RESULTS ----- 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] ----- Detection of spirochaetes by DFA in ticks, after infection Of the 320 infective nymphs placed on the 16 animals, 188 (59%) were either fully (132) or partially (56) engorged with blood. The number that fed on each monkey ranged from two (animal M219) to 18 (animal L971), the mean being 11.8 (Table 1). The rationale for dissecting half of the ticks 4-5 days after their removal from the capsules and the other half 3 weeks after removal entailed the assumption that, if there were quantitative differences in the spirochaete load of ticks that had fed on control and on vaccinated animals, these differences could be detected early, but not late after tick capsule removal, since appreciable differences in spirochaete numbers would no longer exist after 3 weeks, due to spirochaetal multiplication. Table 1 Detection of spirochaetes by direct immunofluorescence in the infected ticks that were used for the challenge infection -------------------------------------------------------------- Vaccine/ Total no. of animal no. DFA+ ticks DFA- ticks ticks engorged(a) -------------------------------------------------------------- Al(OH)3 L457 10 1 11 L549 16 - 16 M021 6 1 9 M581 16 - 17 Lipidated OspA M243 - 5 5 L537 - 12 12 M219 - 2 2 M107 - 9 9 NS1-OspA L458 - 11 12 L594 - 11 11 L971 - 18 18 M585 1 8 11 NS1-OspA/MPL L476 - 11 12 L712 - 13 13 L642 - 15 16 M106 - 14 14 -------------------------------------------------------------- (a)Only live ticks were dissected and examined by DFA. On the other hand, if only very small numbers of spirochaetes were detectable in any tick 4-5 days after tick capsule removal, multiplication over a 3-week period would permit detection by DFA. For each of the animals, no differences were observed in the number of spirochaetes per tick in ticks dissected early or late, thus indicating that spirochaetes may not have multiplied appreciably in the days following engorgement. Hence, only the pooled results are shown for each animal in Table 1. All but two of the ticks that fed on animals that received placebo and that were dissected (43) harboured large numbers of spirochaetes. Conversely, all but one of the ticks that fed on OspA-vaccinated animals and that were dissected (121) harboured no detectable spirochaetes (Table 1). In one of the vaccinated animals (M585), eight blood engorged ticks were DFA negative, but one nymph was DFA positive. This animal was fed upon by 11 nymphs, two of which died before a DFA test could be performed. The spirochaete density on this one preparation was at least tenfold lower (about ten fluorescent spirochaetes on the entire slide) than the number generally observed in the contents of nymphs that fed on control monkeys (Figure 1). [Image] Figure 1 DFA analysis of ticks after the challenge infection. Direct immunofluorescence view of spirochaetes within ticks that fed on (a) a control animal and (b) a vaccinated animal (M585) Western blot analysis of serum samples For the purpose of vaccine efficacy analysis, Western blots were used primarily to investigate whether vaccinated animals had seroconverted to showing antibodies to B. burgdorferi antigens other than OspA. Western blots of whole antigen extracts of B. burgdorferi B31 were incubated with serum samples collected from all animals immediately before and at 4, 6, 9, 15, 27 and 40 or 45 weeks PC. Only the results obtained at weeks 40 or 45 PC are shown (Figure 2). The pattern of reactivity of all serum samples collected after the challenge infection was compared with the reactivity before challenge. Only in one instance, by week 15 PC, one animal, L594, showed one band that was not present before challenge infection. However, by week 45 PC this band had disappeared (Figure 2, tracks 4 and 5). In marked contrast with the essentially complete absence of detectable anti-B. burgdorferi antibodies in OspA- vaccinated animals, serum samples from the four animals that had received Al(OH)3 alone reacted with several antigens by weeks 6 and 15 PC, and with even more numerous bands by weeks 40-45 PC (not shown). Anti-P39 antibodies were detectable in all control animals. To better visualise the gradual increase in the number of bands recognised by serum from control animals over time, which is indirect evidence of an active