Immunization with Oligomannose-Coated Liposome-Entrapped Dense Granule Protein 7 Protects Dams and Offspring from Neospora caninum Infection in Mice

The present study demonstrates that the subcutaneous administration of Neospora caninum dense granule protein 7 (NcGRA7) entrapped in liposomes coated with mannotriose strongly induces the parasite-specific T-helper type 1 immune response and humoral antibody in mice. Although anti-NcGRA7 immunoglobulin G1 antibody production was induced in mice injected with NcGRA7 alone, the dams and offspring were never protected from N. caninum infection. The immunization of mice with liposome-entrapped NcGRA7 before pregnancy resulted in increased offspring survival and decreased the infection rates in the brains of dams after parasite infection at 6 to 9 days of gestation. In conclusion, oligomannose-coated liposome-entrapped NcGRA7 can be used as a new type of effective vaccine to control neosporosis.Neosporosis, caused by an apicomplexan protozoan parasite, Neospora caninum, is a cause of infectious abortion and congenital disease in cattle worldwide (11). Transplacental transmission is the major route of N. caninum infection (7, 39), and the parasite persists in the herd over successive generations, causing significant economic losses due to abortion, decreased milk production, and the resultant culling (6, 10, 21, 46, 47). Horizontal transmission of N. caninum by oocysts that are shed by dogs has also been documented in cattle, but this transmission is not considered a major route of infection (7).Various mouse models have been utilized to understand the host protective immune responses to N. caninum infection. A Th1-type immune response appears to be important in protection against N. caninum infection (2, 34). Gamma interferon (IFN-γ) and interleukin-12 (IL-12), known to be crucial cytokines for the development of Th1-type immunity, are important for protective immunity against acute N. caninum infection (2). Furthermore, CD4⁺ T-cell-depleted BALB/c mice were more highly susceptible to parasite infection than were CD8⁺ T-cell-depleted mice (34, 45). Studies of IFN-γ knockout mice indicated the importance of macrophage activation by IFN-γ for protective immunity (34). On the other hand, a Th2-type immune response with predominant production of humoral antibody specific for the parasite antigens is also capable of mediating protection against neosporosis (17, 18, 30, 38, 40). These observations suggest that a suitable balance in the production of Th1- and Th2-type cytokines has a crucial role in the control of N. caninum infection (33).Oligomannose-coated liposomes have been shown to be a safe adjuvant to induce Th1-type immunity because no skin damage by the liposomes is caused at the injection site (16). A previous study showed that liposomes coated with a neoglycolopid consisting of mannotriose and dipalmitoylphosphatidylethanolamine (Man3-DPPE) were specifically and rapidly incorporated into intraperitoneal macrophages when injected into the peritoneal cavity and that the liposome-incorporating macrophages smoothly accumulated in nearby lymphoid tissue (23). The effect of Man3-coated liposome as an effective adjuvant has been confirmed with Leishmania major infection (41) and with tumors (23, 25). Administration of soluble leishmanial antigens entrapped within the Man3-coated liposomes to BALB/c mice strongly induced the antigen-specific Th1 immune response, as evidenced by a higher level of IFN-γ production and a lower level of IL-4 production than those in mice receiving the antigens alone (41).There is accumulating evidence that some N. caninum-infected cows develop a degree of protective immunity against abortion and/or congenital transmission, indicating the advantage of vaccine development (31). Although the prevention of abortion might be a realistic goal for a vaccine, the ultimate objective for the control of neosporosis must be to prevent the vertical transmission of the parasite. Evidence has shown that cattle which abort due to neosporosis have higher levels of N. caninum-specific antibody than do infected but nonaborting cattle (9). In addition, our previous study showed that a higher level of bovine antibody specific for N. caninum dense granule protein 7 (NcGRA7) was detected in aborting than in nonaborting cows and heifers, while levels of specific antibodies against parasite surface proteins NcSAG1 and NcSRS2 exhibited no significant difference between the aborting and nonaborting cows (22). To control N. caninum infection, a suitable balance of Th1- and Th2-type immune responses is important (33). We speculated that an NcGRA7-specific Th2-type immune response might be predominant in aborting cows. Therefore, induction of the NcGRA7-specific Th1-type immune response could play a crucial role in the control of N. caninum infection, since antibodies against the parasites did not prevent vertical transmission (32). Thus, the present study was conducted to evaluate the vaccine efficacy of oligomannose-coated liposome-entrapped NcGRA7 on N. caninum infection in dams and offspring, using a BALB/c mouse model. Our results suggest that the Th1-type immune response against NcGRA7 plays a crucial role in the control of N. caninum infection.