Toxoplasma gondii HLA-B*0702-restricted GRA7(20-28) peptide with adjuvants and a universal helper T cell epitope elicits CD8(+) T cells producing interferon-γ and reduces parasite burden in HLA-B*0702 mice.

The ability of CD8(+) T cells to act as cytolytic effectors and produce interferon-γ (IFN-γ) was demonstrated to mediate resistance to Toxoplasma gondii in murine models because of the recognition of peptides restricted by murine major histocompatibility complex (MHC) class I molecules. However, no T gondii-specific HLA-B07-restricted peptides were proven protective against T gondii. Recently, 2 T gondii-specific HLA-B*0702-restricted T cell epitopes, GRA7(20-28) (LPQFATAAT) and GRA3(27-35) (VPFVVFLVA), displayed high-affinity binding to HLA-B*0702 and elicited IFN-γ from peripheral blood mononuclear cells of seropositive HLA-B*07 persons. Herein, these peptides were evaluated to determine whether they could elicit IFN-γ in splenocytes of HLA-B*0702 transgenic mice when administered with adjuvants and protect against subsequent challenge. Peptide-specific IFN-γ-producing T cells were identified by enzyme-linked immunosorbent spot and proliferation assays utilizing splenic T lymphocytes from human lymphocyte antigen (HLA) transgenic mice. When HLA-B*0702 mice were immunized with one of the identified epitopes, GRA7(20-28) in conjunction with a universal CD4(+) T cell epitope (PADRE) and adjuvants (CD4(+) T cell adjuvant, GLA-SE, and TLR2 stimulatory Pam(2)Cys for CD8(+) T cells), this immunization induced CD8(+) T cells to produce IFN-γ and protected mice against high parasite burden when challenged with T gondii. This work demonstrates the feasibility of bioinformatics followed by an empiric approach based on HLA binding to test this biologic activity for identifying protective HLA-B*0702-restricted T gondii peptides and adjuvants that elicit protective immune responses in HLA-B*0702 mice.

Towards an immunosense vaccine to prevent toxoplasmosis: protective Toxoplasma gondii epitopes restricted by HLA-A*0201.

The ideal vaccine to protect against toxoplasmosis in humans would include antigens that elicit a protective T helper cell type 1 immune response, and generate long-lived IFN-γ-producing CD8(+) T cells. Herein, we utilized a predictive algorithm to identify candidate HLA-A02 supertype epitopes from Toxoplasma gondii proteins. Thirteen peptides elicited production of IFN-γ from PBMC of HLA-A02 supertype persons seropositive for T. gondii infection but not from seronegative controls. These peptides displayed high-affinity binding to HLA-A02 proteins. Immunization of HLA-A*0201 transgenic mice with these pooled peptides, with a universal CD4(+) epitope peptide called PADRE, formulated with adjuvant GLA-SE, induced CD8(+) T cell IFN-γ production and protected against parasite challenge. Peptides identified in this study provide candidates for inclusion in immunosense epitope-based vaccines.

Human immunome, bioinformatic analyses using HLA supermotifs and the parasite genome, binding assays, studies of human T cell responses, and immunization of HLA-A*1101 transgenic mice including novel adjuvants provide a foundation for HLA-A03 restricted CD8+T cell epitope based, adjuvanted vaccine protective against Toxoplasma gondii.