infection in these animals, serum samples obtained at weeks 4, 8, 12, 16, 20, 24, 28, 33, 36 and 40 PC were compared on the same Western blot (Figure 3(A)). Bands were counted and plotted as a function of time PC (Figure 3(B)). The most pronounced increases in the number of antigens recognised were observed between weeks 4 and 8 PC. However, in some animals, the number of bands continued to increase after this time interval. [Image] Figure 2 IgG antibody responses to whole B. burgdorferi antigens in vaccinated animals before and after the challenge infection. Serum samples were obtained from each animal immediately before and several weeks after the challenge infection. For each animal, a pair of lanes of the Western blot was employed. The first lane of each pair was incubated with the pre-challenge serum and the second lane with the serum obtained post-challenge (PC). Animal L458, lanes 2 and 3; L594, lanes 6 and 7; L971 lanes, 12 and 13; M585, lanes 17 and 18; L476, lanes 4 and 5; L712, lanes 10 and 11; L642, lanes 8 and 9; M106, 15 and 16; L537, lanes 19 and 20; M107, lanes 21 and 22; M243, 23 and 24; M219, lanes 25 and 26. Monoclonal antibodies to P41, P39, OspB and OspA, lanes 1 and 14. Extracts of whole B. burgdorferi B31 were electrophoresed on 15% acrylamide gels. Serum samples from all animals were used at a final dilution of 1:50. L458, week 19 PC; L594, L971, M585, L476, L712, L642, M106, week 45 PC; L537, M107, M243, week 40 PC; M219, week 2 PC. [Image] Figure 3 (A) Longitudinal analysis of the IgG antibody responses of control animals to whole B. burgdorferi antigens. Animal L457, lanes 1- 9 sera from weeks 0 (pre-challenge), 4, 8, 12, 16, 20, 24, 28 and 33 PC, respectively; L549 lanes 10-18, sera from same weeks; M021 lanes 20-30, sera from same weeks, plus weeks 36 and 40 PC; M581, lanes 31-41, sera from same weeks as M021. Monoclonal antibodies to P41, P39, OspB and OspA, lanes 19 and 42. Extracts of whole B. burgdorferi B31 were electrophoresed on 15% acylamide gels. Serum samples from all animals were incubated with the nitrocellulose blot at a final concentration of 1:100. (B) Plot of the number of bands appearing on the Western blot of (A) as a function of time (in weeks) PC In vitro culture of skin biopsy samples Skin samples collected weekly from all animals between 1 and 4 weeks PC were cultured in BSK-H medium as described in the Methods section. Samples were kept for 8 weeks, and checked weekly for presence of B. burgdorferi by darkfield microscopy. No spirochaetes were found. These uniformly negative results were probably an artefact due to the low ratio of medium to tissue volumes, as explained in the Discussion section. Clinical examination of the skin, histopathology, immunohistochemistry and PCR analysis of skin biopsy samples Erythema migrans was not observed clinically in any animal. Skin biopsy samples were obtained, fixed, sectioned, and stained as described in Materials and Methods. Two of the control animals, L457 and M581, showed histopathological and immunohistochemical evidence of infection in skin samples taken at weeks 3 and 4 PC, respectively (Table 2). The dermatitis was scored as 3, which corresponds to a deep perivascular cellular infiltrate involving vascular structures at the panniculus- dermis junction. The infiltrates consisted of lymphocytes, macrophages and plasma cells. B. burgdorferi antigen was detected with the anti-7.5 kDa antibody in both skin biopsies. Three of the animals vaccinated with lipidated OspA (M243, M107 and M219) showed histopathological evidence of infection and M219 also showed reactivity with the anti-7.5 kDa mAb. One of the animals immunised with NS1-OspA/MPL (L712) showed both histopathological and immunohistochemical evidence of infection. The lesion observed in this animal was rated type 2, which corresponds to a dermatitis involving the superficial perivascular structures with an infiltrate of primarily macrophages and the occasional neutrophil. No lesions or evidence of B. burgdorferi antigen were observed in animals immunised with NS1-OspA (Table 2). All skin biopsies obtained before the beginning of the vaccination schedule were negative. Skin biopsy samples collected weekly both from vaccinated and control animals between weeks 1 and 4 PC were processed for PCR as described in Materials and Methods. The results