Differential functional patterns of memory CD4+ and CD8+ T-cells from volunteers immunized with Ty21a typhoid vaccine observed using a recombinant Escherichia coli system expressing S. Typhi proteins.

It is widely accepted that CD4+ and CD8+ T-cells play a significant role in protection against Salmonella enterica serovar Typhi (S. Typhi), the causative agent of the typhoid fever. However, the antigen specificity of these T-cells remains largely unknown. Previously, we demonstrated the feasibility of using a recombinant Escherichia coli (E. coli) expression system to uncover the antigen specificity of CD4+ and CD8+ T cells. Here, we expanded these studies to include the evaluation of 12 additional S. Typhi proteins: 4 outer membrane proteins (OmpH, OmpL, OmpR, OmpX), 3 Vi-polysaccharide biosynthesis proteins (TviA, TviB, TviE), 3 cold shock proteins (CspA, CspB, CspC), and 2 conserved hypothetical proteins (Chp 1 and Chp2), all selected based on the bioinformatic analyses of the content of putative T-cell epitopes. CD4+ and CD8+ T cells from 15 adult volunteers, obtained before and 42 days after immunization with oral live attenuated Ty21a vaccine, were assessed for their functionality (i.e., production of cytokines and cytotoxic expression markers in response to stimulation with selected antigens) as measured by flow cytometry. Although volunteers differed on their T-cell antigen specificity, we observed T-cell immune responses against all S. Typhi proteins evaluated. These responses included 9 proteins, OmpH, OmpR, TviA, TviE, CspA, CspB, CspC, Chp 1 and Chp 2, which have not been previously reported to elicit T-cell responses. Interestingly, we also observed that, regardless of the protein, the functional patterns of the memory T-cells were different between CD4+ and CD8+ T cells. In sum, these studies demonstrated the feasibility of using bioinformatic analysis and the E. coli expressing system described here to uncover novel immunogenic T-cell proteins that could serve as potential targets for the production of protein-based vaccines.

H7N9 T-cell epitopes that mimic human sequences are less immunogenic and may induce Treg-mediated tolerance.

Avian-origin H7N9 influenza is a novel influenza A virus (IAV) that emerged in humans in China in 2013. Using immunoinformatics tools, we identified several H7N9 T cell epitopes with T cell receptor (TCR)-facing residues identical to those of multiple epitopes from human proteins. We hypothesized that host tolerance to these peptides may impair T helper response and contribute to the low titer, weak hemagglutination inhibiting (HI) antibody responses and diminished seroconversion rates that have been observed in human H7N9 infections and vaccine trials. We found that the magnitude of human T effector responses to individual H7N9 peptides was inversely correlated with the peptide's resemblance to self. Furthermore, a promiscuous T cell epitope from the hemagglutinin (HA) protein suppressed responses to other H7N9 peptides when co-administered in vitro. Along with other highly 'human-like' peptides from H7N9, this peptide was also shown to expand FoxP3(+) regulatory T cells (Tregs). Thus, H7N9 may be camouflaged from effective human immune response by T cell epitope sequences that avert or regulate effector T cell responses through host tolerance.

Peptide-pulsed dendritic cells induce the hepatitis C viral epitope-specific responses of naïve human T cells.

Hepatitis C virus (HCV) is a major cause of liver disease. Spontaneous resolution of infection is associated with broad, MHC class I- (CD8(+)) and class II-restricted (CD4(+)) T cell responses to multiple viral epitopes. Only 20% of patients clear infection spontaneously, however, most develop chronic disease. The response to chemotherapy varies; therapeutic vaccination offers an additional treatment strategy. To date, therapeutic vaccines have demonstrated only limited success in clinical trials. Vector-mediated vaccination with multi-epitope-expressing DNA constructs provides an improved approach. Highly-conserved, HLA-A2-restricted HCV epitopes and HLA-DRB1-restricted immunogenic consensus sequences (ICS, each composed of multiple overlapping and highly conserved epitopes) were predicted using bioinformatics tools and synthesized as peptides. HLA binding activity was determined in competitive binding assays. Immunogenicity and the ability of each peptide to stimulate naïve human T cell recognition and IFN-γ production were assessed in cultures of total PBMCs and in co-cultures composed of peptide-pulsed dendritic cells (DCs) and purified T lymphocytes, cell populations derived from normal blood donors. Essentially all predicted HLA-A2-restricted epitopes and HLA-DRB1-restricted ICS exhibited HLA binding activity and the ability to elicit immune recognition and IFN-γ production by naïve human T cells. The ability of DCs pulsed with these highly-conserved HLA-A2- and -DRB1-restricted peptides to induce naïve human T cell reactivity and IFN-γ production ex vivo demonstrates the potential efficacy of a multi-epitope-based HCV vaccine targeted to dendritic cells.

Conservation of HIV-1 T cell epitopes across time and clades: validation of immunogenic HLA-A2 epitopes selected for the GAIA HIV vaccine.

HIV genomic sequence variability has complicated efforts to generate an effective globally relevant vaccine. Regions of the viral genome conserved in sequence and across time may represent the "Achilles' heel" of HIV. In this study, highly conserved T-cell epitopes were selected using immunoinformatics tools combining HLA-A2 supertype binding predictions with relative global conservation. Analysis performed in 2002 on 10,803 HIV-1 sequences, and again in 2009, on 43,822 sequences, yielded 38 HLA-A2 epitopes. These epitopes were experimentally validated for HLA binding and immunogenicity with PBMCs from HIV-infected patients in Providence, Rhode Island, and/or Bamako, Mali. Thirty-five (92%) stimulated an IFNγ response in PBMCs from at least one subject. Eleven of fourteen peptides (79%) were confirmed as HLA-A2 epitopes in both locations. Validation of these HLA-A2 epitopes conserved across time, clades, and geography supports the hypothesis that such epitopes could provide effective coverage of virus diversity and would be appropriate for inclusion in a globally relevant HIV vaccine.

Further progress on defining highly conserved immunogenic epitopes for a global HIV vaccine: HLA-A3-restricted GAIA vaccine epitopes.

Two major obstacles confronting HIV vaccine design have been the extensive viral diversity of HIV-1 globally and viral evolution driven by escape from CD8(+) cytotoxic T-cell lymphocyte (CTL)-mediated immune pressure. Regions of the viral genome that are not able to escape immune response and that are conserved in sequence and across time may represent the "Achilles' heel" of HIV and would be excellent candidates for vaccine development. In this study, T-cell epitopes were selected using immunoinformatics tools, combining HLA-A3 binding predictions with relative sequence conservation in the context of global HIV evolution. Twenty-seven HLA-A3 epitopes were chosen from an analysis performed in 2003 on 10,803 HIV-1 sequences, and additional sequences were selected in 2009 based on an expanded set of 43,822 sequences. These epitopes were tested in vitro for HLA binding and for immunogenicity with PBMCs of HIV-infected donors from Providence, Rhode Island. Validation of these HLA-A3 epitopes conserved across time, clades, and geography supports the hypothesis that epitopes such as these would be candidates for inclusion in our globally relevant GAIA HIV vaccine constructs.