demonstrated to be efficient in the cure of patients suffering American cutaneous leishmaniasis and in the induction of a long-term immune response in healthy vaccinated volunteers, was tested in BALB/c mice infected with Trypanosoma cruzi, in comparison to BCG or Leishmania alone, and a vehicle (PBS) control. BCG-Leishmania vaccination, applied intra-peritoneally 10 and 3 days before T. cruzi trypomastigote inoculation, prolonged the survival, and reduced blood parasitaemia of infected animals. Proliferation studies indicated that splenocytes of mice vaccinated with BCG-Leishmania and harvested in the acute phase of T. cruzi infection displayed stimulation indices higher than cells from PBS-treated mice when stimulated with PHA mitogen, PPD, Leishmania or T. cruzi antigens. Injections of a monoclonal antibody able to neutralise IFN-gamma into BCG-Leishmania vaccinated mice increased parasitaemia to levels similar to those of control animals (treated with PBS) and reversed the beneficial effect of vaccination on the proliferative response to T. cruzi antigen. These results show that vaccination of mice with BCG plus killed Leishmania promastigotes delayed acute T. cruzi infection, stimulated a T-cell response to T. cruzi antigen and promoted IFN-gamma production.

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48.) Immunization of BALB/c mice with mIFN-gamma-secreting Mycobacterium bovis BCG provides early protection against Leishmania major infection.
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Int J Parasitol 1997 Mar;27(3):349-53

Kong D, Belosevic M, Kunimoto DY.

Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Canada.

We developed and tested IFN-gamma-expressing Mycobacterium bovis, strain BCG, for the ability to activate macrophages and protect mice against a heterologous challenge with Leishmania major. One, 2 or 3 weeks after intraperitoneal immunization, mice were challenged with 10(6) L. major amastigotes injected into the right footpad. Recombinant BCG immunization for all 3 challenge time points initially showed greater protection compared to the BCG control, as judged by footpad thickness and number of parasites in the leishmanial lesion. However, at week 4 after challenge, while the 1- and 2-week immunization groups continued to show increased protection, the 3-week immunization group animals exhibited progressive disease. These data suggest that the IFN-gamma-expressing BCG initially activates macrophages more effectively than native BCG, but that late exacerbation of disease can occur, highlighting the complexity of the immune response against leishmaniasis.

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49.) Short report: evaluation of the potency and stability of a candidate vaccine against American cutaneous leishmaniasis.
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Am J Trop Med Hyg 1999 Aug;61(2):294-5

Mayrink W, Pinto J, Da Costa C, Toledo V, Guimaraes T, Genaro O, Vilela L.

Departamento de Parasitologia, Instituto de Ciencias Biologicas/Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.

Availability of a safe, immunogenic, and affordable vaccine would represent the best strategy for control of cutaneous leishmaniasis (CL). Stability in field conditions is a essential property for any candidate vaccine. The stability and immunogenicity of three different preparations (thimerosal-preserved, autoclaved, and lyophilized) of a killed Leishmania amazonensis vaccine were assessed using fresh products and after 12 months of storage at 4 degrees C. Autoclaving was associated with a time-dependent decrease in the immunogenicity of the vaccine, as measured by the leishmanin skin test and production of interferon-gamma. These findings are of importance in the decision of which preparation of candidate killed CL vaccines should move to phase III trials.


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50.) The immunology of susceptibility and resistance to Leishmania major in mice.
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Nat Rev Immunol 2002 Nov;2(11):845-58

Sacks D, Noben-Trauth N.

Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA. [email protected]

Established models of T-helper-2-cell dominance in BALB/c mice infected with Leishmania major -- involving the early production of interleukin-4 by a small subset of Leishmania-specific CD4+ T cells -- have been refined by accumulating evidence that this response is not sufficient and, under some circumstances, not required to promote susceptibility. In addition, more recent studies in L. major-resistant mice have revealed complexities in the mechanisms responsible for acquired immunity, which necessitate the redesign of vaccines against Leishmania and other pathogens that require sustained cell-mediated immune responses.

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51.) Optimization of DNA vaccination against cutaneous leishmaniasis.
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Vaccine 2002 Nov 1;20(31-32):3702-8 Related Articles, Links

Mendez S, Belkaid Y, Seder RA, Sacks D.

Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Room 126, Building 4, Center Dr. MSC 0425, 20892-0425, Bethesda, MD, USA

The present studies were designed to examine the requirements of dose, route of inoculation and constituent antigens for the maintenance of complete and long lasting protection against cutaneous leishmaniasis due to Leishmania major conferred by a cocktail DNA vaccine encoding the Leishmania antigens LACK, LmST11 and TSA. Vaccination of C57Bl/6 mice with LACK DNA alone resulted in partial protection, whereas the combination of LmST11 and TSA provided stronger, though still incomplete protection compared to the combination of all three Ag DNAs. When intradermal (i.d), intramuscular (i.m.), and subcutaneous (s.c.) vaccination routes were compared, i.d. immunization reduced by five-fold the dose necessary to maintain complete protection. In vivo depletion of CD4+ or CD8+ T cells provided direct evidence that both populations are necessary to mediate complete protection. These results establish intradermal vaccination using DNA encoding multiple Leishmania antigens as a way to optimize priming of CD4+ and CD8+ T cells necessary for potent and durable protection against cutaneous leishmaniasis.

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52.) Canine leishmaniasis: epidemiological risk and the experimental model.
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Trends Parasitol 2002 Sep;18(9):399-405

Moreno J, Alvar J.

WHO Collaborating Centre for Leishmaniasis, Servicio de Parasitologi;a, Centro Nacional de Microbiologia, Instituto de Salud Carlos III, Ctra. Majadahonda-Pozuelo km 2, 28220 Majadahonda, Spain.

Increasing risk factors are making zoonotic visceral leishmaniasis a growing public health concern in many countries. Domestic dogs constitute the main reservoir of Leishmania infantum and Leishmania chagasi, and play a key role in the transmission to humans. New reagents and tools allow the detailed investigation of canine leishmaniasis, permitting the monitoring of the immunological status of dogs in both natural and experimental infections. Such studies are essential to determine the basis of the canine protective immune response and to establish a laboratory model, a significant aspect for the development of vaccines against canine leishmaniasis.

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53.) Protective efficacy of a tandemly linked, multi-subunit recombinant leishmanial vaccine (Leish-111f) formulated in MPL adjuvant.
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Skeiky YA, Coler RN, Brannon M, Stromberg E, Greeson K, Crane RT, Campos-Neto A, Reed SG.

Corixa Corporation, 1124 Columbia Street, Suite 200, Seattle, WA 98104, USA. [email protected]

Three immunodominant leishmanial antigens (TSA, LmSTI1 and LeIF) previously identified in the context of host response to infection in infected donors and BALB/c mice, as well as their ability to elicit at least partial protection against Leishmania major infection in the BALB/c mouse model, were selected for inclusion into a subunit based vaccine. This is based on the premise that an effective vaccine against leishmaniasis (a complex parasitic infection) would require a multivalent cocktail of several antigens containing a broader range of protective epitopes that would cover a wide range of MHC types in a heterogeneous population. For practical considerations of vaccine development, we report on the generation of a single recombinant polyprotein comprising the sequences of all three open reading frames genetically linked in tandem. The resulting molecule, Leish-111f, comprises an open reading frame that codes for a 111kDa polypeptide. Evaluation of the immunogenicity and protective efficacy of Leish-111f formulated with IL-12 revealed that the immune responses to the individual components were maintained and as well, rLeish-111f protected BALB/c mice against L. major infection to a magnitude equal or superior to those seen with any of the individual components of the vaccine construct or SLA, a soluble Leishmania lysate. But because rIL-12 is expensive and difficult to manufacture and its efficacy and safety as an adjuvant for human use is questionable, we screened for other adjuvants that could potentially substitute for IL-12. We report that monophosphoryl lipid A (MPL) plus squalene (MPL-SE) formulated with rLeish-111f elicited protective immunity against L. major infection. The demonstrated feasibility to manufacture a single recombinant vaccine comprising multiple protective open reading frames and the potential use of MPL-SE as a substitute for IL-12, takes us closer to the realization of an affordable and safe Leishmania vaccine.

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54.) Molecular biological applications in the diagnosis and control of leishmaniasis and parasite identification.
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Trop Med Int Health 2002 Aug;7(8):641-51 Related Articles, Links

Schallig HD, Oskam L.

Koninklijk Instituut voor de Tropen (KIT), Biomedical Research, Amsterdam, The Netherlands. [email protected]

Molecular biology is increasingly relevant to the diagnosis and control of infectious diseases. Information on DNA sequences has been extensively exploited for the development of polymerase chain reaction-based assays for the diagnosis of leishmaniasis and the identification of parasite species. It has also led to the use of cloned antigen for serodiagnosis. It is expected that the sequencing of the Leishmania major genome and the genomes of other Leishmania species will enable important progress in further improving diagnosis and control. The ability to use genome data to clone and sequence genes, which, when expressed, provide antigens for vaccine development, will increase the possibilities for rational vaccine development. Moreover, DNA on its own will provide the basis for the development of DNA vaccines that may overcome some of the problems encountered with protein-based vaccines. One of the greatest threats to parasite control is the development of drug resistance in parasites. Knowing the molecular basis of drug resistance and the ability to monitor its development with sensitive and specific DNA-based assays for 'resistance alleles' may aid maintaining the effectiveness of available anti-Leishmania drugs. Finally, techniques such as microarrays and nucleic acid sequence-based amplification will eventually allow rapid screening for specific parasite genotypes and assist in diagnostic and epidemiological studies.

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55.) Immunochemotherapy in American cutaneous leishmaniasis: immunological aspects before and after treatment.
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Mem Inst Oswaldo Cruz 2001 Jan;96(1):89-98 Related Articles, Links

Toledo VP, Mayrink W, Gollob KJ, Oliveira MA, Costa CA, Genaro O, Pinto JA, Afonso LC.

Departamento de Analises Clinicas e Toxicologicas, Faculdade de Farmacia, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil.

In this study, we evaluated the immune response of patients suffering from cutaneous leishmaniasis treated with two distinct protocols. One group was treated with conventional chemotherapy using pentavalent antimonium salts and the other with immunochemotherapy where a vaccine against cutaneous leishmaniasis was combined with the antimonium salt. Our results show that, although no differences were observed in the necessary time for complete healing of the lesions between the two treatments, peripheral blood mononuclear cells from patients treated by chemotherapy showed smaller lymphoproliferative responses at the end of the treatment than those from patients in the immunochemotherapy group. Furthermore, IFN-gamma production was also different between the two groups. While cells from patients in the chemotherapy group produced more IFN-gamma at the end of treatment, a significant decrease in this cytokine production was associated with healing in the immunochemotherapy group. In addition, IL-10 production was also less intense in this latter group. Finally, an increase in CD8+ -IFN-gamma producing cells was detected in the chemotherapy group. Together these results point to an alternative treatment protocol where healing can be induced with a decreased production of a potentially toxic cytokine.

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56.) T cell responses to crude and defined leishmanial antigens in patients from the lower Amazon region of Brazil infected with different species of Leishmania of the subgenera Leishmania and Viannia.
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Parasite Immunol 1998 Jan;20(1):19-26

Silveira FT, Blackwell JM, Ishikawa EA, Braga R, Shaw JJ, Quinnell RJ, Soong L, Kima P, McMahon-Pratt D, Black GF, Shaw MA.

