C. David Pendleton

Approaches to improve engineered vaccines for human immunodeficiency virus and other viruses that cause chronic infections

Summary: We used several approaches to develop enhanced vaccines for chronic viral infections such as human immunodeficiency virus (HIV) and hepatitis C virus (HCV), I) Selected epitopes were used to avoid potentially harmful immune responses. 2) Linkage between helper and cytotoxic T‐lymphocyte (CTL) epitopes was found to be important, 3) We developed an “epitope enhancement” approach modifying the sequences of epitopes to make more potent vaccines, including examples for HIV and HCV epitopes presented by murine class II and human class I major histocompatibility complex (MHC) molecules, 4) CTL avidity was found to be important for clearing viral infections in vivo, and the mechanism was examined. High‐avidity CTLs, however, were found to undergo apoptosis when confronted with high‐density antigen, through a mechanism involving tumor necrosis factor (TNF), TNF‐RII, and a permissive state induced through the T‐cell receptor. 5) We employed cytokines in the adjuvant to steer immune responses toward desired phenotypes, and showed synergy between cytokines, 6) Intrarectal immunization with pep‐tide vaccine induced mucosal and systemic CTL, Local mucosal CTL were found to be critical for resistance to mucosal viral transmission and this resistance was enhanced with mucosally delivered interleukin‐12, 7) We used an asymmetry in induction of mucosal and systemic immune responses to circumvent pre‐existing vaccinia immunity for use of recombinant vaccinia vaccines.

Human CTLs to Wild-Type and Enhanced Epitopes of a Novel Prostate and Breast Tumor-Associated Protein, TARP, Lyse Human Breast Cancer Cells

Vaccine therapy for prostate and breast cancer may have potential for treating these major causes of death in males and females, respectively. Critical to the development of tumor-specific vaccines is finding and characterizing novel antigens to be recognized by CD8+ T cells. To define new CD8+ T-cell tumor antigens, we determined two wild-type HLA-A2 epitopes from a recently found tumor-associated protein, TARP (T-cell receptor γ alternate reading frame protein), expressed in prostate and breast cancer cells. We were also able to engineer epitope-enhanced peptides by sequence modifications. Both wild-type and enhanced epitopes induced peptide-specific CD8+ T-cell responses in A2Kb transgenic mice. In vitro restimulation of human CD8+ T cells from a prostate cancer patient resulted in CD8+ T cells reactive to the peptide epitopes that could lyse HLA-A2+ human breast cancer cells (MCF-7) expressing TARP. Epitope-specific human CD8+ T cells were also enumerated in patients’ peripheral blood by tetramer staining. Our data suggest that HLA-A2-binding TARP epitopes and enhanced epitopes discovered in this study could be incorporated into a potential vaccine for both breast and prostate cancer.

Epitope-enhanced conserved HIV-1 peptide protects HLA-A2-transgenic mice against virus expressing HIV-1 antigen.

HIV epitopes may have developed to be poor immunogens. As a counterapproach HIV vaccine strategy, we used epitope enhancement of a conserved HIV reverse transcriptase (RT) epitope for induction of antiviral protection in HLA-A2-transgenic mice mediated by human HLA-A2-restricted CTLs. We designed two epitope-enhanced peptides based on affinity for HLA-A2, one substituted in anchor residues (RT-2L9V) and the other also with tyrosine at position 1 (RT-1Y2L9V), and examined the balance between HLA binding and T cell recognition. CTL lines and bulk cultures in two HLA-A2-transgenic mouse strains showed that RT-2L9V was more effective in inducing CTL reactive with wild-type Ag than RT-1Y2L9V, despite the higher affinity of the latter, because the 1Y substitution unexpectedly altered T cell recognition. Accordingly, RT-2L9V afforded the greatest protection in vivo against a surrogate virus expressing HIV-1 RT mediated by HLA-A2-restricted CTL in a mouse in which all CTL are restricted to only the human HLA molecule. Such antiviral protection has not been previously achieved with an HLA epitope-enhanced vaccine. These findings define a critical balance between MHC affinity and receptor cross-reactivity required for effective epitope enhancement and also demonstrate construction and efficacy of such a component of a new generation vaccine.

