EXPERIMENTAL DESIGN ----- The outer surface protein A (OspA) vaccine against Lyme disease: efficacy in the rhesus monkey [Vaccine 1997 Volume 15 Number 17/18, pages 1872-1887] ----- Distribution of information Personnel responsible for the clinical examinations, sample collection, vaccine administration, and assays described below were blinded with respect to which animal had received a vaccine or a placebo injection. Study population and animal examination schedule The study population consisted of 16 male 2-3 year old Chinese Macaca mulatta (rhesus). It was divided into four groups of four animals each, according to the vaccine formulation received. Animals were examined bi-weekly during the 12-week period before the challenge infection, and weekly thereafter. Vaccine formulations and vaccine administration protocol Vaccine formulations and the vaccine administration protocol were as described previously (4). Animals L458, L594, L971 and M585 received NS1-OspA/Al(OH)3. The NS1-OspA fusion protein is composed of recombinant OspA (cloned from the B. burgdorferi sensu stricto isolate ZS7) that lacks the amino-terminal cysteine and is fused to a fragment of 81 N-terminal amino acids from the nonstructural influenza virus protein NS1. Animals L476, L712, L642 and M106 received NS1-OpsA/4'- monophosphoryl lipid A (MPL)/Al(OH)3; animals M243, L537, M219 and M107 received lipidated OspA/Al(OH)3, a formulation that was used also in the Phase III human trial (14); animals L457, L549, M021, and M581 received only Al(OH)3. Inoculation with B. burgdorferi All animals were inoculated with the B31 strain of B. burgdorferi by the natural route of tick bite using infected Ixodes scapularis nymphal ticks. Assessment of vaccine efficacy Detection of spirochaetes by direct immunofluorescence (DFA) in ticks after infection. Since immunity to B. burgdorferi elicited by OspA appears to be effected chiefly within the tick (20), ticks used for the challenge infection were analysed for the presence of residual spirochaetes by DFA. Engorged ticks that had fed on each animal were divided into two equal groups. Ticks in one of the groups were dissected 4-5 days post-engorgement and ticks in the second group were dissected 3 weeks post-engorgement. Western blot analysis of serum samples collected before and after challenge infection. Seroconversion of the animals was investigated by Western blot using detergent extracts of whole B. burgdorferi as antigen. Serum samples were obtained from all of the animals longitudinally throughout the study period. In virto culture of skin biopsy samples. Skin biopsy samples were collected from each animal before the administration of the first vaccine dose and 1-4 weeks after the challenge infection (post- challenge, PC) and were cultured for 8 weeks. Polymerase chain reaction (PCR) applied to skin samples. PCR of skin biopsies was performed on samples collected as above. Histology and immunohistochemistry of skin biopsy samples. Skin biopsies collected as above were processed for histology and immunohistochemistry. Immune suppression The purpose of the immune suppression (IS) experiment was to assess whether a putative low-level B. burgdorferi infection would be rendered apparent by radically immune suppressing a subgroup of vaccinated monkeys. Five of the vaccinated animals were involved in the experiment. Three had been vaccinated with NS1-OspA/Al(OH)3 (L594, M585, L971) and the other two (L712, M106) with NS1-OspA/Al(OH)3/MPL. Four animals were immune suppressed and one (L971) was left as control and not immune suppressed. IS began at 50 weeks PC and continued for 6 weeks, at which point the animals were sacrificed. IS was assessed longitudinally by performing complete blood cell counts (CBC) and peripheral blood mononuclear cell (PBMC) counts, and by measuring blastogenic responses of PBMCs to concanavalin A (Con A), pokeweed mitogen (PWM), and whole killed B31 B. burgdorferi spirochaetes. The well-being of the animals during IS was assessed by a weekly clinical examination, and through the results of CBC, urinalysis and serum chemistries performed either weekly or bi-weekly. Electrocardiograms (EKG) were also performed, 1 week and 1 month after IS had begun. Possible reactivation of infection subsequent to IS was assessed as above, by in vitro culture, PCR, histology and immunohistochemistry of skin biopsy samples, postmortem analyses and, in addition, in vitro cultivation of urine and bronchoalveolar lavage samples, nerve conduction studies (NCS) and xenodiagnosis. Post-mortem analyses All of the animals from the group that received Al(OH)3, three of the animals that were vaccinated with lipidated OspA/Al(OH)3 and one of the animals that were given NS1-OspA/MPL/Al(OH)3 were sacrificed between week 30 and 40 PC. Animal M219 of the 'lipidated OspA' group and animal L458 of the 'NS1-OspA/Al(OH)3 group died unexpectedly while under anaesthesia, by 2 weeks and 19 weeks PC, respectively. The remaining five animals, which participated in the IS experiment, were sacrificed 56 weeks PC. The following evaluations were performed postmortem. (1) Assessment of gross pathologic changes. This was done in all animals in the following organ systems: thyroids and parathyroids, respiratory system, heart and vessels, spleen, liver, biliary system, pancreas, musculo-skeletal system, gastrointestinal tract, adrenal glands, urinary system, genital system, peripheral lymph nodes, and peripheral and central nervous systems. (2) Assessment of histopathological changes and immunohistochemical evidence of present or past infection with B. burgdorferi-specific monoclonal antibodies. These were assessed in samples taken from skin, synovium, heart, brain, central and peripheral nerves, lungs, kidney, ureter and bladder. (3) In vitro culture and PCR with primers hybridising to a B. burgdorferi chromosomal DNA fragment. Both were performed on a similar set of tissue samples as in (2). (4) Morphometric quantification of cells that bind the anti-Ospa mAb LA31 in heart and lungs. The four control animals and six of the vaccinated animals were involved in this study. (5) Silver-staining (for spirochaetes) of sections from the brain. Brain sections from all of the control monkeys, seven of the vaccinated monkeys and three monkeys that were not challenged with B. burgdorferi were employed in this study. ----- See complete document at: The outer surface protein A (OspA) vaccine against Lyme disease: efficacy in the rhesus monkey [Vaccine 1997 Volume 15 Number 17/18, pages 1872-1887] http://www.geocities.com/HotSprings/Oasis/6455/rhesus-index.html