Instituto Evandro Chagas (FNS), Belem, Para, Brazil.

Amazonian localized cutaneous leishmaniasis (LCL) is caused by parasites of the subgenera Leishmania and Viannia. Respectively, these parasites may cause diffuse cutaneous leishmaniasis (DCL) and mucocutaneous leishmaniasis (MCL). This, together with differing skin test responses, suggests some species-specificity in cell mediated immunity. In this study, T cell responses (proliferative and interferon-gamma) to crude and defined antigens were examined in paired samples pre and post chemotherapy. Untreated L. (L.) amazonensis LCL patients showed lower responses to crude leishmanial antigens than the L. (V.) spp. group. L. (V.) braziliensis antigen was a more potent stimulator of T cell responses than L. (L.) amazonensis antigen in all patient groups. Few positive responses were seen to the L. (L.) amazonensis glycoprotein GP46. A substantial proportion of LCL patients did respond to the L. (L.) pifanoi amastigote antigens A2, and the surface membrane glycoprotein P8. DCL patients were poor responders to all leishmanial antigens, except GP46. In contrast, MCL patients were good responders to all antigens except GP46 and A2. A significant rise in the response to P8 and A2 antigen was seen post treatment across all LCL and MCL patients, indicating that these antigens might provide suitable vaccine candidates.

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57.) Cell-mediated immunity in localized cutaneous leishmaniasis patients before and after treatment with immunotherapy or chemotherapy.
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Parasite Immunol 1989 May;11(3):211-22

Castes M, Moros Z, Martinez A, Trujillo D, Castellanos PL, Rondon AJ, Convit J.

Instituto de Biomedicina, Facultad de Medicina, Universidad Central de Venezuela, Caracas.

In previous studies of the treatment of localized cutaneous leishmaniasis (LCL) we demonstrated that the therapeutic efficiency of immunotherapy (BCG plus promastigotes of Leishmania mexicana) is equal to that of chemotherapy (Glucantime), without causing the serious side-effects of the drug treatment. In the present study, various aspects of cell-mediated immunity, including the lymphoproliferative response, and leucocyte subpopulations were evaluated both before treatment and after cure in 39 LCL patients who had received immunotherapy (IT), in 34 submitted to chemotherapy (CT), and in 14 patients cured by the administration of BCG alone. We demonstrated evident signs of T-cell activation in cured patients who had received either CT or IT. For example, an increased expression of IL-2 receptors was observed in such patients, compared to their pretreatment values. Also, a significant percentage of patients showed augmented in-vitro responses to mitogen, and both in-vitro and in-vivo reactivity to leishmanial antigen. No evidence was found for the development of an exaggerated immune response to Leishmania parasites in the IT group, because the final level of immunological reactivity was comparable to the CT group. Neither was there any difference in terms of the final immune response between the patients cured by BCG treatment alone and the other groups. However, the therapeutic efficiency of BCG was lower and the mean cure time was longer. We conclude that IT is very useful in the treatment of LCL patients because of its high efficiency, low propensity to produce side-effects, and the fact that it does not induce a state of hyper-reactivity.

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58.) Immunotherapy as a treatment of American cutaneous leishmaniasis: preliminary studies in Brazil.
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Parassitologia 1992 Dec;34(1-3):159-65

Mayrink W, Magalhaes PA, Michalick MS, da Costa CA, Lima Ade O, Melo MN, Toledo VP, Nascimento E, Dias M, Genaro O, et al.

Departamento de Parasitologia, Universidade Federal de Minas Gerais, Belo Horizonte, Brasil.

A prophylactic vaccine composed of killed promastigotes of five stocks of Leishmania was tested as an immunotherapeutic agent against American cutaneous leishmaniasis (ACL). The agent was administered by deep intramuscular injection daily for 10 days, followed by a 10-day interval. Out of 62 patients so treated, 47 (76%) were considered clinically cured; 41 required 2-10 treatment courses and the other six 11-19 courses. None of the patients treated by immunotherapy displayed adverse side-effects. Immunotherapy proved to be effective in the treatment of single cutaneous lesions, multiple cutaneous lesions and in cases of mucocutaneous leishmaniasis. In comparison with chemotherapy (Glucantime), immunotherapy is less efficient and more prolonged but can be safely used when antimonials are contra-indicated or are found to be ineffective. Consideration is given to the treatment of victims of ACL living in rural areas remote from a medical centre.

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59.) T-cell responsiveness of American cutaneous leishmaniasis patients to purified Leishmania pifanoi amastigote antigens and Leishmania braziliensis promastigote antigens: immunologic patterns associated with cure.
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Exp Parasitol 1996 Nov;84(2):144-55

Coutinho SG, Oliveira MP, Da-Cruz AM, De Luca PM, Mendonca SC, Bertho AL, Soong L, McMahon-Pratt D.

Department of Protozoology, Oswaldo Cruz Institute-FIOCRUZ, Rio de Janeiro, Brasil.

Patients suffering from American cutaneous leishmaniasis were studied before therapy (active lesion) and at the end of therapy (cured patients). Assays of lymphocyte proliferative responses of peripheral blood mononuclear cells induced in vitro by Leishmania braziliensis promastigote antigens (Lb) or by three proteins (A-2/P-2, P-4, and P-8) derived from Leishmania pifanoi amastigotes were performed. Antigen-stimulated cells were harvested for CD4 and CD8 phenotype analysis and the levels of gamma interferon (IFN-gamma), interleukin 2 (IL-2) and interleukin 4 (IL-4) produced were also determined. Results show two different patterns of Lb-induced T cell responses: (a) predominance of responding CD4+ cells and mixed type 1 and type 2 cytokine production (IFN-gamma, IL-2, and IL-4) during the active disease, (b) similar proportions of responding CD4+ and CD8+ cells and type 1 cytokine production (presence of IFN-gamma and IL-2 and very low IL-4) at the end of therapy (healed lesions). Thus, this last pattern is probably associated with a beneficial T cell response. The A-2/P-2 amastigote cysteine proteinase provided only marginal (s.i. approximately or = 2.5) T cell stimulation in 25% of patients studied; in contrast, the L. pifanoi P-4 and P-8 amastigote antigens induced significant stimulation (s.i. approximately or = 5) in approximately 50% of the patients. In comparison to Lb-stimulated cultures, lower proliferative responses of T lymphocytes to P-4 or P-8 were observed. However, the P-4- or P-8-stimulated cultures had similar percentages of reactive CD4+ and CD8+ cells, as well as type 1 cytokines (presence of IFN-gamma and IL-2, and low levels or absence of IL-4) in the supernatants both before and at the end of therapy. The consistent induction of apparently beneficial T cell responses by the P-4 and P-8 amastigote glycoproteins points to the possibility that these molecules be considered as candidates for future defined vaccines against leishmaniasis.

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60.) Dichotomy of the T cell response to Leishmania antigens in patients suffering from cutaneous leishmaniasis; absence or scarcity of Th1 activity is associated with severe infections.
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Clin Exp Immunol 1995 May;100(2):239-45

Gaafar A, Kharazmi A, Ismail A, Kemp M, Hey A, Christensen CB, Dafalla M, el Kadaro AY, el Hassan AM, Theander TG.

Institute of Tropical Medicine, MRC, Khartoum, Sudan.

The T cell response was studied in 25 patients suffering from cutaneous leishmaniasis caused by Leishmania major with severe (n = 10) and mild (n = 15) disease manifestations. Peripheral blood mononuclear cells (PBMC) from the patients were activated by sonicates of Leishmania promastigotes (LMP) and amastigotes (LDA), and the surface protease gp63. The proliferative responses to Leishmania antigens were lower in patients with severe disease than in patients with mild disease (P = 0.01-0.05), and such a difference was not observed in the response to purified protein derivative of tuberculin (PPD) or tetanus toxoid (TT). LMP-induced interferon-gamma (IFN-gamma) production was lower in patients with severe than in patients with mild disease (P < 0.05). When the IL-4 and IFN-gamma responses of each patient were considered, two response patterns were observed in the cultures activated by the Leishmania sonicates. One response pattern was characterized by high production of IFN-gamma without production of IL-4 (a Th1-like pattern), the other was characterized by low IFN-gamma levels which in most cases were associated with IL-4 production (not a Th1-like pattern). These patterns could not be distinguished when the cells from the same donors were stimulated by TT and PPD. The percentages of patients with a Th1-like response pattern after stimulation by LMP in patients with severe and mild disease manifestations were 30% and 80%, respectively. This difference was statistically significant (P = 0.034).

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61.) Chemotherapy for cutaneous leishmaniasis: a controlled trial using killed Leishmania (Leishmania) amazonensis vaccine plus antimonial.
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Int J Dermatol 2002 Feb;41(2):73-8

Machado-Pinto J, Pinto J, da Costa CA, Genaro O, Marques MJ, Modabber F, Mayrink W.

Department of Dermatology, Santa Casa Hospital, Federal University of Minas Gerais, Belo Horizonte, Brazil. [email protected]

BACKGROUND: Leishmaniasis is endemic in 88 countries in the world, and 350 million individuals are at risk of acquiring the disease. Treatment for American cutaneous leishmaniasis (ACL) is long, expensive, and associated with important side-effects. METHODS: In this double-blind, placebo-controlled study, we treated 102 patients with ACL using either a combination of a single-strain Leishmania amazonensis killed promastigote vaccine plus a half dose of meglumine antimoniate, or placebo plus the same half dose regimen of meglumine antimoniate, in 10-day series followed by 10-day intervals. RESULTS: Of the 47 patients in the experimental arm, 47 (100%) were cured after four series of treatment, compared to four of 49 (8.2%) in the control group (P < 0.0001). Six patients were lost to follow-up. CONCLUSIONS: The combination of a single-strain Leishmania (Leishmania) amazonensis killed promastigote vaccine with a half dose regimen of antimonial is highly effective for the treatment of ACL.

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62.) Immunotherapy versus chemotherapy in localised cutaneous leishmaniasis.
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Lancet 1987 Feb 21;1(8530):401-5 Related Articles, Links

Convit J, Castellanos PL, Rondon A, Pinardi ME, Ulrich M, Castes M, Bloom B, Garcia L.

In a randomised trial a combination vaccine consisting of live BCG together with killed leishmania promastigotes was compared with a standard antimonial regimen in 94 patients with localised cutaneous leishmaniasis. Three vaccinations over 32 weeks gave a similar cure rate (94%) to three 20-day courses of meglumine antimonate. In the immunotherapy group side-effects were few (5.8%) and slight whereas in the chemotherapy group they were frequent (52.4%) and often serious. Immunotherapy is a low-cost, low-risk alternative to chemotherapy in localised cutaneous leishmaniasis, applicable by primary health services in rural areas.
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63.)Immunotherapy of localized, intermediate, and diffuse forms of American cutaneous leishmaniasis.
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J Infect Dis 1989 Jul;160(1):104-15

Convit J, Castellanos PL, Ulrich M, Castes M, Rondon A, Pinardi ME, Rodriquez N, Bloom BR, Formica S, Valecillos L, et al.

Instituto de Biomedicina, Caracas, Venezuela.

