Identification of Vaccine Candidate Peptides in the NcSRS2 Surface Protein ofNeospora caninumby Using CD4⁺Cytotoxic T Lymphocytes and Gamma Interferon-Secreting T Lymphocytes of Infected Holstein Cattle

Abstract

Previously, our laboratory showed that Holstein cattle experimentally infected with Neospora caninum develop parasite-specific CD4 ؉ cytotoxic T lymphocytes (CTL) that lyse infected, autologous target cells through a perforin-granzyme pathway. To identify specific parasite antigens inducing bovine CTL and helper T-lymphocyte responses for vaccine development against bovine neosporosis, the tachyzoite major surface proteins NcSAG1 and NcSRS2 were targeted. In whole tachyzoite antigen-expanded bovine T-lymphocyte lines, recombinant NcSRS2 induced potent memory CD4؉ -and CD8 ؉ -T-lymphocyte activation, as indicated by proliferation and gamma interferon (IFN-␥) secretion, while recombinant NcSAG1 induced a minimal memory response. Subsequently, T-lymphocyte epitope-bearing peptides of NcSRS2 were mapped by using overlapping peptides covering the entire NcSRS2 sequence. Neospora caninum is an intracellular apicomplexan protozoan parasite that causes abortion and congenital infection in cattle (21). Transplacental parasite transmission is the major mode of infection (18, 45) which persists in the herd over successive generations, causing significant economic losses due to abortion, culling, and decreased milk production (13,20,25,52,53). Recrudescence of chronic, congenitally acquired infection due to downregulation of Th1 immune responses during pregnancy may be largely responsible for transplacental N. caninum transmission (10,22,27,42). A vaccine that could control tachyzoite parasitemia and decrease congenital infection or abortion rates is needed.Studies of Toxoplasma gondii, a protozoan parasite closely related to N. caninum in biology, phylogeny, and immunogenicity, unequivocally show the importance of T lymphocytes in resistance to infection by adoptive transfer, gene knockout, and depletion studies with mouse models (19). The presence of parasite-specific CD4 ϩ and CD8 ϩ cytotoxic T lymphocytes (CTL) is also associated with immunity to natural T. gondii infection in humans (14,41). In murine models of neosporosis, a T-lymphocyte-mediated immune response, characterized by increased levels of gamma interferon (IFN-␥) and interleukin 12 (IL-12), correlates with disease resistance (5,30,33), and IFN-␥ alone inhibits tachyzoite growth and cellular invasion (24,38,51,55). In pregnant mice, protection against vertical transmission correlates with induction of parasite-specific Tlymphocyte-mediated immunity (39). Cattle infected with N. caninum produce parasite-specific, major histocompatibility complex (MHC)-restricted, CD4 ϩ CTL (50) that could reduce N. caninum infection indirectly by IFN-␥ activation of mononuclear phagocytes and directly by killing N. caninum-infected cells.To identify vaccine candidate parasite antigens for induction of CTL and IFN-␥ secretion in cattle against neosporosis abortion, this study focused on major tachyzoite glycosylphosphatidylinositol-anchored surface proteins, N. caninum surface antigen 1 (NcSAG1) and N. caninum SAG1-related sequence 2 (NcSRS2) (26, 28). The T. gondii surface pr...

Bovine Immune Response to Inoculation with Neospora caninum Surface Antigen SRS2 Lipopeptides Mimics Immune Response to Infection with Live Parasites

“…The primary purpose of the experiments as designed was not to determine a mechanism of immunity to N. caninum subunits nor compare types of immunization parameters, such as different adjuvants, routes of immunization, or specific immunogen types. Before moving forward with efficacy trials in pregnant cattle, we simply were asking, "Can NcSRS2 subunits be designed and administered that will induce T-lymphocyte activation and IFN-␥ secretion similar to what we and others had observed when experimentally infecting cattle with N. caninum tachyzoites (36,39,43)?" Previous studies with collaborators showed that the DNA immunization procedure used in the present study could induce antigenspecific T-cell responses, optimally detected in short-term Tlymphocyte lines (28).…”

“…Twenty-four nonpregnant female or castrated male yearling Friesian-Holstein cattle (Bos taurus) were selected for MHC-I and MHC-II NcSRS2 responder haplotypes as identified previously (39) ( Table 1). The bovine lymphocyte antigen class I haplotypes and DRB3 alleles were characterized by microarray typing as previously described (31,39). Animals were housed and cared for in accordance with the animal care and use regulations of Washington State University, Pullman, WA, and the animal welfare committee guidelines of the Kimron Veterinary Institute, Bet-Dagan, Israel.…”

“…To detect NcSRS2 subunit-specific T lymphocytes from PBMC without in vitro expansion, the IFN-␥-expressing cells in PBMC were quantified by using an ELISPOT specific for bovine IFN-␥ as previously described (39). Lipopeptides (LP 20-21 and LP 34-36) or lysates of COS cells transfected with DNA vaccine constructs (VR-NcSRS2PI and VR-1055) were added to freshly isolated PBMC (1 ϫ 10 6 cells in 100 l complete RPMI medium) at final concentrations of 10 g per ml.…”

“…caninum SRS2 (NcSRS2), a surface antigen of N. caninum, is a good subunit vaccine candidate because it is highly conserved (15), is associated with immunity to transplacental parasite transmission in mice (15,30) and to infection of sheep placental cells (16), and is an immunodominant antigen in N. caninum-infected cattle (11,39). T-lymphocyte cell lines of cattle infected with N. caninum readily proliferate and secrete IFN-␥ when stimulated with recombinant NcSRS2 (rNcSRS2), and T-lymphocyte epitopes of NcSRS2 have been mapped in cattle infected with N. caninum (39). Peripheral blood mononuclear cells (PBMC) of N. caninum-infected outbred cattle with diverse MHC class II (MHC-II) haplotypes show marked parasite-specific secretion of IFN-␥ and expanded numbers of IFN-␥-secreting T-lymphocytes and cytotoxic T-lymphocytes when stimulated ex vivo with NcSRS2 peptides bearing Tlymphocyte epitopes.…”

Vaccines and drugs against Neospora caninum, an important apicomplexan causing abortion in cattle and other farm animals

“…In cattle, the situation is less polarized, and the classical roles of many cytokines in the laboratory mouse do not extrapolate entirely or at all to cattle. 25 Therefore, a suitable approach for the selection of suitable vaccine candidates would be to perform in vitro assays with bovine CD4(+) cytotoxic lymphocytes, as done by Staska et al 26 The candidate polypeptides inducing the highest level of IFN-γ secretion should then be retained for further assessment.…”