Identification and Epitope Enhancement of a PAX-FKHR Fusion Protein Breakpoint Epitope in Alveolar Rhabdomyosarcoma Cells Created by a Tumorigenic Chromosomal Translocation Inducing CTL Capable of Lysing Human Tumors

Fusion proteins created by chromosomal translocations in tumors can create neoantigenic determinants at the breakpoint, which are unique to the tumor cells but shared by the vast majority of tumors of that histologic type. If the fusion protein is responsible for the malignant transformation, its expression cannot be lost by the tumor to escape immune responses against this tumor antigen. Here, we identify such a fusion protein breakpoint epitope in the PAX-FKHR fusion protein created by the t(2;13) translocation present in 80% of cases of alveolar rhabdomyosarcoma, a highly aggressive pediatric soft-tissue sarcoma. We use autologous dendritic cells pulsed with the RS10 breakpoint fusion peptide to raise a human CTL line from a normal healthy HLA-B7+ blood donor specific for this peptide. These CTLs are CD8+ (CD4-CD56-) and restricted by HLA-B7. These human peptide-specific CTL lyse human HLA-B7+ rhabdomyosarcoma tumor cells. Therefore, the fusion protein is endogenously processed to produce this natural epitope presented by HLA-B7 and thus this peptide is a bone fide human tumor antigen. We also define a substitution that increases the affinity for HLA-B7 without loss of antigenicity. This epitope-enhanced peptide may serve as a candidate cancer vaccine for HLA-B7+ patients with alveolar rhabdomyosarcoma.

Possible Therapeutic Vaccine Strategy against Human Immunodeficiency Virus Escape from Reverse Transcriptase Inhibitors Studied in HLA-A2 Transgenic Mice

ABSTRACT Mutation of human immunodeficiency virus (HIV) leading to escape from anti-HIV drugs is the greatest challenge to the treatment of HIV infection. High-grade resistance to the nucleoside reverse transcriptase (RT) inhibitor lamivudine (also known as 3TC) is associated with a substitution of valine for methionine at position 184 of RT. This amino acid residue is contained within the HLA-A2-restricted epitope VIYQYMDDL (RT-WT). Here, we sought to determine whether a peptide vaccine could be developed using an epitope enhancement strategy that could induce a cytotoxic T-lymphocyte (CTL) response specific for an epitope containing the drug resistance mutation M184V to exert an opposing selective pressure. RT-WT-specific CTLs developed from HLA-A2 transgenic mice did not recognize the M184V mutation of RT-WT (RT-M184V). However, RT-M184V exhibited higher binding affinity for HLA-A2 than RT-WT. Also, both anchor-enhanced RT-WT (RT-2L9V) and RT-2L9V-M184V-specific CTLs recognized RT-M184V and displayed cross-reactivity to RT-WT. Nevertheless, the CTL repertoire elicited by the epitope-enhanced RT-2L9V-M184V appeared more selective for the RT inhibitor-induced M184V mutation. Peptide vaccines based on such strategies may be worth testing for their ability to exert selective pressure against drug-resistant strains and thus delay or prevent the development of HIV with the M184V resistance mutation.

Epitope Enhancement of a CD4 HIV Epitope toward the Development of the Next Generation HIV Vaccine

Virus-specific CD4+ T cell help and CD8+ cytotoxic T cell responses are critical for maintenance of effective immunity in chronic viral infections. The importance of CD4+ T cells has been documented in HIV infection. To investigate whether a stronger CD4+ T cell response can be induced by modifications to enhance the T1 epitope, the first CD4+ T cell epitope discovered in HIV-1-gp120, we developed a T1-specific CD4+ T cell line from a healthy volunteer immunized with a canarypox vector expressing gp120 and boosted with recombinant gp120. This T1-specific CD4+ T cell line was restricted to DR13, which is common in U.S. Caucasians and African-Americans and very frequent in Africans. Peptides with certain amino acid substitutions in key positions induced enhanced specific CD4+ T cell proliferative responses at lower peptide concentration than the original epitope. This relatively conserved CD4 epitope improved by the epitope enhancement strategy could be a component of a more effective second generation vaccine construct for HIV infection.

