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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.

The goal of the present study was to design a vaccine that would provide universal protection against infection of humans with diverse influenza A viruses. Accordingly, protein sequences from influenza A virus strains currently in circulation (H1N1, H3N2), agents of past pandemics (H1N1, H2N2, H3N2) and zoonotic infections of man (H1N1, H5N1, H7N2, H7N3, H7N7, H9N2) were evaluated for the presence of amino acid sequences, motifs, that are predicted to mediate peptide epitope binding with high affinity to the most frequent HLA-DR allelic products. Peptides conserved among diverse influenza strains were then synthesized, evaluated for binding to purified HLA-DR molecules and for their capacity to induce influenza-specific immune recall responses using human donor peripheral blood mononuclear cells (PBMC). Accordingly, 20 epitopes were selected for further investigation based on their conservancy among diverse influenza strains, predicted population coverage in diverse ethnic groups and capacity to recall influenza-specific responses. A DNA plasmid encoding the epitopes was constructed using amino acid spacers between epitopes to promote optimum processing and presentation. Immunogenicity of the DNA vaccine was measured using HLA-DR4 transgenic mice and the TriGrid in vivo electroporation device. Vaccination resulted in peptide-specific immune responses, augmented HA-specific antibody responses and protection of HLA-DR4 transgenic mice from lethal PR8 influenza virus challenge. These studies demonstrate the utility of this vaccine format and the contribution of CD4(+) T cell responses to protection against influenza infection.

Pan-DR epitope (PADRE) peptides have demonstrated the capacity to deliver help for antibody responses in vivo. They were also found, fortuitously, to be able to provide significant helper T-cell activity in vivo. This suggested that linear constructs, containing the PADRE epitope, might be as efficient at generating an immune response as large multivalent antigens. Plasmodium falciparum and P. yoelii PADRE constructs were capable of inducing a high titre IgG antibody response that recognized intact sporozoites. We now report that these antibodies can inhibit sporozoite invasion of hepatocytes in vitro and that mice immunized with the PyCSP-PADRE linear construct were protected when challenged with P. yoelii sporozoites.

Various peptide-based approaches to simultaneous induction of multiple cytotoxic T lymphocyte (CTL) responses were evaluated as part of ongoing efforts to develop immunotherapeutic vaccines for use in humans. To this end, HLA (human histocompatibility leukocyte antigen)-A2-restricted epitopes from several specific viral proteins were tested in an HLA-A2 transgenic mouse model system, which mimics human CTL responses to these viral proteins. Multiple CTL responses were elicited by immunization with either peptides emulsified in incomplete Freund's adjuvant (IFA), or lipidated peptides administered in phosphate buffered saline (PBS). In the case of lipidated peptides, induction of CTL responses was crucially dependent on the presence of helper T lymphocyte (HTL) epitopes, and most efficient in the case of lipidated covalently linked HTL-CTL epitope constructs. CTL could also be induced by immunization with lipidated HTL epitopes simply mixed with CTL epitopes and formulated in PBS. However, this approach was highly dependent on the particular lipidated HTL/CTL combination utilized, and was marginally effective for simultaneous priming of multiple CTL responses. By contrast, all HTL/CTL combinations were potent immunogens when delivered as lipidated, covalently linked molecules. This was the most effective of the approaches analysed in terms of multi-epitope priming, as demonstrated by the induction of simultaneous CTL responses to a pool of five different epitopes.

Induction of humoral immune responses against protein antigen requires that two independent signals be delivered to B cells. It is currently assumed that simple monovalent synthetic peptides would not be effective immunogens for antibody responses because they would not be anticipated to effectively generate the necessary signals unless conjugated to a complex carrier system. In this study, the immunogenicity of short linear peptide constructs comprising Plasmodium vivax B cell epitopes (PVB) and non-natural Pan-DR T helper cell epitopes (PADRE) was assessed in mice and compared to other types of antigen constructs. The 33-residue PADRE-PVB linear constructs were highly immunogenic and induced responses comparable to those obtained with the multiple antigen peptides (MAP) constructs, both in terms of absolute titers and quality of antibody responses. The anti-PVB antibody responses were of long duration, composed mostly of IgG and reactive with intact sporozoites. The PADRE-PVB constructs were immunogenic when formulated in adjuvants such as Alum and Montanide ISA 51 underlining the relevance of these findings for vaccine development.

A novel mechanism for inhibition of T cell responses is described. Using the recognition of the influenza hemagglutinin (HA) 307-319 peptide in the context of DR1 class II major histocompatibility complex molecules, we have found that nonstimulatory analogs of the HA peptide preferentially inhibit HA-specific T cells in inhibition of antigen presentation assays. This antigen-specific effect could be generalized to another DR1-restricted peptide, Tetanus toxoid 830-843. Direct binding and cellular experiments indicated that the mechanism responsible was distinct from competition for binding to DR1 molecules. Likewise, negative signaling and induction of T cell tolerance could also be excluded as effector mechanisms. Thus, the most likely mechanism for this effect is engagement of antigen-specific T cell receptors by DR1-peptide analog complexes, which results in antigen-specific competitive blocking of T cell responses by virtue of their capacity to compete with DR1-antigen complexes for binding to the T cell receptor.

Our strategy to use saturation mutagenesis to produce an unbiased collection of major histocompatibility class I mutants has resulted in unpredicted mutant phenotypes. First, we have shown data supporting our earlier work of the DP20(Y27N) mutant. Allorecognition is altered at the clonal level while no variation in lymphocytic choriomeningitis virus (LCMV)-restricted recognition is observed. The defect does not destroy the integrity of this class I protein on the basis of three observations: (i) LCMV self-restricted recognition is not impaired, (ii) β2 microglobulin still associates with DP20(Y27N) at the cell surface, and (iii) this mutant can stimulate a primary MLR. Thus, we believe DP20(Y27N) specifically affects allorecognition, perhaps by altering self peptide associations. The DP14(A11V;E32Q) mutant appears to interact with T cell receptors (TCR) from a cloned cytotoxic T lymphocyte, but is altered in inducing a wild type signal into the responding cell. This is presumably due to decreased interaction at the cell surface between DP14(A11V;E32Q) and wild type-specific TCR such that variations are detected in how a cell perceives extracellular signals. Analysis of additional mutants suggests that mutant DP163(N66S) alters the binding site for monoclonal antibodies 7–16.10 and 135, while leaving unaltered the binding site for monoclonal antibodies 34-1.2 and 11–20.3. This maps the residue responsible for 7–16.10 and 135 binding to the region of DP163(N66S).