BACKGROUND: Toxoplasmosis causes loss of life, cognitive and motor function, and sight. A vaccine is greatly needed to prevent this disease. The purpose of this study was to use an immmunosense approach to develop a foundation for development of vaccines to protect humans with the HLA-A03 supertype. Three peptides had been identified with high binding scores for HLA-A03 supertypes using bioinformatic algorhythms, high measured binding affinity for HLA-A03 supertype molecules, and ability to elicit IFN-γ production by human HLA-A03 supertype peripheral blood CD8+ T cells from seropositive but not seronegative persons. RESULTS: Herein, when these peptides were administered with the universal CD4+T cell epitope PADRE (AKFVAAWTLKAAA) and formulated as lipopeptides, or administered with GLA-SE either alone, or with Pam2Cys added, we found we successfully created preparations that induced IFN-γ and reduced parasite burden in HLA-A*1101(an HLA-A03 supertype allele) transgenic mice. GLA-SE is a novel emulsified synthetic TLR4 ligand that is known to facilitate development of T Helper 1 cell (TH1) responses. Then, so our peptides would include those expressed in tachyzoites, bradyzoites and sporozoites from both Type I and II parasites, we used our approaches which had identified the initial peptides. We identified additional peptides using bioinformatics, binding affinity assays, and study of responses of HLA-A03 human cells. Lastly, we found that immunization of HLA-A*1101 transgenic mice with all the pooled peptides administered with PADRE, GLA-SE, and Pam2Cys is an effective way to elicit IFN-γ producing CD8+ splenic T cells and protection. Immunizations included the following peptides together: KSFKDILPK (SAG1224-232); AMLTAFFLR (GRA6164-172); RSFKDLLKK (GRA7134-142); STFWPCLLR (SAG2C13-21); SSAYVFSVK(SPA250-258); and AVVSLLRLLK(SPA89-98). This immunization elicited robust protection, measured as reduced parasite burden using a luciferase transfected parasite, luciferin, this novel, HLA transgenic mouse model, and imaging with a Xenogen camera. CONCLUSIONS: Toxoplasma gondii peptides elicit HLA-A03 restricted, IFN-γ producing, CD8+ T cells in humans and mice. These peptides administered with adjuvants reduce parasite burden in HLA-A*1101 transgenic mice. This work provides a foundation for immunosense based vaccines. It also defines novel adjuvants for newly identified peptides for vaccines to prevent toxoplasmosis in those with HLA-A03 supertype alleles.

Identification of T. gondii epitopes, adjuvants, and host genetic factors that influence protection of mice and humans.

Toxoplasma gondii is an intracellular parasite that causes severe neurologic and ocular disease in immune-compromised and congenitally infected individuals. There is no vaccine protective against human toxoplasmosis. Herein, immunization of L(d) mice with HF10 (HPGSVNEFDF) with palmitic acid moieties or a monophosphoryl lipid A derivative elicited potent IFN-gamma production from L(d)-restricted CD8(+) T cells in vitro and protected mice. CD8(+) T cell peptide epitopes from T. gondii dense granule proteins GRA 3, 6, 7, and Sag 1, immunogenic in humans for HLA-A02(+), HLA-A03(+), and HLA-B07(+) cells were identified. Since peptide repertoire presented by MHC class I molecules to CD8(+) T cells is shaped by endoplasmic reticulum-associated aminopeptidase (ERAAP), polymorphisms in the human ERAAP gene ERAP1 were studied and associate with susceptibility to human congenital toxoplasmosis (p<0.05). These results have important implications for vaccine development.

Development of experimental carbohydrate-conjugate vaccines composed of Streptococcus pneumoniae capsular polysaccharides and the universal helper T-lymphocyte epitope (PADRE).

Experimental carbohydrate-conjugate vaccines composed of the 13 amino acid universal Pan HLA-DR Epitope (PADRE) and Streptococcus pneumoniae capsular polysaccharides from serotypes 14, 6B and 9V were produced. Simple carbodiimide-mediated condensation chemistry was used to conjugate the PADRE synthetic peptide to the three chemically different capsular polysaccharides in a 1:1 molar ratio. The immunogenicity of the PADRE peptide component of the conjugate vaccines was confirmed by the induction of PADRE-specific CD4+ helper T cell (HTL) responses following immunization of C57BL/6 mice. High titer antibody responses specific for polysaccharides of S. pneumoniae serotypes 14, 6B and 9V were induced using Complete Freund's Adjuvant (CFA) and alhydrogel Al(OH)3 formulations. The HTL, or carrier, effect of the PADRE synthetic peptide was only evident using the PADRE-polysaccharide conjugates; simple mixtures of the PADRE peptide and polysaccharides were essentially nonimmunogenic. The functional or potential protective value of the polysaccharide-specific antibodies was measured as a function of opsonophagocytic activity for the 6B serotype. High titers of opsonophagocytic activity were measured in sera from mice immunized with formulations containing both adjuvants. These data demonstrate that the PADRE synthetic peptide can induce the HTL responses needed to support the development of antibodies specific for bacterial carbohydrates used in conjugate vaccines.