are shown in Table 2. Only the control animals showed presence of detectable B. burgdorferi DNA. Tubes without template or with lysates of skin collected before the challenge infection did not show a positive signal on Southern blots. Table 2 PCR histopathological and B. burgdorferi-specific immunohistochemical evaluation of skin biopsy samples obtained 1-4 weeks after the challenge infection ------------------------------------------------------------------------ Week 1 Week 2 Week 3 Week 4 ------------ ------------ ------------ ------------ Vaccine/ PCR HP IH PCR HP IH PCR HP IH PCR P H animal no. (a) (b) (c) [sic] ------------------------------------------------------------------------ Al(OH)3 L457 - - - + - - + D3 + - - - L549 - - - - - - + - - - - - M021 - - - - - - - - - + - - M581 - - - + - - - - - + D3 + Lipidated OspA M243 - - - - D2 - - - - - - - L537 - - - - - - - - - - - - M107 - - - - - - - D2 - - - - M219 - - - - D2 + ND ND NS1-OspA L458 - - - - - - - - - - - - L594 - - - - - - - - - - - - L971 - - - - - - - - - - - - M585 - - - - - - - - - - - - NS1-OspA/MPL L476 - - - - - - - - - - - - L712 - - - - D3 + - - - - - - L642 - - - - - - - - - - - - M106 - - - - - - - - - - - - ------------------------------------------------------------------------ (a) Polymerase chain reaction: -, amplicon absent; +, amplicon present. (b) Histopathology: -, normal skin; D2, superficial dermatitis; D3, deep dermatitis, severe. (c) Immunohistochemistry: positive (+) or negative (-), using mAb anti- 7.5 kDa lipoprotein, and alkaline phosphatase-labelled indicator antibodies. ND, not done. Immune suppression of vaccinated animals The number of PBMC decreased rapidly after the initiation of the drug administration protocol in all of the treated animals, and remained relatively low (<6 x 10 superscript(6) PBMC per ml) throughout the study period (Figure 4(A)). Proliferative responses of PBMC to heat-killed B. burgdorferi B31 cells and to Con A also decreased rapidly and uniformly for all suppressed animals (Figure 4(B) and Figure 4(C), respectively), whereas the response to PWM took longer to decrease and in one case (M106), remained virtually unaffected (Figure 4(D)). Post-mortem, the PBMC responses to B. burgdorferi, Con A and PWM remained uniformly depressed (not shown). In contrast, blastogenic responses of mesenteric and inguinal lymph node cells obtained post-mortem did not appear to be uniformly suppressed compared with the control animal that was not immune suppressed (L971). For example, animal M106 had a mesenteric cell response to Con A similar to that of L971 (Figure 5(A)). Responses to PWM appeared to be unaffected (Figure 5(B)). Time points labelled ND (not determined) correspond to those cases where it was not possible to collect a sufficient number of cells. [Image] Figure 4 Evolution of immune suppression as a function of time after the initiation of the immune suppression protocol. Immune suppressed animals: L594, L712, M106, M585. Control (vaccinated but not immune suppressed), L971. Number of PBMCs per ml of blood (A), and blastogenic responses to heat-killed B. burgdorferi (B31) cells (B), Con A (C) and PWM (D). NCPM, net counts per minute (counts per min incorporated by stimulated cells minus counts per min of unstimulated cells). Day 0 is the day the splenectomy was performed [Image] Figure 5 Immune suppression assessed post-mortem in PBMCs, and mesenteric and inguinal lymph node cells. Blastogenic responses to Con A (A) and PWM (B). NCPM, net counts per minute (counts per min incorporated by stimulated cells minus counts per min of unstimulated cells) Physical examinations, clinical laboratory analyses and EKG of immune suppressed animals No abnormal findings were noted throughout in physical examinations performed weekly during the 6-week IS procedure. All animals developed profound lymphopaenia within 2 days after the beginning of immune suppressive drug administration. Lymphopaenia persisted throughout the study in all animals. Absolute leukopaenia followed with onset at 14-27 days after initiation of therapy. Elevation of aspartate aminotransferase and alanine aminotransferase levels occurred in all animals. Onset ranged from day 8 to day 27 post-immunosuppression. These elevations usually occurred during one sampling date only. The same findings have been seen in