DISCUSSION ----- 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] ----- The mechanisms whereby an infection with B. burgdorferi causes Lyme arthritis or neuroborreliosis, the two most morbid manifestations of Lyme disease, are not known. In fact, there is as yet no clear understanding of whether disease is caused by the infection itself, by the immune response to it, to antigens left in the tissues after bacterial demise, or by all of these factors acting in concert. An important and inevitable implication of this lack of understanding is that thus far no bacterial molecules have been incriminated in disease pathogenesis. Vaccines against disease are therefore not feasible yet, and efforts in Lyme disease immunoprophylaxis have focused on the prevention of infection. Unfortunately, the development of a vaccine to prevent infection with B. burgdorferi is constrained by the notion that no less than sterile immunity may be required to avoid long-term sequelae. The scientific literature on syphilis contains accounts of human infections caused by inoculation with as few as ten Treponema pallidum spirochaetes (27). In rabbits, similar low level treponemal infections are too sparse to elicit an antibody response (J. Miller, personal communication) and, as in humans (29), such infections may lie quiescent for a long time, only to become clinically apparent much later (J. Miller, personal communication and ref. 29). B. burgdorferi infections can be caused in mice by intradermal injection of very few organisms (ID subscript(50)=10) (30), and once an infection becomes chronic, spirochaetes are present in the skin and other organs at much lower densities than during the acute phase (30). Similarly, spirochaetes may be cultivated from the skin of recently infected humans, but very rarely from the cerebrospinal fluid of patients with chronic neuroborreliosis (31). Notwithstanding such low level infections, disease is clinically apparent. Could a low level infection occur in an OspA-vaccinated host? The mechanism whereby B. burgdorferi spirochaetes succumb to the immunity elicited by OspA has been under intense scrutiny. A two-tiered mode of protection by anti-OspA antibody, the key mediator of protective immunity, had initially been postulated, one acting on the spirochaetes within the tick prior to transmission and the second upon delivery within the host skin (32). Evidence gathered subsequently, indicated that the expression of OspA is interrupted when spirochaetes reach the tick's salivary gland, en route to the vertebrate host (20, 33). A corollary of this finding is that anti-OspA antibody can kill only within the tick midgut, while OspA is still expressed, but not thereafter (20). One can envisage that within the midgut of an infected nymph that is feeding on an OspA-vaccinated host, a process of selection may ensue whereby spirochaetes with the least surface density of OspA molecules, and endowed perhaps with other attributes such as enhanced motility and ability to penetrate the tick midgut wall, may escape antibody-dependent killing. Two recent discoveries make this scenario more than just formally possible. First, I. scapularis saliva has the ability to inactivate complement (34). Hence, killing within the midgut could occur via a mechanism that involves antibody alone, a mechanism that appears to be slower in effecting spirochaetal death than that enacted by antibody and complement together. Second, although OspA is an abundant B. burgdorferi protein, only a minor fraction of OspA molecules is exposed on the outer surface of the spirochaete (35). This entails that small variations in the absolute number of OspA surface molecules could cause significant differences in the spirochaete killing rate. Thus, one can argue that low level infections may occur in an OspA-vaccinated host even in the absence within the tick of variant spirochaetes that do not express OspA at all (36). By analogy with syphilis, such low level infections could be pathogenic. We therefore built into this efficacy trial multiple ways of finding evidence of infection in the vaccinated animals and, moreover, we attempted to uncover putative low-level infections by radically immune suppressing a portion of the study population. DFA analysis of the ticks that were used for the challenge infection indicated that spirochaetal survival in the face of the anti-OspA serum antibody titre present at the time of challenge was minimal. Only 0.8% of the engorged ticks that were dissected and that had fed on vaccinated animals (1/121) contained detectable spirochaetes, whereas 95% (41/43) of such ticks that fed on control animals contained spirochaetes (Table 1). This result indicates that most, but not all, of the spirochaetes that remained in the ticks which fed on vaccinated animals were destroyed within the first week of exposure to anti-OspA antibody. No estimate is possible, of course, of the number of spirochaetes that were 'missing' from the midgut because they escaped the initial onslaught of anti-OspA antibody, but it is reasonable to assume that this number must be either very small, or zero. The latter alternative, which is equivalent to sterile immunity, is supported by the results of PCR applied to skin biopsy samples collected during the first 4 weeks PC. During this period, spirochaete DNA was detectable in the skin of the four control monkeys but in none of the vaccinated animals (Table 2). This result is a strong indication of absence of infection in the vaccinated animals. Moreover, by the time of the challenge infection all of the vaccinated animals had an LA2 antibody titre, i.e. a titre of anti-OspA antibody that is capable of effecting antibody-dependent killing of B. burgdorferi, that was between 100-fold (NS1-OspA/Al(OH)3 group) and 400-fold (lipidated OspA/Al(OH) group), the titre known to be effective in killing the spirochaete in vivo, which is 1-5 LA2 µg equivalents per ml 4 [sic]. On the other hand, two vaccinated monkeys (and two of the control animals) had immunohistochemically detectable spirochaetal antigens in the skin during the early localised phase of infection (Table 2) and in several organs post-mortem (Table 3). More convincingly, several of the vaccinated animals and all of the control monkeys exhibited post-mortem, spirochaetal DNA in organs such as the lungs, brain, heart, bladder and peripheral nerves (Table 3). These remote sites could have been reached only after spirochaetal dissemination. Although the frequency of appearance of PCR amplicons was higher in control than in vaccinated animals, the