The clinical efficacy of immunotherapy for localized American cutaneous leishmaniasis with a combination of heat-killed Leishmania mexicana amazonensis promastigotes and viable BCG (bacille Calmette Guerin) has been compared with meglumine antimoniate chemotherapy and with BCG alone in a controlled clinical study in 217 patients. The results in the first two groups were comparable, with greater than 90% clinical cures with an average time of 16-18 w required for healing. The cure rate was considerably lower (42%) and more prolonged in the group receiving BCG alone. Secondary effects were observed in less than 5% of the patients receiving combined immunotherapy or BCG alone. In contrast, 49% of the patients receiving chemotherapy showed side effects. High therapeutic efficacy was also observed using combined immunotherapy in patients with intermediate and diffuse cutaneous leishmaniasis who were previously unresponsive to chemotherapy. Cure or clinical improvement was seen in all 11 patients with intermediate forms of the disease, and marked clinical improvement was observed in 9 of 10 patients with diffuse disease. The results on the efficacy of the combined vaccine in immunotherapy for American cutaneous leishmaniasis provide a strong rationale for studying its effectiveness in prophylactic trials.

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64.) Characterization of the immune response in subjects with self-healing cutaneous leishmaniasis.
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Am J Trop Med Hyg 1995 Sep;53(3):273-7

Carvalho EM, Correia Filho D, Bacellar O, Almeida RP, Lessa H, Rocha H.

Servico de Imunologia do Hospital Universitario Prof. Edgard Santos, Faculdade de Medicina da Universidade Federal da Bahia, Salvador, Bahia, Brazil.

In patients with cutaneous leishmaniasis in areas of Leishmania braziliensis transmission, ulcers may heal without therapy. In the present study, we evaluated the T cell responses of 10 subjects who two years earlier had a rapidly (less than three months) self-healing cutaneous disease. The immunologic responses of these cases were determined by intradermal skin test, measurements of antibodies, lymphocyte proliferative responses, and interferon-gamma (IFN-gamma) production in cultures stimulated with Leishmania antigens. These data were compared with those observed in 10 other patients with active cutaneous and mucosal leishmaniasis. Evidence of strong lymphocyte blastogenesis and IFN-gamma production was observed in eight of 10 patients with self-healing cutaneous leishmaniasis, with stimulation indices ranging from 32 to 506, and IFN-gamma levels ranging from 500 to 2,900 pg/ml. The mean +/- SD stimulation index of the lymphocyte proliferative responses (288 +/- 247) and the mean +/- SD of IFN-gamma production after stimulation with Leishmania antigen (970 +/- 960 pg/ml) in subjects with self-healing cutaneous leishmaniasis were similar (P > 0.05) to those observed in patients with mucosal disease (stimulation index = 308 +/- 282 and IFN-gamma level = 838 +/- 819 pg/ml). These responses were higher (P < 0.01) than those observed in patients with active cutaneous leishmaniasis (stimulation index = 50 +/- 82 and IFN-gamma level = 264 +/- 336 pg/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

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65.) Antigen provoking gamma interferon production in response to Mycobacterium bovis BCG and functional difference in T-cell responses to this antigen between viable and killed BCG-immunized mice.
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Infect Immun 1994 Oct;62(10):4396-403

Kawamura I, Yang J, Takaesu Y, Fujita M, Nomoto K, Mitsuyama M.

Department of Bacteriology, Niigata University School of Medicine, Japan.

It has been shown that gamma interferon (IFN-gamma)-producing CD4+ T cells, which are generated only by immunization with viable bacteria, exert a significant role in protective immunity against mycobacteria in mice. In this study, we have tried to determine the antigen recognized by the T cells in search of a possible protective antigen. T cells from viable Mycobacterium bovis BCG-immunized mice were stimulated with several antigens, and IFN-gamma production was measured. Purified protein derivative and viable and killed BCG lysates caused significant IFN-gamma production, and almost the same level of IFN-gamma activity was detected in both groups stimulated with viable and killed BCG lysates. However, heat shock protein (HSP) 65 and HSP 70 were not a major antigen for IFN-gamma production. The antigen provoking IFN-gamma production is localized mainly in the membrane fraction of BCG cells, and the approximate molecular size was 18 kDa. On the other hand, T cells from killed BCG-immunized mice never responded to this antigen for IFN-gamma production, whereas they could mount a delayed-type hypersensitivity response. These results showed that the antigen provoking IFN-gamma production was present in killed as well as viable BCG. In addition to the antigen presentation by antigen-presenting cells, some kinds of differentiation factor (such as monokines) that are produced only by stimulation with viable cells seemed to be necessary for the development of IFN-gamma-producing T cells.

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66.) Autoclaved Leishmania major vaccine for prevention of visceral leishmaniasis: a randomised, double-blind, BCG-controlled trial in Sudan.
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Lancet 2000 Nov 4;356(9241):1565-9

Khalil EA, El Hassan AM, Zijlstra EE, Mukhtar MM, Ghalib HW, Musa B, Ibrahim ME, Kamil AA, Elsheikh M, Babiker A, Modabber F.

Leishmaniasis Research Group/Sudan, Institute of Endemic Diseases, Khartoum. <[email protected]

BACKGROUND: Visceral leishmaniasis is a major cause of morbidity and mortality in the Sudan. Drug treatment is expensive, and drug resistance is becoming increasingly common. Safe, effective, and cheap vaccines are needed. We report the results of a vaccine trial against human visceral leishmaniasis. METHODS: We undertook a double-blind randomised trial to test the safety and efficacy of an autoclaved Leishmania major (ALM) promastigote vaccine (1 mg per dose). Of 5093 volunteers screened, 2306 had negative leishmanin skin tests and reciprocal titres of less than 6400 in the direct agglutination test. They were randomly assigned two doses of ALM mixed with BCG or BCG alone. Volunteers were followed up for 2 years. The primary endpoint was clinical visceral leishmaniasis or post-kala-azar dermal leishmaniasis. Analyses were by intention to treat. FINDINGS: Side-effects were confined to the injection site. The cumulative frequency of visceral leishmaniasis at 2 years did not differ significantly between the group assigned ALM plus BCG and that assigned BCG alone (133/1155 [11.5%] vs 141/1151 [12.3%], p=0.6). The vaccine efficacy was 6% (95% CI -18 to 25). The proportion of individuals showing leishmanin skin conversion was significantly higher in the ALM plus BCG group than in the BCG alone group throughout follow-up (303 [30%] vs 72 [7%] at 42 days). Individuals whose leishmanin test converted after vaccination (induration > or =5 mm) had a significantly lower frequency of visceral leishmaniasis than non-responders (27/375 [7.2%] vs 210/1660 [12.7%], p=0.003). INTERPRETATION: We found no evidence that two doses of ALM plus BCG offered significant protective immunity against visceral leishmaniasis compared with BCG alone. Leishmanin skin conversion with an induration of 5 mm or more in either group was associated with protection from the disease.

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67.) Leishmania donovani p36(LACK) DNA vaccine is highly immunogenic but not protective against experimental visceral leishmaniasis.
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Infect Immun 2001 Aug;69(8):4719-25

Melby PC, Yang J, Zhao W, Perez LE, Cheng J.

Medical Service, Department of Veterans Affairs Medical Center, South Texas Veterans Health Care System, University of Texas Health Science Center, San Antonio, Texas 78229-3900, USA. [email protected]

The acquisition of immunity following subclinical or resolved infection with the intracellular parasite Leishmania donovani suggests that vaccination could prevent visceral leishmaniasis (VL). The LACK (Leishmania homolog of receptors for activated C kinase) antigen is of interest as a vaccine candidate for the leishmaniases because of its immunopathogenic role in murine L. major infection. Immunization of mice with a truncated (24-kDa) version of the 36-kDa LACK antigen, delivered in either protein or DNA form, was found previously to protect against cutaneous L. major infection by redirecting the early T-cell response away from a pathogenic interleukin-4 (IL-4) response and toward a protective Th1 response. The amino acid sequence of the Leishmania p36(LACK) antigen is highly conserved, but the efficacy of this vaccine antigen in preventing disease caused by strains other than L. major has not been determined. We investigated the efficacy of a p36(LACK) DNA vaccine against VL because of the serious nature of this form of leishmaniasis and because it was unclear whether the LACK vaccine would be effective in a model where there was not a dominant pathogenic IL-4 response. We demonstrate here that although the LACK DNA vaccine induced a robust parasite-specific Th1 immune response (IFN-gamma but not IL-4 production) and primed for an in vivo T-cell response to inoculated parasites, it did not induce protection against cutaneous or systemic L. donovani challenge. Coadministration of IL-12 DNA with the vaccine did not enhance the strong vaccine-induced Th1 response or augment a protective effect.

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68.) Vaccination of Balb/c mice against experimental visceral leishmaniasis with the GP36 glycoprotein antigen of Leishmania donovani.
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Vaccine 2001 Apr 30;19(23-24):3104-15

Paraguai de Souza E, Bernardo RR, Palatnik M, Palatnik de Sousa CB.

Instituto de Microbiologia, Prof. Paulo de Goes, Universidade Federal do Rio de Janeiro (UFRJ), CCS, Cidade Universitaria, Ilha do Fundao, CP 68040. CEP 21941-590., Rio de Janeiro, Brazil.

Leishmania donovani GP36 glycoprotein is the main antigen of the FML Fucose Mannose Ligand (FML) complex specifically recognized by sera of kala-azar human patients. The GP36 was isolated by chemical elution + sonication and used for Balb/c mouse vaccination in combination with saponin, by the s.c. route, inducing a strong and specific protective effect against experimental visceral leishmaniasis shown by the increase of: specific IgG antibodies (82.6%), mainly IgG2a, the delayed type of hypersensitivity to promastigote lysate (37.8%, P < 0.001), the in vitro cellular proliferative response to GP36 of ganglia lymphocytes (53.5%, P < 0.005) and the decrease of liver parasite burden (68.1%, P < 0.025). Saponin treated controls reacted significantly differently from GP36 vaccinated animals at all the assayed variables (P < 0.05). GP36 induced significant protection against murine visceral leishmaniasis at concentrations commonly used for vaccination with recombinant antigens.

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69.) Vaccination of langur monkeys (Presbytis entellus) against Leishmania donovani with autoclaved L. major plus BCG.
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Parasitology 1998 Mar;116 ( Pt 3):219-21

Dube A, Sharma P, Srivastava JK, Misra A, Naik S, Katiyar JC.

Division of Parasitology, Central Drug Research Institute, Lucknow, India. [email protected]

The protective potential of killed Leishmania major (ALM) along with BCG was evaluated against L. donovani in Indian langur monkeys in single and triple dose schedules. A delayed protection was observed in monkeys after a single dose schedule of ALM (3 mg)+BCG (3 mg) given intradermally 2 months before intravenous challenge with L. donovani. Triple dose schedule each of 1 mg ALM + 1 mg BCG was more effective. The status remained unchanged until the end of the experiment (approximately 8 months). The study indicates that a combination of ALM + BCG may be a good candidate vaccine for exploiting against human Kala-azar.

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70.) Successful vaccination against Leishmania donovani infection in Indian langur using alum-precipitated autoclaved Leishmania major with BCG.
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Vaccine 2001 May 14;19(25-26):3485-92

Misra A, Dube A, Srivastava B, Sharma P, Srivastava JK, Katiyar JC, Naik S.