other animals similarly immune suppressed and probably result from a drug effect. All electrocardiographic findings were within normal limits. Evidence of reactivation of a residual B. burgdorferi infection In vitro culture. Urine samples and cells collected from lung lavages were cultivated over an 8-week period. No B. burgdorferi spirochaetes were recovered. The rationale for culturing lung lavage specimens in the immune suppressed animals was arrived at after noticing that all of the controls and several of the vaccinated animals showed immunohistochemical evidence of B. burgdorferi antigens in the lungs (see below). Xenodiagnosis. An attempt was made to recover spirochaetes from immune suppressed animals by xenodiagnosis. Engorged nymphs were dissected 3 weeks after removal of the tick capsules and analysed by DFA. An average of 18 nymphs fed on each of the five animals participating in the immune suppression experiment. None contained spirochaetes that were detectable by DFA. In contrast, of the 18 ticks that engorged while feeding on the positive control animal, nine were dissected and all nine were DFA positive. Nerve conduction studies. The baseline values of nerve conduction remained unchanged in all animals throughout the study, indicating that if immune suppression had led to a reactivation of infection, this had not affected the peripheral nerves investigated. Histology and immunohistochemistry of skin biopsy samples. Skin biopsy samples were collected weekly after the initiation of the IS protocol. Two animals, L594 and L971 (IS control) showed a superficial dermatitis on one occasion. In these perivascular lymphocytic infiltrates there were macrophage-type cells that stained positive with the anti-7.5 kDa mAb. Skin biopsies of animals L712, M585 and M106 were normal at all sampling periods. Post-mortem analyses Gross pathology. Neither control nor vaccinated animals showed abnormalities attributable to the B. burgdorferi infection in any organ system at the gross (macroscopic) level. The only exception to this generalisation is perhaps the finding of a slight meningeal congestion in vaccinated animal M107. However, no immunohistochemical or PCR- derived evidence of infection of the central nervous system was obtained for this animal post-mortem (see below). All organ systems of animals participating in the immune suppression experiment were grossly normal, with exception of the lungs, which showed pneumonia presumably related either to a cytomegalovirus infection secondary to immune suppression or to the weekly lung lavages. Histopathology and immunohistochemistry. Histopathology and immunohistochemistry of sections stained with B. burgdorferi-specific mAbs were assessed in samples taken from the skin, lungs (anterior, middle and posterior lung lobes), urinary system (kidney, ureter and bladder), heart (left and right heart, and septum), spleen, nervous system (brain and central and peripheral nerves), and joints and synovial membranes (Table 3). In contrast with what had been observed in skin biopsy samples taken between weeks 1 and 4 PC, the skin of both vaccinated and control animals had no detectable type 3 dermatitis (deep perivascular cellular infiltrate at the panniculus-dermis junction) by the time the animals were sacrificed. Similarly, no spirochaetes or spirochaetal antigens were detectable by immunohistochemistry, except in samples from animal M219. It should be noted, however, that these samples were obtained 2 weeks PC, at the time this animal died by anaesthesia (Table 3). Sections of the anterior, middle and posterior lung lobes of all of the sacrificed/dead animals were explored for presence of histopathological changes and for immunohistochemical evidence of B. burgdorferi. Lymphocytic hyperplasias (LH) were prevalent both in control and vaccinated animals (Table 3). In addition, most of the animals with LH also showed immunohistochemical evidence of B. burgdorferi, including four of the vaccinated animals that were not immune suppressed and all of the controls. Among the immune suppressed animals, the only immunopositive lung samples were those of L971 and L594 (Table 3). However, all of these monkeys showed pneumonia, which was identified by intranulcear basophilic inclusion bodies. As mentioned above, pneumonia may have been caused by the weekly lung