Daniel Rivera

Identification of immunogenic HLA-B7 “Achilles’ heel” epitopes within highly conserved regions of HIV

Genetic polymorphisms in class I human leukocyte antigen molecules (HLA) have been shown to determine susceptibility to HIV infection as well as the rate of progression to AIDS. In particular, the HLA-B7 supertype has been shown to be associated with high viral loads and rapid progression to disease. Using a multiplatform in silico/in vitro approach, we have prospectively identified 45 highly conserved, putative HLA-B7 restricted HIV CTL epitopes and evaluated them in HLA binding and ELISpot assays. All 45 epitopes (100%) bound to HLA-B7 in cell-based HLA binding assays: 28 (62%) bound with high affinity, 6 (13%) peptides bound with medium affinity and 11 (24%) bound with low affinity. Forty of the 45 peptides (88%) stimulated a IFN-gamma response in PBMC from at least one subject. Eighteen of these 40 epitopes have not been previously described; an additional eight epitopes have not been previously described as restricted by B7. The HLA-B7 restricted epitopes discovered using this in silico screening approach are highly conserved across strains and clades of HIV as well as conserved in the HIV genome over the 20 years since HIV-1 isolates were first sequenced. This study demonstrates that it is possible to select a broad range of HLA-B7 restricted epitopes that comprise stable elements in the rapidly mutating HIV genome. The most immunogenic of these epitopes will be included in the GAIA multi-epitope vaccine.

Epitope-Driven TB Vaccine Development: A Streamlined Approach Using Immuno-Informatics, ELISpot Assays, and HLA Transgenic Mice

New vaccine candidates that might better control the worldwide prevalence of Mycobacterium tuberculosis (Mtb) have yet to be described. Strong CD4+ T cell-mediated immune response (CMI) is correlated with protection from the development of TB disease; however, the selection of suitable vaccine antigens has been thwarted by the size and complexity of the (Mtb) proteome, and by the relative difficulty of delivering these antigens in the right immunological context. One possible solution is to develop immunotherapeutic vaccines for TB that are based on T cell epitopes representing multiple antigens. This text illustrates the stepwise development of epitope-driven vaccines from in silico epitope mapping to testing the vaccine in a live Mtb challenge model. First, we used the whole genome Mtb microarray to identify bacterial proteins expressed under the conditions thought to model Mtb survival and replication in human macrophages. Eighteen of these proteins were also found by Behr et al. to be absent from at least one strain of BCG; the sequences of these eighteen proteins were then screened for T-cell epitopes using the immuno-informatics algorithm, EpiMatrix. Of the seventeen representative epitopes evaluated in ELISpot assays, all seventeen were confirmed to elicit interferon (IFN)-gamma secretion by PBMC from Mtb-exposed subjects. A parallel live Mtb challenge study in mice showed prototype epitope-based TB vaccines to be robustly immunogenic but not as effective as BCG. These experiments illustrate the use of immuno-informatics tools for vaccine development and describe a pathway for the development of a more effective, epitope-driven, immunotherapeutic vaccine for TB.

Confirmation of immunogenic consensus sequence HIV-1 T-cell epitopes in Bamako, Mali and Providence, Rhode Island.

The design of epitope-driven vaccines for HIV has been significantly hampered by concerns about conservation of vaccine epitopes across clades of HIV. In previous work, we have described a computer-driven method for a cross-clade HIV vaccine comprised of overlapping, highly conserved helper T-cell epitopes or “immunogenic consensus sequence epitopes” (ICS epitopes). Here, we evaluated and compared the immunogenicity of 20 ICS HIV epitopes in ELISpot assays performed using peripheral blood monocytes (PBMC) from HIV-infected donors in Providence, Rhode Island, USA and in Bamako, Mali, West Africa. Each core 9-mer HIV sequence contained in a given consensus peptide was conserved in at least 105 to as many as 2,250 individual HIV-1 strains. Nineteen of the 20 ICS epitopes (95%) were confirmed in ELISpot assays using PBMC obtained from 13 healthy, HIV-1 infected subjects in Providence, and thirteen of the epitopes (65%) were confirmed in ELISpot assays using PBMC derived from 42 discarded blood units obtained at the Central Blood Bank in Bamako. Twelve of the epitopes were confirmed in ELISpot assays performed both in Providence and Bamako. These data confirm the utility of bioinformatics tools to select and design novel vaccines containing “immunogenic consensus sequence” T-cell epitopes for a globally relevant vaccine against HIV; a similar approach may also be useful for any pathogen that exhibits high variability (influenza, HCV, or variola for example). An HIV vaccine containing these immunogenic consensus sequences is currently under development.

Novel function of complement C3d as an autologous helper T-cell target

The C3d fragment of complement component C3 has been shown to enhance immune responses to antigens that lack T‐cell epitopes such as bacterial polysaccharides. C3d binds to the B‐cell complement receptor 2 (CR2 or CD21); this binding serves as a co‐activation signal to the B cell when the polysaccharide antigen portion binds simultaneously to the B‐cell receptor (surface Ig). Bringing together receptor‐associated signal transduction molecules CD19 and Igα/β, respectively, results in a lower threshold of activation. Paradoxically, C3d has also been shown to enhance antibody titers in the CD21 knockout (KO) mouse model as well as increase Th1 and Th2 cytokine secretion, suggesting that that an auxiliary CR2‐independent pathway of immune activation may exist. We hypothesized that in addition to its molecular adjuvant property that enhances signal 1 during B‐cell activation (co‐signal 1), C3d also contains T‐cell epitopes that are able to stimulate autoreactive C3d peptide‐specific helper T cells which we term ‘co‐signal 2’. Using the EpiMatrix T‐cell epitope‐mapping algorithm, we identified 11 putative T‐cell epitopes in C3d, a very high epitope density for a 302 amino‐acid sequence. Eight of these epitope candidates were synthesized and shown to bind a variety of class II HLA‐DR molecules of different haplotypes, and to stimulate C3d peptide‐specific T cells to secrete pro‐inflammatory cytokines in vitro. Further, we demonstrate a C3d‐peptide specific increase in CD4+ intracellular IFN‐γ+ T cells in peripheral blood mononuclear cells (PBMCs) exposed to C3d peptides in vitro. We believe that the discovery of these autologous T cells autoreactive for C3d provides evidence supporting the ‘co‐signal 2’ hypothesis and may offer a novel explanation of the CD21 KO paradox.

Immunoinformatics Applied to Modifying and Improving Biological Therapeutics

Protein therapeutics have recently emerged as a viable means of treating chronic diseases and are beginning to rival small-molecule drugs in market share. Although their promise of targeted therapy is a major medical advance, repeated administrations in many cases lead to development of antitherapeutic antibodies that compromise treatment. Multiple sources of immunogenicity are considered in this chapter with a focus on the T-cell-dependent immune response. Development of high-affinity antibodies depends on activation of T helper cells by antigen presentation. Disruption of antigen presentation in an antigen-specific manner would be a rational solution to this problem. Here we present the powerful combination of recombinant protein expression and immunoinformatic and molecular modeling tools as a means of reducing immunogenicity by modification of T-cell epitopes. This approach promises to bring to the clinic safer protein therapeutics both as first- and second-generation products.