Division of Parasitology, Central Drug Research Institute, P.O. Box 173, 226001, Lucknow, India.

Autoclaved Leishmania major (ALM) along with BCG, presently undergoing phase II clinical trial by WHO for its vaccine potential against cutaneous leishmaniasis, has been successfully evaluated in single and triple dose schedules against L. donovani in Indian langurs (Presbytis entellus). Encouraged with the results, another formulation alum-precipitated ALM (provided by WHO) along with BCG has been evaluated in this system. Eight monkeys were vaccinated with alum-precipitated ALM + BCG (1 mg of each per animal) while four were kept as unvaccinated controls. All were challenged with 100 x 10(6) amastigotes i.v. on day 60 post vaccination. Parasitic assessment in splenic tissue was performed on day 45, 90 and 180 p.c. Initially, seven of the eight vaccinated monkeys developed infection (two to six amastigotes per 1000 cell nuclei), which resolved by day 180 p.c., while the eighth monkey had a parasite burden of 14 amastigotes per 1000 cell nuclei on day 45 p.c. and died on day 130 p.c. On the other hand, there was progressive infection in unvaccinated control animals and three out of four died between days 110 and 120 p.c., and one monkey, which had low parasite burden, died on day 178 p.c. Prior to challenge, there was an initial rise in antileishmanaial antibodies in the vaccinated group compared to the unvaccinated control group, which later came down to normal level, while it remained higher in the unvaccinated control group. An increasing pattern of antigen-specific proliferative responses and interferon-gamma level to the two antigens--autoclaved L. donovani (ALD) and ALM--was observed in vaccinated monkeys throughout the experiment. There was a good correlation between parasite burden and IFN-gamma level on days 90 and 180 p.c., indicating IFN-gamma response as a sensitive parameter of immune status. The findings suggest alum-precipitated ALM+BCG as a potential vaccine against visceral leishmaniasis and warrants clinical trials.

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71.) [Protective effects of leishmanial antigens against Leishmania infantum infection in Lagurus lagurus]
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Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi 1999;17(4):237-40

[Article in Chinese]

Chai J, Chang KP, Zuo X, Yan L, Hou Y, Zhang S, Ruziguli, Jiang W, Zhang L.

National Hydatid Disease Centre of China, Institute for Endemic Diseases Control and Research of Xinjiang Uygur Autonomous Region, Urumqi, Xinjiang 83000.

AIM: To determine the protective effect of leishmanial surface antigens against experimental visceral leishmaniasis in Lagurus lagurus caused by Leishmania infantum. METHODS: Recombinant surface protein (rGP63) and lipophosphoglycan (LPG) of Leishmania were used with Corinebacterium parum vaccine as ajuvant to immunize Lagurus lagurus against a challenge with virulent strain of Leishmania infantum. The efficacy of immunoprotection was observed. RESULTS: When challenged with up to 2 x 10(7) promastigotes, the number of LD on the liver printing sections in the rGP63 + LPG + CP immunised animals was significantly decreased, the parasite reduction rate being 89.79%. LPG + CP gave a parasite reduction rate of 60.6% and rGP63/beta-galactosidase fusion protein + CP showed a parasite reduction rate of 42.45%. Purified rGP63 showed no protection. Immunization with rGP63 + LPG + CP followed by challenge inifection with 1 x 10(6), 5 x 10(6) and 1 x 10(7) promastigotes also showed significantly reduced infection rates. CONCLUSION: A combination of rGP63 + LPG + CP antigens could provide significant immunoprotection against L. infantum challenge in L. lagurus.

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72.) Vaccination with DNA encoding ORFF antigen confers protective immunity in mice infected with Leishmania donovani.
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Vaccine 2003 Mar 7;21(11-12):1292-9

Sukumaran B, Tewary P, Saxena S, Madhubala R.

School of Life Sciences, Jawaharlal Nehru University, 110067, New Delhi, India

The gene ORFF is part of the multigenic LD1 locus on chromosome 35 that is frequently amplified in Leishmania. The function of ORFF is unknown. The gene encoding ORFF was cloned into a eukaryotic expression vector downstream to the cytomegalovirus (CMV) promoter. BALB/c mice were injected intramuscularly with ORFF DNA and challenged with Leishmania donovani promastigotes. Vaccination with ORFF gene induced both humoral and cellular immune response against ORFF, which provided significant level of protection against challenge with L. donovani. A qualitative PCR was used to determine whether activation of Th1 cells develops selectively in response to this ORFF DNA vaccine. The results indicated that mRNA for IFN-gamma was significantly induced in immunized mice. No significant change in IL-4 mRNA expression was observed in mice immunized with ORFF DNA vaccine versus mice immunized with control plasmid. Thus, DNA immunization may offer an attractive alternative strategy against leishmaniasis.


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73.) Intradermal infection model for pathogenesis and vaccine studies of murine visceral leishmaniasis.
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Infect Immun 2003 Jan;71(1):401-10

Ahmed S, Colmenares M, Soong L, Goldsmith-Pestana K, Munstermann L, Molina R, McMahon-Pratt D.

Department of Epidemiology and Public Health, Yale University School of Medicine, New Haven, Connecticut 06520-8034, USA.

The levels of protection found in vaccine studies of murine visceral leishmaniasis are significantly lower than for cutaneous leishmaniasis; whether this is due to the high-challenge murine model employed and/or is a consequence of differences required in tissue-specific local immune responses is not understood. Consequently, an intradermal murine model of visceral leishmaniasis has been explored. Intradermal inoculation established a chronic infection in susceptible mice which was associated with a pattern of parasite clearance with time postinfection in the liver and skin; in contrast, parasite persistence and expansion was observed in lymphoid tissue (spleen and draining lymph node). The course of disease found appears to be similar to those reported for subclinical canine and human visceral leishmaniasis. Clearance of parasites from the skin was correlated with an inflammatory response and the infiltration and activation of CD4(+) and CD8(+) T cells. In contrast, in lymphoid tissue (lymph node or spleen), the production of Th1/Th2 cytokines (interleukin-4 [IL-4], IL-10, and gamma interferon) appeared to correlate with parasite burden and pathogenesis. In vaccination experiments employing the Leishmania infantum D-13 (p80) antigen, significantly higher levels of protection were found with the intradermal murine model (29 to 7,500-fold more than naive controls) than were found with a low-dose intravenous infection model (9 to 173-fold). Thus, this model should prove useful for further investigation of disease pathogenesis as well as vaccine studies of visceral leishmaniasis.

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74.) Saponins, IL12 and BCG adjuvant in the FML-vaccine formulation against murine visceral leishmaniasis.
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Vaccine 2002 Nov 22;21(1-2):30-43

Santos WR, de Lima VM, de Souza EP, Bernardo RR, Palatnik M, de Sousa CB.

Instituto de Microbiologia, "Professor Paulo de Goes" Universidade Federal do Rio de Janeiro (UFRJ), CCS, Cidade Universitaria, Ilha do Fundao, Caixa Postal 68040, CEP 21941-590, RJ, Rio de Janeiro, Brazil

The FML antigen of Leishmania donovani, in combination with either Riedel de Haen (R), QuilA, QS21 saponins, IL12 or BCG, was used in vaccination of an outbred murine model against visceral leishmaniasis (VL). Significant and specific increases in anti-FML IgG and IgM responses were detected for all adjuvants, and in anti-FML IgG1, IgG2a and IgG2b and delayed type of hypersensitivity to L. donovani lysate (DTH), only for all saponins and IL12. The QS21-FML and QuilA-FML groups achieved the highest IgG2a response. QuilA-FML developed the strongest DTH and QS21-FML animals showed the highest serum IFN-gamma concentrations. The reduction of parasitic load in the liver in response to each FML-vaccine formulation was: 52% (P<0.025) for BCG-FML, 73% (P<0.005) for R-FML, 93% (P<0.005) for QuilA-FML and 79.2% (P<0.025) for QS21-FML treated animals, respectively. Protection was specific for R-FML and QS21-FML while the QuilA saponin treatment itself induced 69% of LDU reduction. The FML-saponin vaccines promote significant, specific and strong protective effects against murine visceral leishmaniasis. BCG-FML induced minor and non-specific protection while IL12-FML, although enhancing the specific antibody and IDR response, failed to reduce the parasitic load of infected animals.

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75.) COMPONENT OF FLY SALIVA MAKES PROMISING LEISHMANIASIS VACCINE
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Source: www.niaid.nih.gov/ 06 august 2.001

Researchers seeking to make a vaccine against a serious parasitic infection have discovered a dose of fly saliva might be just what the doctor ordered. Leishmaniasis, a disabling and sometimes deadly tropical illness, is caused by a parasite transmitted to people through the bite of a sand fly. In a report released today, scientists from the National Institute of Allergy and Infectious Diseases (NIAID) describe how a vaccine they developed against a component of sand fly saliva prevents leishmaniasis in mice.


Leishmaniasis, a major health problem in many tropical and desert climates, has resisted efforts to develop an effective vaccine. "Today's report describes a novel vaccine," says NIAID Director Anthony S. Fauci, M.D. "Rather than targeting the parasite, as is typical, our researchers produced a vaccine to the saliva of the insect that transmits the parasite. This approach could potentially be used to develop vaccines for other insect- or tick-borne diseases."


Leishmaniasis refers to a group of related diseases. Different species of the single-celled parasite Leishmania can cause flesh-eating nose, throat and mouth infections (mucosal leishmaniasis); painful skin lesions (cutaneous leishmaniasis); or fatal infestations of the internal organs (visceral leishmaniasis). An estimated 12 million people currently are affected by one or more of these diseases, most of whom live in South or Central America, Africa and the Middle East.


NIAID's José Ribeiro, M.D., Ph.D., an expert on the biochemistry of blood-feeding bugs, has spent more than 30 years studying how components of saliva not only help insects and ticks obtain their blood meals but also modulate the immune response. He and others have previously shown that laboratory animals immunized with sand fly saliva often resist infection when later bitten by a Leishmania-carrying insect, or challenged with parasites in the presence of sand fly saliva. In the new study reported August 6 in the "Journal of Experimental Medicine", Dr. Ribeiro directed a research team that sought to use this information to produce a novel vaccine against the disease. Jesus Valenzuela, Ph.D., Dr. Ribeiro and colleagues examined saliva from the sand fly carrier of Leishmania major, a parasite species that causes cutaneous leishmaniasis. The researchers separated the proteins of the saliva and identified one, dubbed SP15, which appeared to be the target of natural immune responses in mice. They then worked backwards, using the protein to help them find its underlying gene.


Once they identified the SP15 gene, Dr. Ribeiro's team used it to construct a DNA vaccine, which they used to immunize mice. When the immunized mice were later injected with L. major parasites mixed with fly saliva, the infection was markedly milder compared to infection in mice that had not been vaccinated. The immunized mice had much smaller skin lesions, and their infections cleared within six weeks. Unvaccinated mice developed large skin ulcers and did not eliminate the parasite.


Because sand fly bites produce both antibodies and T-cell responses, the researchers analyzed the mice to see which type of immune response was keeping the parasites in check. When the NIAID team vaccinated "knockout" mice, genetically engineered not to produce antibodies, the mice were still protected by the vaccine, suggesting T cells were protecting the animals from disease.