lavages or as a consequence of CMV infection secondary to IS. Two of the control animals, L457 and M021, showed lymphocytic hyperplasias in the ureter and kidney, respectively, that stained positive for B. burgdorferi (Table 3). In contrast, only one of the vaccinated animals (L971) had detectable pathologic changes in the urinary system. This animal had ureteritis, and the lesion contained cells that stained positive for the 7.5 kDa B. burgdorferi antigen (Table 3). Perivascular lymphocytic infiltrates of the renal pelvis were also observed in animal L971. Three of the four control animals and one of the vaccinated animals (M219) had myocarditis (Table 3). Perivascular lymphocytic infiltrates of the renal pelvis were also observed in animal L971. Three of the four control animals and one of the vaccinated animals (M219) had myocarditis (Table 3). Perivascular lymphocytic infiltrates were present, often with immunohistochemical evidence of the 7.5 kDa B. burgdorferi antigen. All heart sections were normal in all of the animals that participated in the immune suppression experiment. The spleen showed evidence of infection but only in the control animals, with macrophages staining positively with the anti-B. burgdorferi mAb. The spleens of animals that participated in the immune suppression experiment were not analysed (Table 3). Sections of joint and synovium samples from the hip, knee, tarsus, shoulder, elbow and carpus (left and right, in all cases) of all of the dead/sacrificed animals were assessed for histopathological changes and for immunohistochemical evidence of the presence of B. burgdorferi organisms (or their antigens). One control animal, M021, showed arthritis, which also showed positive staining for B. burgdorferi. Microscopic joint lesions of the synovial membrane were observed in two of the animals that participated in the immune suppression experiment (L971 and L594) (Table 3). These lesions consisted of synovial cell hyperplasia with perivascular lymphocyte infiltrates. Synovial membrane sections from both animals stained with anti-B. burgdorferi mAbs. Several parts of the central and peripheral nervous system were sectioned and analysed for histopathological changes and for immunohistochemical evidence of the presence of B. burgdorferi organisms (or their antigens). The brain cortex, midbrain and stem were investigated, as well as the entire spinal cord, spinal nerves and ganglia. Peripheral nerves examined were the sciatic, median and facial nerves. The most frequent changes observed were in the peripheral nerves of the control animals, which showed perivascular lymphocytic infiltrates ranging from mild to marked and immunohistochemical evidence of the presence of B. burgdorferi (Table 3). In addition, one of the control animals also had a moderate cellular infiltrate and positive immunostaining for B. burgdorferi in the spinal cord. Three of the vaccinated animals, M219, M107 (lipidated OspA),, and L971 (NS1-OspA) also showed histopathological evidence of infection in central (ganglioneuritis, M219, L971) and in peripheral nerves (perivascular lymphocytic infiltrate, M107). Macrophages within the infiltrate of animal M107 stained positive for B. burgdorferi (Table 3). Morphometric quantification of cells that bound the anti-OspA mAb LA31. We observed previously (16) that some mononuclear cells from perivascular infiltrates that were formed in tissues concomitantly with the presence of B. burgdorferi would stain either with the anti-7.5 kDa mAb or with the LA31 (anti-OspA) mAb. Antigen that bound to either mAb appeared to be present within the cytoplasm of the stained mononuclear cells (16). Since the number of stained cells was larger the more intense the cellular infiltrates, we sought to quantify inflammation in the heart and lungs indirectly, by counting the mononuclear-type cells that bound the anti-OspA LA31 mAb. These cells were quantified on sections of the heart and right lung of all of the control animals, all of the animals vaccinated with lipidated OspA and one animal of each of the NS1-OspA and NS1-OspA/MPL groups. There were marked differences in the numbers of stained cells (Table 4), as well as in the severity of the mycoarditis (Figure 6) in control vs. vaccinated animals. All of the control animals had between 24 and 106 cells per cm² (mean of 63) in