A major step towards prevention of infection with B. burgdorferi
has been the development of rOspA vaccines for humans (28
) and dogs (33
). Vaccination of susceptible individuals in areas where Lyme borreliosis is endemic most likely will reduce the morbidity associated with infection with B. burgdorferi
. The vaccines, however, will not entirely eliminate the risk of becoming infected with B. burgdorferi
even after repeated vaccinations. One currently available rOspA vaccine was less than 50% effective in preventing infection with B. burgdorferi
after two injections and only 76% effective after a third injection (32
). Most importantly, the duration of protective immunity and the number of subsequent yearly booster vaccinations required to maintain sustained high levels of protective borreliacidal antibody remains unknown.
The purpose of this investigation was to develop an in vitro system to determine the effects of cytokines on the production of borreliacidal antibody. We showed that borreliacidal antibody, specifically anti-OspA borreliacidal antibody, was readily detected in cultures of lymph node cells obtained from C3H/HeJ mice vaccinated with formalin-inactivated B. burgdorferi in aluminum hydroxide. Anti-OspA borreliacidal antibody was detected on day 7 of vaccination, peaked on day 17, and thereafter rapidly declined. By contrast, vaccination with rOspA with or without aluminum hydroxide induced less anti-OspA borreliacidal antibody. When anti-OspA borreliacidal antibody-producing cells were treated with rIL-4, borreliacidal activity was inhibited. In addition, treatment of anti-OspA borreliacidal antibody-producing cells with anti-murine IL-4 failed to alter the production of cidal antibody.
Our results show that rOspA is a poor immunogen for inducing production of borreliacidal antibody, even when administered with aluminum hydroxide. Although our rOspA was not lipidated, vaccination of mice with rOspA in aluminum hydroxide should have enhanced borreliacidal antibody production. Instead, low titers of borreliacidal antibody were produced by immune lymph node cells cocultured with macrophages and B. burgdorferi
. These results confirm and extend our previous findings that humans and hamsters launch a poor anti-OspA borreliacidal antibody response, even after multiple vaccinations with lipidated rOspA (22
). By contrast, vaccination of mice with formalin-inactivated B. burgdorferi
with aluminum hydroxide induced high levels of borreliacidal antibody. Anti-OspA borreliacidal antibody was detected in cultures of lymph node cells 7 days after vaccination and peaked on day 17 of vaccination. The enhanced production of borreliacidal antibody may be due to better recognition and processing of native OspA on the formalin-inactivated B. burgdorferi
. Taken together, these results suggest that a recombinant protein may lose the conformational epitopes necessary to induce high levels of borreliacidal antibody.
Despite production of high levels of anti-OspA borreliacidal antibody (range, 64 to 1,024), the anti-OspA borreliacidal antibody response waned rapidly. Borreliacidal antibody decreased 8- to 16-fold within days after the period (17 days) of maximum production. Once borreliacidal antibody production was down-regulated (21 days after vaccination), immune lymph node cells failed to produce borreliacidal antibody, even in the presence of antigen-presenting cells and borrelial antigen. These in vitro results suggest that the rapid decrease of circulating anti-OspA borreliacidal antibody in serum detected in vaccinated humans and animals (22
) is the result of limited production of borreliacidal antibody by immune cells. It is possible that immune lymph node cells become less responsive to or more tolerant of epitopes of OspA that are responsible for the induction of borreliacidal antibody. In support of this argument, we showed previously (22
) that production of nonborreliacidal anti-OspA antibody remained elevated for many months after vaccination of humans and hamsters with rOspA, while the level of anti-OspA borreliacidal antibody was undetectable within weeks after vaccination.
This selective down-regulation of borreliacidal antibody production is important because it affects the duration of protection against infection with B. burgdorferi
. In this study we attempted to enhance the production of borreliacidal antibody by treatment of immune cells with rIL-4. IL-4 has been shown to enhance B-lymphocyte survival (14
), increase the rate of B-lymphocyte proliferation (12
), and augment secondary antibody responses (12
). Unexpectedly, treatment with rIL-4 inhibited borreliacidal responses. When borreliacidal antibody producing cells were treated immediately (10 min) with various concentrations of rIL-4, borreliacidal activity was greatly inhibited (range, 4- to 16-fold) compared to control lymph node cells. In addition, a treatment delay of 4 days did not affect the production of borreliacidal antibody. Furthermore, treatment of immune cells capable of producing borreliacidal antibody immediately or on day 4 of cultivation with anti-murine IL-4 also did not affect the production of borreliacidal antibody. These combined results suggest that IL-4 plays a minor role in the regulation of the secondary borreliacidal antibody response in vitro.
Our in vitro results, however, show that anti-OspA borreliacidal antibody is due primarily to the production of IgG1 antibody. This antibody is IL-4-dependent (30
). It is puzzling why addition of rIL-4 to cultures of immune lymph node cells did not enhance production of borreliacidal antibody. An explanation may be that IL-4 is more involved with initial processing and production of the primary antibody response. Our in vitro antibody system uses immune cells that are restimulated with B. burgdorferi
. Although IgG1 anti-OspA borreliacidal antibody is produced, this response may now be IL-4 independent (10
What then accounts for the production of anti-OspA borreliacidal antibody if IL-4 plays a minor role? We hypothesize that gamma interferon (IFN-γ) is involved. It is interesting that production of borreliacidal antibody occurred only when antigen-processing cells (macrophage) were added to cultures of immune lymph node cells. It is known that IFN-γ mediates apoptosis of T and B lymphocytes in the absence of accessory cells (1
). When accessory cells are present, IFN-γ promotes progression of T-lymphocyte activation (18
) and antibody isotype switching (34
). In addition, we detected lysis of B. burgdorferi
when supernatants obtained from immune lymph node cells contained IgG2a antibodies. Production of IgG2a antibody is IFN-γ dependent (23
). Additional studies are needed to define the role of IFN-γ, especially if its antagonist, IL-4 (15
), does not play a major role in the regulation of secondary borreliacidal antibody responses.
Our results also show that borreliacidal activity occurs by different functions of subclasses of IgG antibody. IgG1 borreliacidal antibody kills B. burgdorferi organisms by clumping, while IgG2a and IgG2b kill individual spirochetes by lysis. Although both activities result in the death of B. burgdorferi cells, enhanced production of clumping antibodies (IgG1) could adversely affect the effectiveness of a vaccine. If rOspA induces primarily IgG1 borreliacidal antibody, the antibody may be effective only when large numbers of B. burgdorferi cells are located at a site of infection. By contrast, IgG2a and IgG2b borreliacidal antibody can kill individual spirochetes in the presence of complement. More studies are needed to determine if rOspA vaccines induce primarily IgG1 antibody. A more effective vaccine would produce primarily IgG2a and IgG2b borreliacidal antibody.
In conclusion, we developed an in vitro system to study the role of cytokines on production or inhibition of protective borreliacidal antibody. The present study shows that IL-4 does not play a major role in production of a protective borreliacidal antibody response generated by lymph node cells in vitro. More studies are needed to determine means to prolong the borreliacidal antibody response for protection against infection with B. burgdorferi.