The results demonstrate a vaccine containing a component of sand fly saliva can protect mice from the severe symptoms associated with cutaneous leishmaniasis, perhaps by mimicking natural immunity to the infection. "People get bitten by infected sand flies all the time without developing leishmaniasis," says Dr. Ribeiro. "It could be that those who develop disease are merely unlucky; they are bitten by a Leishmania-carrying fly before uninfected flies have had time to naturally immunize them."


Dr. Ribeiro next plans to test his vaccine in dogs-natural reservoirs of the parasite-and monkeys. His team also will look at other Leishmania species and the sand fly species that transmit them, hoping to develop vaccines for other forms of leishmaniasis. In addition, he has begun to study people who are naturally exposed to Leishmania to see which components of fly saliva might protect them from disease. "Different sand fly species, each with its unique collection of salivary proteins, transmit different Leishmania species," he explains. "If anti-saliva vaccines are to work in people, they will have to be specifically engineered for the problem insects of each region."



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76.) Effort to Target Clinical Development of Corixa's Leishmaniasis Vaccine
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Source: www.gatesfoundation.org
Infectious Disease Research Institute receives $15 million donation from the Bill & Melinda Gates Foundation
Contact:

Jesse Ciccone
FitzGerald Communications Inc.
Phone: (415) 986-9500, Email: [email protected]

Jim DeNike
Corixa Corporation
Phone: (206) 754-5716 , Email: [email protected]


SEATTLE, Washington -- A vaccine for leishmaniasis – a parasitic disease that kills up to 500,000 adults and children every year – is one step closer to reality. The Infectious Disease Research Institute (IDRI) today announced receipt of a $15 million grant from the Bill & Melinda Gates Foundation to fund their ongoing effort with Corixa Corporation (Nasdaq: CRXA) to develop a vaccine to prevent leishmaniasis.

Leishmaniasis is a skin and visceral disease endemic in the Indian sub-continent, Africa, South America, and the Middle East. According to the World Health Organization (WHO), an estimated 15 million new cases of the disease appear each year. Leishmaniasis is caused by the parasite leishmania, which is carried by sand flies.

"We are excited to receive funding for our leishmaniasis program from the Bill & Melinda Gates Foundation," stated Cynthia Healy, Ph.D., chair of the board of IDRI. "This generous donation represents a serious commitment to treating and preventing diseases of developing countries. IDRI and Corixa have been collaborating on leishmaniasis research for the past several years, which has resulted in a promising candidate vaccine. Support from the Bill & Melinda Gates Foundation will allow us to optimize the vaccine and begin applying it in areas of the world where it is needed most.

"IDRI's mission is to develop vaccines for diseases of developing countries, and our collaboration with Corixa has been instrumental in these efforts. Our aim is to produce the world's first successful vaccine against a parasitic disease."

Already in Phase I clinical trials in Brazil, IDRI has tested a prototype leishmaniasis vaccine comprising Corixa's novel antigens on a compassionate-use basis on a small number of leishmaniasis patients. The funding will allow IDRI to pursue additional studies, including advanced clinical trials in several countries. As part of the collaboration, Corixa anticipates the company will manufacture and supply product formulations for further clinical studies. They also will manage required, multi-site clinical trials and product registration efforts. Corixa's adjuvant technology also is expected to be a key component of the vaccine.

"Corixa's collaboration with IDRI brings complementary skills, resources and technology together to attempt to resolve diseases that affect major patient populations in less developed nations," said Steven Reed, Ph.D., chief scientific officer and executive vice president of Corixa. "A vaccine powered by both Corixa's antigens and adjuvants, combined with IDRI's efforts to conduct vaccine testing in developing countries, will be a dramatic breakthrough for this important unmet medical need."

Corixa has developed a prototype vaccine for leishmaniasis, which has been tested in patients in Brazil. A similar vaccine has been licensed to Heska, Inc., for vaccination of dogs against Leishmania infection. Dogs can serve as a host for the sand fly that harbors the parasite responsible for the disease.

About IDRI
IDRI is a not-for-profit corporation dedicated to the study of infectious diseases and cancer and to the development of vaccines, therapeutics and diagnostics for their treatment and prevention. IDRI's mission is to promote such research through its internal programs and, occasionally, by supporting research at other organizations with expertise in infectious disease and cancer research consistent with these purposes and with its tax exempt status under Section 501(c)(3) of the Internal Revenue Code. IDRI research is supported by the National Institutes of Health.

About Corixa
Corixa is a research- and development-based biotechnology company committed to treating and preventing autoimmune diseases, cancer and infectious diseases by understanding and directing the immune system. Corixa is focused on immunotherapeutic products and has a broad technology platform enabling both fully integrated vaccine design and the use of its separate, proprietary vaccine components on a standalone basis. The company partners with numerous developers and marketers of pharmaceuticals and diagnostic products with the goal of making its potential products, Powered by Corixa, available to patients around the world. Corixa was founded in 1994 and is headquartered in Seattle, Wash., with additional operations in Hamilton, Mont. and Redwood City, Calif. For more information, call the company's investor relations information line at 1-877-4CORIXA or 1.877.426.7492 or visit Corixa's website.


Forward-Looking Statements
Except for the historical information presented, certain matters discussed in this press release are forward-looking statements that are subject to certain risks and uncertainties that could cause actual results to differ materially from any future results, performance or achievements expressed or implied by such statements. Such risks and uncertainties include adverse events resulting from further clinical trials of the leishmaniasis vaccine being developed by IDRI and Corixa and other risks detailed from time to time in Corixa's public disclosure filings with the U.S. Securities and Exchange Commission (SEC), including the Annual Report on Form 10-K for the fiscal year ended December 31, 1999, and the Company's Registration Statement on Form S-3, filed on March 7, 2000. Copies of Corixa's public disclosure filings with the SEC are available from its investor relations department.


###
The Bill & Melinda Gates Foundation is dedicated to improving people's lives by sharing advances in health and learning with the global community. Led by Bill Gates' father, William H. Gates, Sr., and Patty Stonesifer, the Seattle-based foundation has an endowment of approximately $24 billion.

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77.) Report on the fourth TDR/IDRI meeting on second generation vaccine against Leishmaniasis. Merida, Yucatan, Mexico, May 1-3, 2001.
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Source: www.uady.mx/~biomedic/biblio.htm REVISTA BIOMEDICA
Revista Biomedica, 2002 Vol_ 13 Num 1.htm

Eric Dumonteil1, Diane McMahon-Pratt2, Virginia L. Price3.

1Universidad Autónoma de Yucatán, Mérida, Yucatán, Mexico, 2Yale University School of Medicine, New Haven, CT, USA, 3UNDP/ World Bank/WHO Special Programme in Training and Research in Tropical Diseases (TDR) Geneva, Switzerland.

A meeting entitled "Second generation vaccines against leishmaniasis" was held in Merida, Mexico on May 1-3, 2001. The meeting was hosted by the Universidad Autónoma de Yucatán in Merida and sponsored by the WHO/World Bank/UNDP Special Programme for Training and Research in Tropical Disease (TDR) and the Infectious Disease Research Institute (IDRI). The meeting consisted of two parts: 1) a review of the evaluation of recombinant Leishmania antigen testing in two independent labs and 2) individual presentations regarding second generation Leishmania vaccine candidates. [The first generation Leishmania vaccine is considered to be the killed whole Leishmania parasite vaccine that has been tested in clinical trials in Iran, Sudan, and Latin America (1-3). A new formulation of this vaccine including bacillus Calmette-Guérin(BCG) and alum as adjuvants will be tested in 2001 in Iran and Sudan]

In an effort to organize a systematic way of testing candidate antigens as potential vaccine candidates, comparative studies were done in two different laboratories; Universidade Federal de Bahia, Salvador, Brazil, and the University of Copenhagen, Copenhagen, Denmark. Nine different antigens (plus soluble Leishmania antigen as a positive control) were tested as vaccine candidates in murine models for Leishmania. Adjuvants used were monophosphoryl lipid (MPL, donated by Corixa corporation) and interleukin 12 (IL-12 , donated by Genetics Institute). The results of these studies were, however, inconclusive largely due to technical problems regarding the stability and potency of the antigens. The need for guidelines concerning the characterization and testing of recombinant antigens were discussed (see conclusions).

The individual presentations on second generation Leishmania vaccine candidates suggested several promising candidates that have shown protection in animal studies. Some of these presentations are summarized below.

Antigens that show protection in animal models

Nicolas Fasel of the University of Lausanne, Lausanne, Switzerland and Sima Rafati of the Institute Pasteur in Tehran, Iran, described vaccination studies against cutaneous Leishmania (L. major) done in mice and African green monkeys. The antigen used was L. major histone H1, a nuclear antigen that has no homology to human H1. Antigen was made either as a recombinant glutathione S-transferase (GST)-fusion protein in E. coli or as a long synthetic peptide representing the complete histone H1 sequence. Montanide ISA 721 was used as adjuvant. Elisa data showed that both antigens elicited an increase in specific antibodies after vaccination, some (but not all) monkeys had increase gamma interferon (IFN-gamma) levels, and all exhibited a positive delayed type hypersensitivity (DTH) response.

A DNA vaccine was also prepared encapsulated in microspheres, small particles of poly-lactide-co-glycolide. DNA encoded antigens were histone H1, cysteine proteinase B (CPB), and cysteine proteinase A (CPA), alone or in combination. A single injection of 20 µg DNA was used for vaccination of mice. Only the cocktail containing CPA and CPB showed protection.

J. Alexander (University of Strathclyde, Glasgow, Scotland) studied the vaccine potential of enzymatically active and inactive CPB to protect against L. mexicana, and showed that vaccination with CPB alone may stimulate interleukin 4 (IL-4) production and exacerbate infection. However, the addition of IL-12 to these active and inactive CPB recombinant antigens resulted in significant inhibition of lesion growth in CBA/Ca, BALB/c and C57BL/6 mice following challenge infection. A DNA vaccine consisting of genes for the antigens CPB, GP63 and GP46 has been tested in a murine model against L. mexicana by E. Dumonteil of the Universidad Autónoma de Yucatán, Merida, Mexico. The individual antigens showed limited protection, but a high dose (50 µg each) of the combination of all three antigens showed strong protection in initial studies.

G. Grimaldi and R. Porrozzi (FIOCRUZ, Rio de Janeiro, Brazil) and A. Campos-Neto (Infectious Disease Research Institute, Seattle, WA, USA) have examined rhesus macaques (Macaca mulatta) as an experimental model for study of cutaneous leishmaniases (CL) induced by either L. major, or L. amazonensis, or L. braziliensis infection. The rhesus macaques as a model for human disease is thought to be appropriate in that the outcome of Leishmania infection in primates is similar to that in humans in that it largely depends on the virulence of the infecting parasite strain and the immune responsiveness of the host. Lesion development, histopathology of CL and the Th1-type profile are similar to the human disease.

Several antigen/adjuvant combinations were tested for the induction of protection in both mice and monkeys to Leishmania challenge. The L. major antigens tested were: LeIF (ribosomal initiation factor), STI1 (a stress and temperature inducible protein), and TSA (a thiol-specific antioxidant).