the right heart, the section of the heart with the most cells, whereas vaccinated animals had a range of 0-25 (mean of 8), and only two of the six animals assessed showed such cells. In the lung's anterior lobe, the range was 12-155 (mean of 77) in control animals, and 0-36 (mean of 7) in vaccinated monkeys. A similar distribution of cells between control and vaccinated animals was seen in the left heart, septum and posterior lung lobe (Table 4). A comparison of the mean number of stained cells in control and vaccinated animals by organ (Kruskal-Wallis ANOVA by ranks) or overall (MANOVA) indicated that the difference between the two groups was either significant (P<0.05, right heart, septum and lung lobes) or highly significant (P<0.01, left heart and overall). [Image] Figure 6 Myocarditis in vaccinated and in control monkeys. (A) Right heart from monkey M107 (lipidated OspA group). Note the one cell staining red with the mAb that recognises the 7.5 kDa lipoprotein of B. burgdorferi. When compared with the right heart of monkey M021 (B) which is a control monkey, there is a greatly reduced inflammatory infiltrate, as well as fewer stained cells in the myocardium of the vaccinated animal. Magnification x 400 (A and B) Silver staining of brain sections. Brain sections from six vaccinated animals, the four controls and three animals that had not been challenged with B. burgdorferi were stained as described in Materials and Methods. One of the four control monkeys (M021) and one of the four animals vaccinated with lipidated OspA (L537) showed stained filaments consistent with the appearance of silver-stained B. burgdorferi spirochaetes. The remainder of the brain sections either stained negative or showed stained filaments that did not unequivocally resemble the morphology of silver stained spirochaetes (Table 3). One of the three animals that had not been exposed to B. burgdorferi also showed a staining pattern consistent with that of spirochaetes. PCR of tissue samples. B burgdorferi DNA could be amplified by PCR from the organs of all four control animals. The lung and the bladder were the organs most frequently positive. Of a total of 114 determinations, 11 (9.6%) were found to be positive in control animals. Of the six vaccinated, non immune suppressed animals whose organs were analysed by PCR, four showed one or two positive results. The lung was also the most frequently positive organ in these animals. Of a total of 150 determinations, seven (4.7%) were positive. In the four immune suppressed vaccinated animals, all but one (M106) had one or two organs from which an amplicon could be detected. Of a total of 64 determinations in these animals, four (6.3%) were positive (Table 3). Tubes without template did not show a positive signal on Southern blots. Although the frequency of appearance of PCR amplicons was higher in control than in vaccinated animals, comparison of values by a nonparametric X² [chi-square] test indicated that the difference was not significant (P>0.05). Similarly, the frequency of appearance of PCR amplicons in the immune suppressed animals did not differ significantly from that of the other two groups. In vitro culture. Tissue samples from the same organs that were analysed by PCR were subjected to in vitro culture. No spirochaetes were recovered. Table 3 Evaluation of tissue samples obtained post-mortem from several organ systems by PCR, histopathology (HP), B. burgdorferi-specific immunohistochemistry (IH), and silver staining (SS) ------------------------------------------------------------------------ Skin Lung Heart Kidney Vaccine/ ------------ ------------ ------------ ------------ animal no. PCR HP IH PCR HP IH PCR HP IH PCR HP IH ------------------------------------------------------------------------ Al(OH)3 L457 0/3 - - 3/3 LH + 0/3 M + 0/3 - - L549 0/3 D2 - 0/3 LH + 0/3 M + 0/3 - - MO21 0/3 - - 0/3 LH + 0/3 M + 1/3 LH + M581 0/3 - - 1/3 LH + 0/3 - - 0/3 - - Lipidated OspA M243 0/3 - - 0/3 LH - 0/3 - - 0/3 - - L537 0/3 - - 1/3 LH + 0/3 - - 0/3 - - M219 ND D2 + ND LH + ND M - ND - - M107 0/3 - - 2/3 LH + 0/3 - - 0/3 - - NS1-OspA L458 0/2 - - 0/2 - - 0/2 - - 0/2 - - L594 0/2 D2 + 0/2 P + 0/2 - - 0/2 - - L971 0/2 D3 + 0/2 P + 0/2 - - 0/2 ND - M585 0/2 D2 - 0/2 P - 1/2 - - 0/2 - - NS1-OspA/MPL L476 0/3 - - 1/3 LH + 0/3 - - 0/3 - - L712 0/2 - - 0/2 P - 0/2 - - 0/2 - - L642 ND ND ND ND ND ND ND ND ND ND ND ND M106 0/2 D2 - 0/2 P - 0/2 - - 0/2 - - ------------------------------------------------------------------------ ------------------------------------------------------------------------ Ureter Bladder Liver Spleen Vaccine/ ------------ ------------ ------------ ------------ animal no. PCR HP IH PCR HP IH PCR HP IH PCR HP IH ------------------------------------------------------------------------ Al(OH)3 L457 ND - + 0/3 - - 0/3 ND ND 0/3 - + L549 ND - - 1/3 - - 0/3 ND ND 0/3 - + MO21 ND - - 2/3 - - 0/3 ND ND 0/3 - + M581 ND - - 1/3 - - 0/3 ND ND 0/3 - - Lipidated OspA M243 ND - - 0/3 - - 0/3 ND ND 0/3 - - L537 ND - - 0/3 - - 0/3 ND ND 0/3 - - M219 ND - - ND - - ND ND ND ND - - M107 ND - - 0/3 - - 1/3 ND ND 0/3 - - NS1-OspA L458 ND - - ND - - ND ND ND 0/2 - - L594 ND - - 0/2 - - ND - - ND ND ND L971 ND U + 0/2 - - ND - - ND ND ND M585 ND - - 0/2 - - ND - - ND ND ND NS1-OspA/MPL L476 ND - - 0/3 - - 0/3 ND ND 0/3 - - L712 ND - - 0/2 - - ND H - ND ND ND L642 ND ND ND ND ND ND ND ND ND ND ND ND M106 ND - - 0/2 - - ND - - ND ND ND ------------------------------------------------------------------------ ------------------------------------------------------------------------ Spinal nerves+ Synov./Joint Brain Spinal cord ganglia Vaccine/ ------------ --------------- ------------ ----------- animal no. PCR HP IH PCR HP IH SS PCR HP IH PCR HP IH ------------------------------------------------------------------------ Al(OH)3 L457 0/3 - - 0/3 - - - ND - - ND - - L549 0/3 - - 0/3 - - - ND - - ND - - MO21 0/3 SH + 0/3 - - + ND - - ND - - M581 0/3 - - 2/3 - - ? ND I(3)+ ND - - Lipidated OspA M243 0/3 - - 0/3 - - ? ND - - ND - - L537 0/3 - - 0/3 - - + ND - - ND - - M219 ND - - ND - - ? ND - - ND GN* - M107 0/3 - - 0/3 - - ? ND - - ND - - NS1-OspA L458 0/2 - - 0/2 - - ND ND - - ND - - L594 1/2 SH* + 1/2 - - ND ND - - ND - - L971 0/2 SH* + 0/2 - - - ND - - ND GN* - M585 0/2 - - 0/2 - - ND ND - - ND - - NS1-OspA/MPL L476 0/3 - - 0/3 - - - ND - - ND - - L712 0/2 - - 1/2 - - ND ND - - ND - - L642 ND ND ND ND ND ND ND ND ND ND ND ND ND M106 0/2 - - 0/2 - - ND ND - - ND - - ------------------------------------------------------------------------ ------------------------------------------------------------------------ Periph. nerves Vaccine/ ----------------- animal no. PCR HP IH ------------------------------------------------------------------------ Al(OH)3 L457 ND I(2) + L549 ND I(4) + MO21 0/3 - - M581 0/3 I(2) + Lipidated OspA M243 1/3 - - L537 1/3 - - M219 ND - - M107 0/3 I(2) + NS1-OspA L458 ND - - L594 0/2 - - L971 0/2 - - M585 0/2 - - NS1-OspA/MPL L476 0/3 - - L712 0/2 - - L642 ND ND ND M106 0/2 - - ------------------------------------------------------------------------ LH, lymphocytic hyperplasia; M, myocarditis; SH, synovial cell hyperplasia; SH*, SH with perivascular lymphocyte infiltrate; I(2) mild perivascular lymphocytic infiltrate; I(3), moderate; I(4), marked; N, nephritis; U, ureteritis; P, pneumonitis; D2, superficial dermatitis; D3, deep dermatitis; H, hepatitis; PCR, denominator, number of aliquots amplified from a given tissue lysate; numerator, number of PCR-positive aliquots; SS, silver staining; ?, probably positive. Animal numbers in italics correspond to animals that were immune suppressed. The mAb anti-7.5 kDa was used for the immunohistochemistry. Table 4 Morphometric quantification of cells that bind anti-OspA mAb LA31 in the heart and lungs ------------------------------------------------------------------------ Lung* Lung* Vaccine/ anterior posterior animal no Right heart Left heart Septum lobe lobe ------------------------------------------------------------------------ Al(OH)3 L457 40/35 5/153 2/126 12/25 3/83 L549 24/197 5/99 24/107 75/110 34/230 M021 106/57 15/166 12/105 155/116 100/145 M581 80/81 29/100 20/91 64/59 22/252 Lipidated OspA M219 0/51 0/97 0/83 0/41 0/131 M243 0/62 0/77 0/113 0/48 0/101 L537 22/62 0/112 0/63 36/126 19/157 M107 25/58 2/94 3/90 8/45 17/168 NS1-OspA L458 0/81 0/120 0/148 0/94 0/176 NS1-OspA/MPL L476 0/51 0/76 0/69 0/89 0/120 ------------------------------------------------------------------------ Numerator: number of B. burgdorferi-positive cells per cm²; denominator, cross-sectional area measured (mm²); mAb, LA31 anti-OspA. *Right lung. ----- 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