OspC has been suggested as an alternative vaccine to OspA (14
). Previous studies have shown that the levels of OspC increase on the outer surface of B. burgdorferi
during tick attachment (28
). Concomitantly, the levels of OspA and OspB are down-regulated, with little or no expression of OspA at the time of passage into the mammalian host (3
). Moreover, high levels of anti-OspC antibody (2
), including anti-OspC borreliacidal antibody (27
), are detected in sera of patients with early Lyme borreliosis. Even after several months of infection of the host, expression of OspC is not down-regulated on B. burgdorferi
). Furthermore, passive transfer of anti-OspC antibody results in resolution of established arthritis and carditis (3
). These findings suggest that rOspC should be considered for inclusion in a second generation of Lyme borreliosis vaccines.
There are, however, concerns about the heterogeneity (12
) and especially the immunogenicity (34
) of OspC. Vaccination with rOspC can induce antibodies, but many of these antibodies are not involved in protection (4
). Only a few epitopes of rOspC, perhaps just one, are responsible for the induction of antibodies that can kill B. burgdorferi
). Production of sustained high levels of anti-OspC borreliacidal antibody is paramount for determining the potential of an OspC vaccine. Although other mechanisms of protection may be involved (24
), we investigated only the borreliacidal antibody response induced by vaccination with OspC (5
Our results demonstrate that rOspC is a poor immunogen, even in the presence of alum, for inducing borreliacidal antibody. A low titer (<320) of anti-OspC borreliacidal antibody was produced at the peak interval of the borreliacidal antibody response after rOspC vaccination of mice. By contrast, consistently higher and sustained levels of borreliacidal antibody were produced in mice vaccinated with B. burgdorferi
. When mice were vaccinated with B. burgdorferi
in alum, even higher levels of borreliacidal antibody (titer, 5,120) were produced. In addition, the anti-OspC borreliacidal antibody (titer, 1,280) was maintained for 70 days. An explanation of these differences may be that native OspC on B. burgdorferi
is processed more efficiently by antigen-processing cells than is rOspC. Recombinant OspC may have lost conformational epitopes necessary to induce high levels of borreliacidal antibody. Another explanation may be that OspC of B. burgdorferi
50772 is lipidated while the rOspC is not. In support, Erdile et al. (9
) demonstrated that lipidation significantly increased the immunogenicity of OspA. Regardless of the mechanism of immunogenicity, rOspC needs to be improved before field trials in humans are considered.
Our results also showed that the anti-OspC borreliacidal antibody response waned rapidly. Low titers (range, 20 to 320) of borreliacidal antibody were detected in sera 2 months or less after vaccination of mice with all four preparations of OspC, despite the use of an adjuvant. Similarly, the borreliacidal antibody titer waned rapidly in supernatants obtained from cultures of immune lymph node cells. The peak anti-OspC borreliacidal antibody level (titer, 2,048) was obtained with lymph node cells obtained on day 28 after vaccination. By day 35 after vaccination, the lymph node cells produced only a low level (titer, 64) of borreliacidal antibody, despite being cultured with B. burgdorferi expressing high levels of OspC. Our findings suggest that immune cells become progressively less responsive to the epitope(s) of OspC responsible for the induction of borreliacidal antibody. This dampening of the protective borreliacidal antibody response may partially explain the ability of B. burgdorferi to persist in vivo. Vaccination with rOspC may be of limited value if this inhibitory mechanism is not neutralized or modified.
The mechanism responsible for production of anti-Osp C borreliacidal antibody does involve IL-6: When lymph node cells obtained from mice vaccinated 35 days earlier were exposed to different concentrations of IL-6, borreliacidal antibody production increased two- to eightfold. Furthermore, anti-IL-6 treatment of lymph node cells obtained 28 days after vaccination decreased the production of borreliacidal antibody by 10-fold or more. In addition, anti-OspC borreliacidal antibody was not detected in supernatants from lymph node cells obtained from mice vaccinated 28 or 35 days earlier and treated with 100 μg of anti-IL-6.
We also showed that endogenous IL-6 affects the production of anti-OspC borreliacidal antibody. Episodes of waxing and waning of IL-6 production occurred in cultures of lymph node cells obtained from vaccinated mice. The highest levels of IL-6 occurred in cultures of lymph node cells beginning to produce anti-OspC borreliacidal antibody. Once maximum production of borreliacidal antibody had occurred (day 28 after vaccination), a low level of IL-6 was detected. By day 35 after vaccination, the level of IL-6 had increased in lymph node cultures to maintain a low level of production of anti-OspC borreliacidal antibody. The number of spirochetes in the lymph node cell cultures did not influence the levels of IL-6 production. Lymph node cultures demonstrating low levels of borreliacidal antibody production contained the same number of spirochetes as did cultures of lymph node cells that caused elevated levels of killing antibody. These studies demonstrate the significance of IL-6 in the development of OspC specific humoral responses.