Mice were immunized with a di-fusion comprised of TSA-LmSTI1 and a tri-fusion comprised of TSA-LmSTI1-LeIF. Protection was seen with either the di-fusion or the tri-fusion with IL12 as adjuvant but not with several other adjuvants tested.

Monkey immunizations were performed with TSA and LmSTI11 as antigens and IL-12 plus alum as the adjuvant. (Alum with IL-12 does not appear to change the Th1 pattern normally seen with IL-12 as adjuvant but seems to make the antigen more immunogenic.) Three immunizations of 25 µg each antigen were given with a challenge six weeks after the last immunization. Protective immunity followed immunization, in that no lesions developed in any of vaccinated monkeys (n = 6) after infection. (All controls developed lesions.) Monkeys vaccinated with LmSTI1-TSA+IL-12+alum were also protected against rechallenge with L. major (4 months following the first challenge), but no cross-protection was found against tertiary challenge 2 months following the second challenge with L. braziliensis. The data point to a long-lasting anti-L. major immunity induced in primates by the recombinant antigens LmSTI1 and TSA.

G. Matlashewski of McGill University (Montreal, Canada) described the A2 genes of Leishmania, which encode proteins of unknown function. The A2 genes are part of a multigene family of at least 11 genes and the protein is composed largely of multiple repeats of a 10 amino acid sequence. The protein is abundant in amastigotes and absent in promastigotes. It is present in L. infantum, L. chagasi, L. mexicana, L. amazoniensis, and L. donovani but not in L. tropicana and L. major. (L. major has been shown to have the A2 genes but in truncated form.)

The role of the A2 proteins has been studied both by creating knock-out strains (8 of the 11 genes were knocked out) and by using anti-sense technology. It was then shown that the A2-deficient strains of L. donovani were avirulent in mice. Thus, A2 can be considered an amastigote specific virulence factor and therefore a good vaccine candidate for L. donovani.

A2 was tested as a vaccine (recombinant protein or DNA vaccine) in a murine model of visceral leishmaniasis. Mice vaccinated with A2 protein showed greatly reduced parasite numbers in the spleen and a mixed Th1/Th2 type of response (increased IFN-gamma by spleen cells and no increase of IL4 but increased IgG2a and IgG1 levels). A2 as a DNA vaccine gave partial protection (measured by reduced LDU).

C.B. Palatnik de Sousa (University of Rio de Janeiro, Rio de Janeiro, Brazil) studied the fucose mannose ligand (FML) antigen complex of L. donovani and its GP36 component as a vaccine.

After demonstrating the protective capacity of FML and GP36 with saponins as adjuvant in mice, a dog vaccination trial was initiated (4). The FML-vaccine was examined in dogs naturally exposed in an endemic area in Natal, Brazil. Ninety-seven percent of the vaccinated dogs produced antibodies and 100% demonstrated a DTH response to the FML antigen seven months post-vaccination. After 2 years of monitoring the dogs, data indicate that significant protection was achieved: only 8% of vaccinated dogs showed mild signs of visceral leishmaniasis, with no deaths, while in the control group 33% of the dogs developed clinical symptoms or fatal disease. After 41 months, 3/4 of the control dogs were infected (as determined by PCR and bone marrow puncture) and the remaining vaccinated dogs were all negative for parasites and continued to maintain positive antibody and DTH responses. Thus, the FML-vaccine induced significant protection against canine visceral leishmaniasis in the field.

D. Smith (Imperial College, London, UK) described the hydrophilic acylated surface antigens (HASPs) as vaccine candidates. HASPs are dually acetylated at their N-termini, a modification required for subcellular targeting within the parasite to the membrane. Although the precise function(s) of these proteins are not known, genetic experiments using knock-out and over-expression mutants clearly indicate a role in virulence of L. major. Although all of the proteins family members are expressed in metacyclic promastigotes, only HASPA1 and B are detectable in amastigotes.

HASPs, expressed as unmodified recombinant proteins in E.coli, were employed for immunization of BALB/c mice (with or without IL-12). Immunization resulted in protection against infection with L. donovani in the animal model of visceral leishmaniasis (5). HASPB1 induces a low level of IL-12 production in murine dendritic cells. Protection correlates with the level of IFN-gamma producing CD8 T cells and IgG1 production. Interestingly, rHASPB1 fails to confer protection in IL-4 and IL-4 receptor alpha (IL-4R) deficient mice. These data suggest further complexity in the regulation of vaccine-induced immunity.

Understanding mechanisms of protection

P. Scott of the University of Pennsylvania (Philadelphia, PA, USA) discussed studies on the mechanism of protection from leishmaniasis which were done in a Vervet monkey model (6). The antigen used was ALM (autoclaved L. major) with IL-12 as adjuvant. The immunizations resulted in increased specific IFN-gamma levels and a typical Th1-type response. Specific IFN-gamma levels were as high as the positive controls, which were animals that had already healed and were resistant. However, the vaccine did not protect. The conclusion from this study was that IFN-gamma production by PBLs to crude leishmanial antigen does not always correlate with immunity.

He then discussed the role of IL-12 as one of several parameters that might be required for eliciting and maintaining immunity (7). It was shown that IL-12 knockout mice, if treated with IL-12 exogenously, are resistant to L. major infection. However, upon rechallenge after cessation of IL-12 treatment, they became susceptible. In addition, adoptive transfer of Th1 cells from healed mice into IL-12 knockout mice failed to protect against challenge. Moreover, the IFN-gamma response is not recovered in the IL-12 knockout mice. Thus, IL-12 seems to be needed continuously to maintain resistance. This might be due to a requirement for IL-12 to optimize IFN-gamma production from Th1 cells, to protect Th1 cells from apoptosis, to promote Th1 cells survival, and/or to recruit additional Th1 cells from a naïve or undifferentiated T cell population.

D. McMahon-Pratt (Yale University, New Haven, CT, USA) discussed severity of disease as a function of the infecting Leishmania species together with host genetics and consequent inflammatory and immune responses. In the case of cutaneous murine leishmaniasis, infection caused by L. amazonensis appears to be readily distinguished from that induced by L. major in a number of significant immunologic features. For L. amazonensis, the mechanisms involved in pathogenesis in the murine model appear to singularly involve immune subversion and require the participation of both CD4+ T cells and B cells (immunoglobulin). A low level of IFN-gamma (with little/no IL-4) production seems to be a feature of disease. Antigen presentation and monocyte recruitment at the local site of infection appears to be critical for the development of pathology. The roles of immune T and B cells in pathology as well as resolution of disease could impact the efficacy of vaccines against New World leishmaniasis, especially potentially as the immune response wanes after vaccination.

A. Campos-Neto (Infectious Disease Research Institute, Seattle, WA, USA), pointed out that for the past 20 years, it has been generally accepted that immunity against experimental infection of mice with Leishmania is strictly dependent of the generation of a Th1 type of immune response against the parasite's antigens. However, they have observed that several leishmanial recombinant antigens, selected on this criteria and expressed as recombinant proteins, do not necessarily turn out to be protective antigens. For example, BALB/c mice infected with L. major develop low antibody titers to the antigen Ldp23, and to many others tested. In addition, Ldp23 induces preferentially Th1 response in lymph node cells from infected mice. However, in combination with adjuvants that preferentially induce Th1 responses such as IL-12 and MPL-SE, despite stimulating strong antigen specific Th1 response in the absence of any detectable Th2 response, Ldp23 does not confer protection. In contrast, the antigen LACK stimulates strong and preferentially Th2 responses in lymph node and spleen cells from L. major-infected mice. In addition the sera of these animals contain high titers of IgG1 anti-LACK antibodies. In spite of this, LACK induces substantial protection in BALB/c mice if administered in conjunction with adjuvants that stimulate Th1 responses. In another situation, antigens like LmSTI1 stimulate strong and preferentially Th1 responses in lymph node cells of BALB/c mice infected with L. major and the sera of these animals contain high titers of both IgG1 and IgG2a anti-LmSTI1 antibodies. Moreover, LmSTI1 induces excellent protection in these animals if used with IL-12 or MPL-SE as adjuvants.

Therefore, for antigen selection purposes in vaccine development against leishmaniasis these results do not support the Th1 paradigm, although a Th1 response may well be essential for protection against leishmaniasis. In conclusion, for vaccine development against leishmaniasis, the polarization to Th1/Th2 antigen-specific immune response that is developed against the parasite antigens during the infectious process may be irrelevant. Rather, immunogenicity and perhaps the amount of antigen expressed or secreted by the parasite in vivo, are more important factors reflecting protective anti parasite immune response. Antigens that fulfill these criteria should therefore be more successful in inducing protection as long as they are administered with adjuvants that in combination with them modulated a strong Th1 response.

M. Barral-Neto (Universidade Federal de Bahia, Bahia, Brazil) described a priming in-vitro (PIV) system that was used to evaluate the immune response of naïve individuals following in-vivo vaccination with parasite. IFN-gamma levels were followed as an indicator of the type of response to Leishmania antigen. Peripheral blood mononuclear cells (PBMC) of volunteers who were DTH-negative and anti-Leishmania antibody-negative were used for in-vitro stimulation with Leishmania antigen and IFN-gamma was monitored. This in-vitro stimulation experiment identified two different types of responders: those that produced low amounts of IFN-gamma and those that produced high levels of IFN-gamma. The IFN-gamma levels were directly proportional to tumor necrosis factor- alpha (TNF-alpha) and interleukin-10 (IL-10) levels but not to interleukin-5 (IL-5) levels. Those volunteers who produced low amounts of IFN-gamma in vitro remained low producers 40 days after vaccination, whereas high producers exhibited increased IFN-gamma production after vaccination. However, at 6 months post-vaccination, all individuals tested were producing similarly high levels of IFN-gamma upon stimulation of their PBMC with Leishmania promastigotes, indicating that the low in-vitro producers were slow in-vivo responders to vaccination. High IFN-gamma producers exhibited an increased frequency of activated CD8+T cells both in-vitro and in-vivo, as compared to low IFN-gamma producers. This delay in the cell mediated immune (CMI) response may influence resistance to leishmaniases.

New antigen discovery

T. Aebischer, from the Max Plank Institute in Tubingen, Germany, discussed the generic characteristics of potential new protective antigens. Previous work (4) indicated that protective antigens require a minimal level of expression of 105-6 molecules per organism throughout the parasite lifecycle. In addition, protection seemed to be dependent upon antigen accessibility, i.e. a molecule expressed on the surface membrane or secreted.

Studies of the kinetics of antigen presentation in infected mice, examined using green fluorescent protein (GFP)-labeled parasites, confirm these observations. They have found two waves of antigen presenting cells in high dose infection in the draining lymph node with only the second harboring live parasites. Therefore, in natural low dose infection protective antigens presented from live parasites should be accessible throughout infection and thus would most likely be secreted or surface components. These results strengthen the conclusion that in the rational search for candidate vaccine antigens, they should be abundant, preferably secreted or surface exposed protein molecules.

J. Blackwell (Wellcome Trust Centre for Molecular Mechanisms in Disease, Cambridge, UK) described a microarray analysis of the Leishmania life cycle as a mechanism for identifying new potential vaccine antigens. In the vertebrate host, metacyclic promastigotes rapidly enter macrophages and transform to amastigote forms. They used DNA microarrays for simultaneous monitoring of expression profiles for 2183 unique Leishmania genes as the parasite undergoes the developmental transition from logarithmic promastigotes to metacyclic forms and host-derived amastigotes. From this analysis they have identified over 100 previously unknown genes which are upregulated in amastigotes. These are presently being tested as new vaccine candidates. Some cocktails of these antigens appear to be effective as DNA vaccines in mice.