Identification and Enhancement of HLA-A2.1-Restricted CTL Epitopes in a New Human Cancer Antigen-POTE

Identification of CD8+ T cell epitopes that can induce T cells to kill tumor cells is a fundamental step for development of a peptide cancer vaccine. POTE protein is a newly identified cancer antigen that was found to be expressed in a wide variety of human cancers, including prostate, colon, lung, breast, ovary and pancreas. Here, we determined HLA-A2.1-restricted cytotoxic T lymphocyte (CTL) epitopes in the POTE protein, and also designed enhanced epitopes by amino acid (AA) substitutions. Five 9-mer peptides were first selected and their binding affinity to HLA-A2 molecules was measured by the T2 binding assay. POTE 272–280 and POTE 323–331 showed the strongest HLA-A2 binding affinity. AA substituted peptides POTE 252-9V (with valine at position 9), POTE 553-1Y (with tyrosine at position 1) and POTE 323-3F (with phenylalanine at position 3) conferred higher affinity for HLA-A2, and induced CTL responses cross-reactive with wild type antigens. While POTE 252-9V was the strongest in this respect, POTE 323-3F had the greatest increase in immunogenicity compared to wild type. Importantly, two modified epitopes (POTE-553-1Y and POTE-323-3F) induced CTLs that killed NCI-H522, a POTE-expressing HLA-A2+ human non-small cell lung cancer cell line, indicating natural endogenous processing of these epitopes. In conclusion, the immunogenicity of POTE epitopes can be enhanced by peptide modification to induce T cells that kill human cancer cells. A combination of POTE 553-1Y and POTE 323-3F epitopes might be an attractive vaccine strategy for HLA-A2 cancer patients to overcome tolerance induced by tumors and prevent escape.

Characterization of a helper T cell epitope recognized by mice of a low responder major histocompatibility type

Most known helper T cell (Th) epitopes studied have naturally been immunodominant epitopes recognized by T cells from animals of high responder major histocompatibility complex (MHC) haplotype. We have previously found that most such immunodominant Th epitopes tend to be amphipathic alpha helices, that is, helices with hydrophobic residues on one side and hydrophilic residues on the other, and the corresponding peptide can usually elicit a response to the native protein. However, very few epitopes seen by MHC low responder T cells have been identified. Within the CNBr fragment of residues 1-55 of sperm whale myoglobin (SwMb), a Th epitope is known to exist that stimulates T cells from low responder H-2k mice, but it has not yet been localized to a length of 8-12 residues, the usual length of a Th epitope. To determine whether this low responder epitope would have similar properties, we located it using 10 evenly overlapping 15-residue peptides that span the region. Analysis of this region by the computer program predicted the site covered by two peptides (residues 26-40 and 31-45 which overlap by 10 residues) to be the most likely site for a Th epitope. Of the 10 peptides tested experimentally, only one peptide (residues 26-40) was able to stimulate two low responder Th clones that are specific for the 1-55 region. The peptide was able to prime T cells of low responder B10.BR mice in vivo for in vitro response to the native SwMb as well as to the peptide fragment of residues 1-55. Immunization of low responder mice with SwMb showed that, of the 10 overlapping peptides, the major site of response within the 1-55 region is to the identified peptide. Finally, an extended peptide of residues 24-42 was made to increase the amphipathic score. This extended peptide induced greater proliferation of the clones. Thus, this low responder epitope has properties similar to those of immunodominant epitopes recognized by high responders.