difference was not significant (nonparametric X² [chi-square] test, P>0.05). Unfortunately, silver staining of brain tissues yielded a result from which no conclusion may be drawn at this point, for spirochaete-like filaments also were observed in samples from an uninfected animal, in addition to one of the controls and one vaccinated monkey. If there was a low-level disseminated infection, it remained below the threshold of seroconversion, for no vaccinated animal seroconverted, as assessed by Western blot. In contrast, all of the control animals seroconverted within the first 6 weeks PC and exhibited a gradual increase in the number of serum antibody specificities that is consistent with an active infection. All of the control monkeys satisfied the Dressler criteria for Western blot diagnosis of a B. burgdorferi infection in humans (37). The putative low-level infection was not increased by the immune suppression protocol employed, as evidenced by the following findings: (1) the frequency of post-mortem detection of spirochaetal DNA by PCR was not significantly different from that obtained in the vaccinated animals that were not immune suppressed; (2) no spirochaetes were recovered from the immune suppressed animals by xenodiagnosis, whereas a recently infected and otherwise normal animal was successfully xenodiagnosed using a similar procedure; (3) no changes were detected in peripheral nerve conduction velocity or in amplitude of the response to stimulation during nerve conduction studies, whereas five of eight B. burgdorferi-infected rhesus monkeys (and none of ten uninfected controls) showed such changes (19); (4) cultivation of bronchoalveolar lavage and urine samples yielded no B. burgdorferi spirochaetes. It is doubtful that these results arose from insufficient immune suppression. T-cell function was clearly diminished, insofar as the blastogenic responses of PBMC and lymph node cells to Con A were severely reduced. The failure to affect B-cell function, as indicated by the persistence of the response to PWM, is probably irrelevant since the 'low-level infection' was serologically non-immunogenic and thus unlikely to have been under the control of antibody. However, surveillance of infection by anti-OspA antibodies may have occurred if OspA is re-expressed by the spirochaete in an organ-specific manner (10), as the frequent finding of anti-OspA antibodies in patients with Lyme arthritis (38) appears to suggest. The apparent failure to enhance the putative low-level infection admits, of course, an explanation other than absence of the infection per se. The working hypothesis of the IS experiment, that the B. burgdorferi infection burden was kept low by an immune surveillance mechanism, may be wrong. Indeed, the notion that spirochaetes localise to immune-privileged sites, a notion often invoked to explain why it is that a chronic B. burgdorferi infection can coexist with bactericidal serum antibodies elicited during the infection process, is inimical to the concept of immune surveillance. It is most unlikely that a low-level infection could have been detected by in vitro cultivation of tissue specimens. In any case, our inability to recover spirochaetes from the control animals implies that the in vitro culture results are moot. This outcome contrasted with results from two previous studies in which we had been able to cultivate spirochaetes from skin samples of nine out of nine infected animals (15) and four out of five infected animals (16). A potentially relevant change had been introduced in the in vitro culture procedure used in the current study, in that skin samples of a volume eight to ten times larger were cultured in the same volume of medium as before, under the naive expectation that this would increase the chances of recovering spirochaetes. As we tried to troubleshoot the culture procedure we assessed whether spirochaetes could grow in BSK-H medium in the presence of added normal skin at a low ratio of medium to skin sample volume (100:1) and also at higher ratios. Live spirochaetes were observed only in tubes where the ratio of medium to skin volume was at least 3000:1 (Y. Gu et al., unpublished observation). Skin biopsy samples obtained from several rhesus macaques infected with B. burgdorferi (strains JD1 and NT1) subsequently to the present study, unfailingly yielded cultivable spirochaetes when incubated at a medium to skin volume ratio of about 3000:1 (Y. Gu et al., unpublished observation). Could a non-immunogenic B. burgdorferi infection be pathogenic? Some of the vaccinated animals showed dermatitis shortly after the challenge infection, and lung lymphocytic hyperplasia, myocarditis and PNS and CNS involvement post-mortem. On a quantitative basis, however, pathologic changes were significantly less intense, as demonstrated by the morphometric quantification of inflammation in the heart and lungs (P<0.05) (Table 4). The results of our efficacy study thus appear to fit the following interpretation. As a consequence of their exposure to infected ticks, vaccinated animals, regardless of the vaccination protocol, were probably infected by a small number of spirochaetes. These spirochaetes, in contrast to those received by control animals, were too sparse to be immunogenic, but sufficient to disseminate, become detectable by PCR and, in some organs, by immunohistochemisty. The putative low-level infection received by the vaccinated animals was clearly less pathogenic than the full infectious burden given to the control monkeys. Since the low-level infection appeared not to be under T (or B)-cell immune surveillance, it is puzzling that it remained at a low level, unless, as mentioned before, OspA was re-expressed selectively by B. burgdorferi in certain organs. If so, one possible solution to the riddle is that the infection was transient, terminated eventually by remaining anti-OspA antibodies. Another possible, even probable explanation is that the spirochaetes that putatively evaded the anti- OspA antibody in the tick midgut were low-virulence mutants that do not express, or express only a portion of, OspA. An infection with such mutants could be self-limiting. We are currently investigating the infectivity of OspA escape mutants in mice to assess this hypothesis. Thus, while our results do not allow us to discriminate unequivocally between transient and still-exant low-level infections, they underscore more convincingly the former possibility and thus suggest that sterile immunity was ultimately achieved. ----- 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