Conclusions.

Based on these presentations, it was concluded that several promising Leishmania vaccine candidates exist that warrant further development, eventually towards human clinical testing. However, it was recommended that strict quality control of recombinant antigens should be included in pivotal animal studies (as well as for monkey and human trials). Antigen characterization should include: SDS-PAGE (reduced and non-reduced), Western blot analysis, endotoxin levels, sterility and amino acid analysis for determination of protein concentration. It was agreed that the minimum criteria for moving a candidate vaccine to phase I studies must include the consistent (i.e. repeated in a second independent laboratory) induction of significant protection in an animal model, and a formulation with an adjuvant than can currently be used in clinical trials. It was felt that collaboration and coordination of efforts in the testing of vaccine candidate antigens in more than one laboratory is warranted.

Additionally, though there have been important development in this field, we still do not understand completely what constitutes a protective response in humans. It is thus considered important to encourage continued effort towards understanding what constitutes long-term protection to leishmaniases both in animal models as well as in humans. Defining the correlates of protection and development of predictive assays for the evaluation of vaccine candidates are important to the progress of the field.

References.

1.- Sharifi I, FeKri A R, Aflatonian M R, Khamesipour A, Nadim A, Ahmadi-Mousavi M R, et al Randomised vaccine trial of single dose of killed Leishmania major plus BCG against anthroponotic cutaneous leishmaniases in Bam, Iran. Lancet 1998; 351: 1540-3.

2. Modabber F. First generation leishmaniases vaccines in clinical development: Moving, but what next? Curr Opin Anti-infect Invest Drugs 2000; 2: 35-9.

3.- Velez I D, del Pilar Agudelo S, Arbelaez M P, Gilchrist K, Robledo S M, Puerta J A, et al. Safety and immunogenicity of a killed Leishmania (L.) amazonensis vaccine against cutaneous leishmaniasis in Colombia: a randomized controlled trial. Trans R Soc Trop Med Hyg 2000; 94: 698-703.

4.- da Silva VO, Borja-Cabrera G P, Correia Pontes N N, de Souza E P, Luz K G, Palatnik M, et al. A phase III trial of efficacy of the FML-vaccine against canine kala-azar in an endemic area of Brazil (Sao Goncalo do Amaranto, RN). Vaccine 2000; 19: 1082-92.

5.- Stager S, Smith D F, Kaye PM. Immunization with a recombinant stage-regulated surface protein from Leishmania donovani induces protection against visceral leishmaniasis. J Immunol 2000; 165: 7064-71.

6.- Gicheru M M, Olobo J O, Anjili C O, Orago A S, Modabber F, Scott P. Vervet monkeys vaccinated with killed Leishmania major parasites and IL-12 develop a type 1 response, but are not protected against challenge infection. Infect Immun 2001; 69: 245-51.

7.- Park A Y, Hondowicz B D, Scott P. IL-12 is required to maintain a Th1 response during Leishmania major infection. J Immunol 2000; 165: 896-902.

8.- Aebischer T, Wolfram M, Patzer S I, Ilg T, Wiese M, Overath P. Subunit vaccination of mice against new world cutaneous leishmaniasis: comparison of three proteins expressed in amastigotes and six adjuvants. Infect Immun 2000; 68:1328-36.

El artículo completo en formato PDF Rev Biomed 2002; 13:53-58

Corresponding address: Eric Dumonteil, Ph.D. Laboratorio de Parasitología, Centro de Investigaciones Regionales, Universidad Autónoma de Yucatán, Ave. Itzaes # 490 x 59, C.P. 97000, Mérida, Yucatán, Mexico. Tel/Fax: (52-9) 924-59-10 Email: [email protected]

Received October 18, 2001. Acepted December 19, 2001.

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78.) Drug development: LEISHMANIASIS
====================================================
Source: The WHO 1999 -2.000

Miltefosine promises to be the first oral treatment for visceral leishmaniasis. Dose-finding and efficacy confirmation trials have been completed and the drug will shortly be submitted for registration.

Miltefosine

Miltefosine, an anticancer drug that TDR and ASTA Medica (now Zentaris) have been developing for visceral leishmaniasis, could be the first oral treatment to become available for this disease (see TDR Programme Report, 1997-1998). During the biennium, Phase II (dose-finding) and Phase III (efficacy confirmation) trials in adult patients were successfully completed. In mid-2001, the drug will be submitted for registration in India and Germany for use in patients aged over 12 years. As well, a Phase II trial in children was completed, and a Phase III trial in children has begun (expected to finish end of October 2001), an indication which could later be added to the criteria for use. A Phase IV (post registration, in a real field situation) trial has been planned for India, Nepal and Bangladesh, which will feed into the control programme. This Phase IV trial will be supported by the WHO Regional Office for South-East Asia and the Government of India.

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79.) Vaccines for cutaneous leishmaniasis
====================================================
Source: The WHO 1997-1998


A number of first-generation vaccines[i] have produced encouraging results when used in single dose in Phase III field trials[i]. They are now being tested in two and three doses against cutaneous leishmaniasis in Iran, in Ecuador (outside TDR), Colombia, Brazil (outside TDR) and Venezuela.

Iran

Results from a vaccine trial in Bam, using a single dose of killed Leishmania major promastigotes[i] + BCG[i], show that boys were protected more than girls and that BCG alone may have a short-lived protective effect. After two years, 78% protection was seen in boys who had received the vaccine.

In a trial in Isfahan, only 35% of children people given a single-dose vaccine of killed L. major promastigotes + BCG, responded to the vaccine. The incidence of disease was 35% lower in those who responded to the vaccine compared to those who did not respond.

Ecuador (outside TDR)

Over 70% protection against cutaneous leishmaniasis was recorded during the first year of a small-scale vaccine trial. The vaccine was given in two doses, and consisted of whole killed Leishmania promastigotes native to the New World plus BCG. An ongoing study will determine how long this protective immunity will last.

A placebo-controlled trial is being conducted using a vaccine of whole killed promastigotes from L. amazonensis + BCG.

Colombia

Phase II trials have compared two vaccines consisting of L. amazonensis with and without BCG. Plans for Phase III trials will involve only the preparation without BCG.

Other trials

Phase II trials in Brazil (outside TDR) have shown that a preparation of merthiolate L. amazonensis without BCG is immunogenic.

A trial in Venezuela of a killed L. mexicana + BCG vaccine for cutaneous leishmaniasis, is being guided, but not funded by TDR.


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80.) Vaccine development: LEISHMANIASIS
====================================================
Source: The WHO

Results of the first trial of a vaccine against visceral leishmaniasis showed it to be associated with a lower incidence of disease


Sudan: leishmaniasis vaccine trial The first trial of a vaccine against visceral leishmaniasis in humans was completed.[8] The trial was conducted by scientists of the Institute of Endemic Diseases, University of Khartoum, Sudan, supported by TDR and assisted by Médecins sans Frontières-Holland, using a vaccine composed of autoclaved Leishmania major promastigotes (Fesharki et al, at Razi Vaccine and Serum Institute, Iran) mixed with a low dose of bacillus Calmette-Guérin (BCG) (as adjuvant), which was compared with BCG alone. The presence of extensive cross-reactivity between different species of Leishmania was the rationale behind this trial of a vaccine, made from L. major, which had proved almost 100% effective in langur monkeys against L. donovani infection.[9]


The trial was carried out in the Sudan where visceral leishmaniasis is a major cause of morbidity and mortality (a prevalence of 80-130 per 1000 in the study area). Here, as in many other endemic countries, the development of a safe, effective and cheap vaccine would be a long-term solution for controlling visceral leishmaniasis. Drug treatment does exist, but is prohibitively expensive, not easy to administer, not always available, and resistance to it develops rapidly. Vector control is also a possibility, but requires infrastructure that is not available.

In the double-blind study, no evidence was found that two injections of Leishmania + BCG offered significant protective immunity against visceral leishmaniasis compared with BCG alone. However, the Leishmania + BCG vaccine did induce significantly higher rates of leishmanin skin test (LST) conversion (30%, vs. 7% by BCG alone) at 42 days, which was associated with a significantly lower incidence of disease – responders had a 43% lower incidence of leishmaniasis as compared to LST non-responders (7.2% vs. 12.7%, p<.003). Similar results, i.e. lower incidence of disease in LST converted than non-converted individuals, have been obtained in other studies. As BCG alone might have some protective activity against leishmaniasis, as seen in the therapeutic trials of Convit et al in Venezuela, the real prophylactic effect of the vaccine, compared to no vaccine (and not to BCG as in this trial), might be higher. In this trial, BCG was used for ease of keeping the trial blind.

To improve the immunogenicity of killed Leishmania vaccines, different adjuvants used in humans are being sought. Safety and efficacy trials of alum-precipitated Leishmania + BCG have begun in the Sudan. Preliminary results showed that a single injection produces a dramatic immune response – a strong LST conversion – in every recipient (better than three injections without alum). Multiple doses of L. major + BCG vaccine did not prove to be more effective than a single injection in preventing cutaneous leishmaniasis, so development of this vaccine is not being pursued without the addition of alum. Following a successful comparative safety and immunogenicity trial of different formulations of Mayrink’s vaccine (killed L. amazonensis, produced by Biobras, Brazil), with or without adjuvant,[10] a trial of three injections without adjuvant has begun in Colombia (Velez ID, Universidad de Antioquia, Medellin).

A dozen or so recombinant antigens were recently evaluated as potential second generation vaccine candidates in independent testing coordinated by TDR and conducted in two laboratories in Brazil and Denmark. Unfortunately the results were inconclusive, as not all the control preparations were active. Several second generation vaccines (synthetic peptides and multi-fusion recombinant proteins) are being developed outside TDR, and efforts are being made to create a collaborative programme for these activities, ideally through a consortium which will coordinate the definition, testing and advanced development of promising candidate antigens.

[8] Khalil EA et al. Autoclaved Leishmania major vaccine for prevention of visceral leishmaniasis: a randomised, double-blind, BCG-controlled trial in Sudan. Lancet, 2000, 356(9241):1565-9. [PMID: 11075771 [PubMed - indexed for MEDLINE]]

[9] Misra A, Dube et al. Successful vaccination against Leishmania donovani infection in Indian langur using
alum-precipitated autoclaved Leishmania major with BCG. Vaccine, 2001, 19(25-26):3485-92. [PMID: 11348715 [PubMed - indexed for MEDLINE]]

[10] Velez ID et al. Safety and immunogenicity of a killed Leishmania (L.) amazonensis vaccine against cutaneous leishmaniasis in Colombia: a randomized controlled trial. Transactions of the Royal Society of Tropical Medicine and Hygiene, 2000, 94: 698-703. [PMID: 11198661 [PubMed - indexed for MEDLINE]]

====================================================
81.) Vaccine from fly spit
====================================================
Fly saliva could protect us from a dangerous disease.
7 August 2001

JOHN WHITFIELD
Source: www.nature.com/

Worth spit: a new vaccine works against the fly that carries Leishmania.
© SPL


An injection of fly spit sounds like medieval quackery, but could be the vanguard of modern medicine. Researchers have used sand fly saliva to develop a vaccine that protects mice against leishmaniasis, a disease spread by the insect1.