The decreased production of anti-OspC borreliacidal antibody by immune lymph node cells, even in the presence of high concentrations of borrelial antigen, has important implications for developing strategies to prevent Lyme borreliosis. Protracted low levels of anti-OspC borreliacidal antibody may not be adequate to eliminate B. burgdorferi
in host tissues, especially those not readily accessible to antibody, thus promoting the chronic nature of the disease. In support, Zhong et al. (34
) showed that passive transfer of antiserum to OspC resulted in resolution of infection as well as of arthritis and carditis. Since high and sustained levels of anti-OspC borreliacidal antibody are likely to constitute a critical factor in the elimination of B. burgdorferi
from the host, the mechanism responsible for the rapid waning of borreliacidal antibody production needs to be investigated. Cytokines or other immune modulators play an important role in establishing the effectiveness and duration of protection against infection with B. burgdorferi
). We found that IL-6 directly or indirectly plays an important role in the induction of anti-OspC borreliacidal antibody. Modulation of the IL-6 cytokine response or that of other cytokines that affect IL-6 may enhance the level and duration of borreliacidal antibody production.
At present we do not know which cells are responsible for the production of IL-6. IL-6 is a multifunctional cytokine secreted by a wide variety of cells including macrophages, dendritic cells, B and T lymphocytes, endothelial cells, hepatocytes, and germinal center cells (33
). IL-6 receptors have been also found on B and T-lymphocytes as well as macrophages (33
). Interestingly, T cells down-regulate IL-6 receptors on activation, whereas B cells acquire IL-6 receptors only at the final stages of maturation (19
) Ricon et al. (26
) found that IL-6 was consumed during the differentiation of Th2 cells. These findings may explain our results. Thus, B. burgdorferi
is processed by antigen-presenting cells, which release IL-6 and cause the expansion of B lymphocytes, either directly or by T-cell help, into plasma cells which produce borreliacidal antibody. Once T cells are fully activated, they may down-regulate IL-6 and the production of borreliacidal antibody. We found low levels of IL-6 in supernatants of lymph node cells with high levels of borreliacidal antibody. In contrast, high levels of IL-6 occurred only during periods (7 to 21 days after vaccination) of initial production of borreliacidal antibody and periods (35 to 63 days after vaccination) when low levels of production of killing antibody were maintained. The mechanisms by which IL-6 is regulated need to be elucidated if the rapid waning of the borreliacidal antibody response is to be prevented.
The borreliacidal antibody response detected in vitro was due to the production of anti-OspC borreliacidal antibody. Absorption of supernatant from cultures of lymph node cells from mice vaccinated 28 days earlier with rOspC decreased the borreliacidal antibody titer from 2,048 to 8. Similarly, the rOspC ELISA titer was decreased in absorbed supernatant from 512 to less than 10. Most importantly, the anti-OspC borreliacidal antibody was due primarily to the production of IgG2b antibody. These findings suggest that OspC expresses borreliacidal epitopes that preferentially promote the IgG2b subclass. Typically, antibodies produced during a secondary immune response are of the IgG1, IgG2b and IgG3 subclasses (17
). An association between IgG2b production and IL-6 has been shown (1
). An advantage of IL-6-induced borreliacidal IgG2b antibody is that IgG2b fixes complement and bind Fc receptors (17
). Thus, OspC induces antibody with the potential to eliminate spirochetes by complement-dependent antibody-mediated lysis and by Fc-mediated phagocytosis. In contrast, Munson et al. (22
) demonstrated that vaccination with OspA induces primarily IgG1, which does not bind complement. These findings suggest that OspC would be a more effective immunogen, since vaccination with OspC induces predominantly IgG2b, complement-fixing borreliacidal antibody.
In summary, we found that IL-6 plays a major role in the induction of a secondary anti-Osp C borreliacidal IgG2b antibody response. Our studies further support OspC as an alternate vaccine candidate; however, modification of the cytokine response, particularly IL-6, may be necessary to increase the duration of high levels of borreliacidal anti-OspC production. It is possible that other cytokines may interfere with IL-6 and the borreliacidal antibody response, since cytokines can act antagonistically (16
). Characterization of the cytokine-induced mechanisms induced by B. burgdorferi
or subunit vaccines will facilitate the development of a Lyme borreliosis vaccine with a prolonged duration of protection.