Considerable research has focused on the development of an effective Lyme borreliosis vaccine. Early studies demonstrated that vaccination with whole B. burgdorferi
spirochetes induced protective antibodies in experimental animals (19
). These protective antibodies are borreliacidal (8
), and the level of borreliacidal antibody is related to the duration of protection (7
). Several Osps, especially OspA, of B. burgdorferi
have been shown to induce protective borreliacidal antibody in humans and experimental animals (3
). To date, OspA has received the most intense evaluation as a potential vaccinogen (12
). However, the anti-OspA borreliacidal antibody response wanes rapidly in OspA-vaccinated humans and hamsters (32
). These latter results are discouraging.
In this report, we established that T cells affected the production of borreliacidal antibody. Borreliacidal antibody was detected only in supernatants of cultures containing T and B cells obtained from hamsters 14 days after vaccination. When T cells from hamsters 7 or 21 days after vaccination were coincubated with immune T cells and borreliacidal antibody-producing cells, borreliacidal antibody production was abrogated. These results suggest that immune T cells rapidly lose the ability to promote borreliacidal antibody production, despite the presence of antigen-processing cells and borrelial antigen in the cultures. In addition, T cells obtained 1 week after vaccination suppressed the production of borreliacidal antibody. These findings support our previous study (32
) showing that borreliacidal antibody, specifically anti-OspA borreliacidal antibody, develops slowly and wanes rapidly in vaccinated humans and hamsters. We addressed this further by vaccinating C3H/HeJ mice with several whole-cell vaccines (unpublished data). Again, borreliacidal antibody production developed slowly, peaked at weeks 4 to 6 after vaccination, and then decreased rapidly. Our in vitro results showed that the borreliacidal antibody production was directed primarily against OspA because absorption of the cell-free supernatants of 14-day immune T and B cells lost nearly all of their borreliacidal activity. Collectively, these results suggest that T cells play a major role in the ability of OspA to induce and maintain a sustained level of borreliacidal antibody.
Creson et al. (10
) reported that borreliacidal activity waned with the elimination of spirochetes from the host by immune clearance or therapy. Our in vitro results extend these findings by demonstrating that borreliacidal antibody production can also wane in the presence of high concentrations of B. burgdorferi
spirochetes. The mechanism responsible for the delay in production and waning of anti-OspA borreliacidal antibody involves T cells. T cells from hamsters 7 or 21 days after vaccination prevented anti-OspA borreliacidal antibody production when cocultured with T and B cells obtained from hamsters 14 days after vaccination. This effect was also detected when either 7- or 21-day T cells were added in reduced concentrations to the borreliacidal antibody-producing 14-day immune T and B cells. In support, 2 × 108
spirochetes were present throughout the duration of the in vitro cultivation of suspensions of T and B cells obtained from hamsters 14 days after vaccination and cocultured with T cells obtained from hamsters 7 or 21 days after vaccination. These findings demonstrate that T cells or their products play a major role in influencing the induction and more importantly the decline of protective borreliacidal antibody, despite the presence of B. burgdorferi
antigens. Vaccine strategies, therefore, must place more emphasis on defining the role that immune cells play in these responses.
Previously, we showed that B. burgdorferi
-specific T lymphocytes were also responsible for the induction of severe destructive Lyme arthritis (22
). When naive hamsters were infused with T lymphocytes from vaccinated hamsters, they developed severe destructive arthritis after challenge with B. burgdorferi
unless high levels of borreliacidal antibody were present at the time of infection. The ability of T cells or their products to decrease borreliacidal production will likely make frequent boosters or the addition of a safe adjuvant necessary to prolong the high levels of borreliacidal antibody. However, repeated vaccinations and use of an adjuvant may increase the potential for side effects (1
), such as severe destructive arthritis (21
). Thus, additional experiments are needed to determine the immunologic mediator(s) responsible for maintaining sustained high levels of borreliacidal antibody.
Our findings are significant for an additional reason. Although anti-OspA borreliacidal antibodies were readily detected in human volunteers after primary and booster vaccination, the levels of borreliacidal activity varied widely and decreased rapidly (32
). Only one vaccinee had detectable borreliacidal activity 6 months after vaccination. Keller et al. (21
) suggested that an anamnestic response would provide protection against infection even in the absence of circulating antibodies. Our in vitro results suggest that the circulating antibody is the result of a limited production of borreliacidal antibody by immune cells obtained 14 days after vaccination. Once borreliacidal antibody production is down-regulated (21 days after vaccination), immune cells failed to produce borreliacidal antibody, even in the presence of antigen-processing cells and borrelial antigen. This finding suggests that immune T cells become less responsive or tolerant to epitopes of OspA responsible for the induction of borreliacidal antibody. In support, we showed previously (32
) that production of anti-OspA borreliacidal antibody did not correlate with production of total OspA antibody after vaccination of humans or hamsters with OspA. Furthermore, Foley et al. (15
), Straubinger et al. (48
), and Schutzer et al. (37
) showed that infection with B. burgdorferi
occurred in OspA-vaccinated rabbits, dogs, and a human, respectively. Again, OspA vaccination may be of limited value considering the heterogeneity of OspA (28
) and the inability of the protective epitope of OspA to induce high and sustained levels of borreliacidal antibody.
Our results also show that other specific anti-B. burgdorferi
antibodies, other than anti-OspA borreliacidal antibody, can be detected in cultures of macrophages and B. burgdorferi
containing T and B cells from hamsters 14 days after vaccination. When cell-free supernatants from these cultures were absorbed with recombinant OspA, little borreliacidal activity was detected. However, anti-B. burgdorferi
antibodies against the 22- and 34-kDa proteins were detected in the absorbed samples by Western immunoblotting. Although the 22- and 34-kDa proteins have been shown to induce protective antibodies (14
), our in vitro results indicate that the protective or borreliacidal epitopes of these Osps were not processed in vitro or in vivo by macrophages for presentation to immune T and B cells. One would expect that in vitro exposure of B. burgdorferi
to antigen-presenting cells (macrophages) and immune T and B cells obtained from hamsters 14 days after vaccination would augment or induce a protective borreliacidal response to the 22- and 34-kDa proteins. Therefore, our results indicate that the putative protective borreliacidal epitopes, including those of OspA, are difficult for the host’s immune system to recognize, despite vaccination of hamsters with B. burgdorferi
contained in an adjuvant and subsequent exposure in vitro of these immune T cells to antigen-processing cells and high concentrations of spirochetes.
In conclusion, we showed that anti-OspA borreliacidal antibody was produced in vitro only when T and B cells were obtained from hamsters 14 days after vaccination. Production of the anti-OspA borreliacidal antibody was prevented by the addition of T cells obtained from hamsters 7 or 21 days after vaccination. Further studies are needed to delineate the mechanism(s) of induction and waning of borreliacidal activity. These studies will improve the immunogenicity of OspA and other Osps and aid in developing an efficacious and safe vaccine against infection with B. burgdorferi.