This, the first potential vaccine against an insect-borne disease derived from the carrier, rather than the parasite itself, is based on chemicals that help the fly feed, preventing blood clotting and dilating blood vessels.

Leishmania, a single-celled protozoon, infects about 12 million people worldwide. Different types of leishmaniasis erode the mucus membranes of the mouth, nose and throat, or internal organs. This latter condition is often fatal.

Jose Ribeiro, of the US National Institute of Allergy and Infectious Diseases (NIAID) in Bethesda, Maryland, focused on the skin-attacking Leishmania major, which is spread by the Middle Eastern sand fly (Phlebotomus papatasi).

The bites of uninfected sand flies seem to protect mice from Leishmania, the group had previously found. Apparently, the same thing applies to people in South America, Africa and around the Mediterranean, where the disease is common. "Newcomers and children get sick, but they're the tip of the iceberg of those who are infected," says Ribeiro.

Ribeiro's team isolated a protein from the fly's saliva that provoked a particularly strong mouse immune response to Leishmania. They identified the gene for this protein, and injected it into mice. The mouse's cells made the protein from the DNA, triggering their immune response.

When subsequently injected with a mixture of fly saliva and Leishmania, the mice developed none of the disease symptoms. They did not eliminate the parasite, but suppressed it to one-hundredth of the level found in untreated mice. This is similar to the response of humans who are 'immunized' by fly bites, says Ribeiro.

"This is a very promising vaccine candidate," says immunologist Heidrun Moll of the University of Wurzburg, Germany. The importance of the carrier in the immune response to insect-borne disease is widely acknowledged, says Moll, but technical difficulties - such as breeding the insects and keeping them contained - has slowed progress along this avenue.

About half a dozen potential leishmaniasis vaccines have been derived from the parasite over the past decade, including one by Moll herself. Eventually, people could be immunized using a cocktail of vaccines against both fly and parasite, she says.

As well as different vaccines for different Leishmania species, we may also need vaccines based on different species of sand fly - salivary proteins vary a great deal between species, says Ribeiro. "But this is not a big deal," he assures - it takes less than two months to go from fly spit to vaccine candidate.

Researchers are also targeting the insects that carry other vaccines. One possible way to fight malaria is to develop vaccines that "do harm" to mosquitos that suck them up in human blood, says Filip Dubovsky of the Malaria Vaccine Initiative in Washington DC. As yet, there are no candidate malaria vaccines that exploit mosquito saliva.


References
Valenzuela, J. G. et al. Towards a defined anti-Leishmania vaccine targeting vector antigens: characterization of a protective salivary protein. Journal of Experimental Medicine, 194, 1 - 13, (2001).

====================================================
82.)Toward a defined anti-Leishmania vaccine targeting vector antigens: characterization of a protective salivary protein.
====================================================
J Exp Med. 2001 Aug 6;194(3):F7-F9.

Valenzuela JG, Belkaid Y, Garfield MK, Mendez S, Kamhawi S, Rowton ED, Sacks DL, Ribeiro JM.

Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 4 Center Dr., Rm. 4/126, Bethesda, MD 20892, USA.

Leishmania parasites are transmitted to their vertebrate hosts by infected phlebotomine sand fly bites. Sand fly saliva is known to enhance Leishmania infection, while immunity to the saliva protects against infection as determined by coinoculation of parasites with vector salivary gland homogenates (SGHs) or by infected sand fly bites (Kamhawi, S., Y. Belkaid, G. Modi, E. Rowton, and D. Sacks. 2000. Science. 290:1351-1354). We have now characterized nine salivary proteins of Phlebotomus papatasi, the vector of Leishmania major. One of these salivary proteins, extracted from SDS gels and having an apparent mol wt of 15 kD, was able to protect vaccinated mice challenged with parasites plus SGH. A DNA vaccine containing the cDNA for the predominant 15-kD protein (named SP15) provided this same protection. Protection lasted at least 3 mo after immunization. The vaccine produced both intense humoral and delayed-type hypersensitivity (DTH) reactions. B cell-deficient mice immunized with the SP15 plasmid vaccine successfully controlled Leishmania infection when injected with Leishmania plus SGH. These results indicate that DTH response against saliva provides most or all of the protective effects of this vaccine and that salivary gland proteins or their cDNAs are viable vaccine targets against leishmaniasis.

====================================================
83.) Otros estudios y referencias Bibliograficas
====================================================
Source; The net

1, CHARACTERIZATION OF SECRETED/SHED LEISHMANIA ANTIGENS FOR USE IN SUB-UNIT VACCINE AND DIAGNOSTIC DEVELOPMENT, Brannon M., U.S.A.

2, COMPARATIVE STUDY OF CELLULAR RESPONSES AFTER STIMULATION OF HUMAN T LYMPHOCYTES BY DEFINED RECOMBINANT LEISHMANIAL ANTIGENS, Meddeb-Garnaoui A., TUNISIA

3, CHARACTERIZATION OF HEALTHY DONORS AND CL PATIENTS' T CELL RESPONSES TO LEISHMANIAL ANTIGENS, Turgay N., TURKEY

4, THE EFFICACY OF IMMUNIZATION WITH MIXED LIVE/KILLED LEISHMANIA MAJOR PROMASTIGOTES, Marufov A., UZBEKISTAN

5, MULTIPLE DOSES OF AUTOCLAVED LEISHMANIA MAJOR VACCINE AGAINST ANTHROPONOTIC CUTANEOUS LEISHMANIASIS IN BAM, IRAN, Sharifi I., IRAN

6, PROTECTIVE EFFICACY OF MULTIPLE DOSES OF A LEISHMANIA MAJOR VACCINE MIXED WITH BCG IN ZOONOTIC CUTANEOUS LEISHMANIASIS (ZCL) ENDEMIC AREA, Khamesipour A., IRAN

7, IMMUNE RESPONSES MEASURED IN HUMAN VOLUNTEERS VACCINATED WITH AUTOCLAVED LEISHMANIA MAJOR PLUS BCG, Mahmoodi M., IRAN

8, THE ROLE OF BCG IN IMMUNE RESPONSES INDUCED BY MULTIPLE DOSES OF AUTOCLAVED LEISHMANIA MAJOR VACCINE INJECTION, Alimohammadian M.H., IRAN

9, PHASE III EFFICACY FIELD TRIAL OF MULTIPLE DOSES OF AUTOCLAVED LEISHMANIA MAJOR VACCINE MIXED WITH BCG IN CHILDREN IN ZOONOTIC CUTANEOUS LEISHMANIASIS (ZLC) ENDEMIC AREA , Khamesipour A., IRAN

10, DEDTRITIC CELL VACCINATION AGAINST EXPERIMENTAL CUTANEOUS LEISHMANIOSIS, Dotsika E., GREECE

11, DNA VACCINATION AGAINST LEISHMANIA MAJOR IN THE MURINE MODEL WITH DIFFERENT CANDIDATE, Bahloul C., TUNISIA


12, PROCYCLIC PROMASTIGOTES OF LEISHMANIA MAJOR CONFER MORE EFFICIENT IMMUNITY THAN METACYCLIC ONE IN BALB/C MICE, Alimohammadian M.H., IRAN

13, INDUCING CELL MEDIATED IMMUNITY IN BALB/C MICE USING LIPOSOMES ENCAPSULATED WITH CRUDE LEISHMANIA ANTIGENS (CLA), Jaafari M.R., IRAN

14, ORAL IMMUNIZATION(BALB/C MICE): A NEW STRATEGY FOR PROTECTION AGAINST LEISHMANIASIS, Rossi-Bergmann B., BRAZIL

15, BCG, IL12 AND SAPONIN IN THE FML-VACCINE AGAINST VISCERAL LEISHMANIASIS IN MICE, Palatnik-De-Sou.S.A. C.B., BRAZIL

16, T-SUPPRESSIVE COMPONENTS IN L. AMAZONENSIS, Rossi-Bergmann B., BRAZIL

17, COMPARISON BETWEEN THREE VACCINES AGAINST CANINE LEISHMANIASIS: IN VITRO EVALUATION OF MACROPHAGE KILLING ABILITY, Trotta T., ITALY

18.) Identification and characterization of T cell-stimulating antigens from Leishmania by CD4 T cell expression cloning
2001
Probst P, Stromberg E, Ghalib HW, Mozel M, Badaro R, Reed SG, and Webb JR
Journal of Immunology
166/498-505


19.) Leishmaniasis vaccination: Targeting the source of infection
2001
Reed SG
Journal of Experimental Medicine
194/F7-F9


20.) Potent stimulation of the innate immune system by a Leishmania brasiliensis recombinant protein
2001
Borges MM, Campos-Neto A, Sleath P, Grabstein KH, Morrisey PJ, Skeiky YAW, and Reed SG
Infection and Immunity
69/5270-5277


21.) Protection against cutaneous leishmaniasis induced by recombinant antigens in murine and nonhuman primate models of the human disease
2001
Campos-Neto A, Porrozzi R, Greeson K, Coler RN, Webb JR, Seiky YAW, Reed SG, and Grimaldi G.
Infection and Immunity
69/4103-4108


22.) A novel protein K26 of L. chagasi in combination with K39 upgrading the sensitivity of the serodiagnosis of Visceral Leishmaniasis
2000
Badaro R, Nakatani M, Houghton R, Skeiky Y, Bhatia A, Ghalib H, Brites C, and Reed S
Clin. Infect. Disease
31/293


23.) Canine visceral leishmaniasis on Margarita Island Nueva Esparta, Venezuela)
2000
Zerpa, O, Ulrich, M, Negron, E, Rodriguez, N, Centeno, M, Rodriguez, V, Barrios, RM, Belizario, D, Reed, S, and Convit, J
Transactions of the Royal Society of Tropical Medicine and Hygiene
94/484-487

24.) Characterization of secreted/shed antigens of Leishmania for the development of recombinant antigen based sub-unit vaccine and diagnostic cocktails
1999
Skeiky YAW, Bhatia A, Daifalla NS, Jen S, Benson DB, Badaro R, Campos-Neto A, Houghton RL, and Reed SG
American Journal of Tropical Medicine and Hygiene
61/266


25.) Cloning, characterization and serological evaluation of K9 and K26: two related hydrophilic antigens of Leishmania chagasi
1999
Bhatia, A, Daifalla, NS, Jen, S, Badaro, R, Reed, SG, and Skeiky, YA
Mol. Biochem. Parasitol.
102/249-261

26.) A cloned antigen (recombinant K39) of Leishmania chagasi diagnostic for visceral leishmaniasis in human immunodeficiency virus type 1 patients and a prognostic indicator for monitoring patients undergoing drug therapy
1998
Houghton, RL, Petrescu, M, Benson, DR, Skeiky, YA, Scalone, A, Badaro, R, Reed, SG, and Gradoni, L
Journal of Infectious Disease
177/1339-1344


27.) Human and murine immune responses to a novel Leishmania major recombinant protein encoded by members of a multicopy gene family
1998
Webb, JR, CamposNeto, A, Ovendale, PJ, Martin, TI, Stromberg, EJ, Badaro, R, and Reed, S
Infection and Immunity
66/3279-3289

 

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