Claire Crimi

Optimization of epitope processing enhances immunogenicity of multiepitope DNA vaccines

Experimental DNA vaccines comprised of multiple minimal cytotoxic T lymphocytes (CTL) epitopes can effectively induce broad CTL responses; however, such constructs frequently exhibit significant variation in epitope immunogenicity. Antigenicity assays utilizing human cells transfected with one such multiepitope construct revealed that the epitopes with poor immunogenicity were inefficiently processed in transfected cells. Compilation of a database of 94 epitope/flanking region combinations, for which immunogenicity was measured experimentally, revealed that the type of residue immediately following the carboxyl-terminus of the epitope exerted a prominent effect on immunogenicity. Experiments utilizing a variety of HBV-specific vaccine constructs demonstrated epitope immunogenicity could be modulated by the insertion of a single amino acid and the effect on immunogenicity could be ascribed to modulation of processing efficiency. These findings demonstrate that multiepitope DNA vaccines can be engineered to enhance CTL immunogenicity by increasing processing efficiency.

A Rational Strategy to Design Multiepitope Immunogens Based on Multiple Th Lymphocyte Epitopes

Four HLA-DR-restricted HIV-derived Th lymphocyte (HTL) epitopes cross-reactive with the murine I-Ab class II molecule were used to evaluate different vaccine design strategies to simultaneously induce multiple HTL responses. All four epitopes were immunogenic in H-2b mice, demonstrating the feasibility of murine models to evaluate epitope-based vaccines destined for human use. Immunization with a pool of peptides induced responses against all four epitopes; illustrating immunodominance does not prevent the induction of balanced multispecific responses. When different delivery systems were evaluated, a multiple Ag peptide construct was found to be less efficient than a linear polypeptide encompassing all four epitopes. Further characterization of linear polypeptide revealed that the sequential arrangement of the epitopes created a junctional epitope with high affinity class II binding. Disruption of this junctional epitope through the introduction of a GPGPG spacer restored the immunogenicity against all four epitopes. Finally, we demonstrate that a GPGPG spacer construct can be used to induce HTL responses by either polypeptide or DNA immunization, highlighting the flexibility of the approach.

Development of a DNA vaccine designed to induce cytotoxic T lymphocyte responses to multiple conserved epitopes in HIV-1.

Epitope-based vaccines designed to induce CTL responses specific for HIV-1 are being developed as a means for addressing vaccine potency and viral heterogeneity. We identified a set of 21 HLA-A2, HLA-A3, and HLA-B7 restricted supertype epitopes from conserved regions of HIV-1 to develop such a vaccine. Based on peptide-binding studies and phenotypic frequencies of HLA-A2, HLA-A3, and HLA-B7 allelic variants, these epitopes are predicted to be immunogenic in greater than 85% of individuals. Immunological recognition of all but one of the vaccine candidate epitopes was demonstrated by IFN-γ ELISPOT assays in PBMC from HIV-1-infected subjects. The HLA supertypes of the subjects was a very strong predictor of epitope-specific responses, but some subjects responded to epitopes outside of the predicted HLA type. A DNA plasmid vaccine, EP HIV-1090, was designed to express the 21 CTL epitopes as a single Ag and tested for immunogenicity using HLA transgenic mice. Immunization of HLA transgenic mice with this vaccine was sufficient to induce CTL responses to multiple HIV-1 epitopes, comparable in magnitude to those induced by immunization with peptides. The CTL induced by the vaccine recognized target cells pulsed with peptide or cells transfected with HIV-1 env or gag genes. There was no indication of immunodominance, as the vaccine induced CTL responses specific for multiple epitopes in individual mice. These data indicate that the EP HIV-1090 DNA vaccine may be suitable for inducing relevant HIV-1-specific CTL responses in humans.

Rational Design of a Multiepitope Vaccine Encoding T-Lymphocyte Epitopes for Treatment of Chronic Hepatitis B Virus Infections

ABSTRACT Protein sequences from multiple hepatitis B virus (HBV) isolates were analyzed for the presence of amino acid motifs characteristic of cytotoxic T-lymphocyte (CTL) and helper T-lymphocyte (HTL) epitopes with the goal of identifying conserved epitopes suitable for use in a therapeutic vaccine. Specifically, sequences bearing HLA-A1, -A2, -A3, -A24, -B7, and -DR supertype binding motifs were identified, synthesized as peptides, and tested for binding to soluble HLA. The immunogenicity of peptides that bound with moderate to high affinity subsequently was assessed using HLA transgenic mice (CTL) and HLA cross-reacting H-2bxd (BALB/c × C57BL/6J) mice (HTL). Through this process, 30 CTL and 16 HTL epitopes were selected as a set that would be the most useful for vaccine design, based on epitope conservation among HBV sequences and HLA-based predicted population coverage in diverse ethnic groups. A plasmid DNA-based vaccine encoding the epitopes as a single gene product, with each epitope separated by spacer residues to enhance appropriate epitope processing, was designed. Immunogenicity testing in mice demonstrated the induction of multiple CTL and HTL responses. Furthermore, as a complementary approach, mass spectrometry allowed the identification of correctly processed and major histocompatibility complex-presented epitopes from human cells transfected with the DNA plasmid. A heterologous prime-boost immunization with the plasmid DNA and a recombinant MVA gave further enhancement of the immune responses. Thus, a multiepitope therapeutic vaccine candidate capable of stimulating those cellular immune responses thought to be essential for controlling and clearing HBV infection was successfully designed and evaluated in vitro and in HLA transgenic mice.

In vitro induction of primary, antigen-specific CTL from human peripheral blood mononuclear cells stimulated with synthetic peptides

A protocol for in vitro induction of primary, antigen-specific CTL from human peripheral blood mononuclear cells (PBMCs) was developed. Antigen presenting cells (APCs) consisted of Staphylococcus aureus Cowan-I (SAC-I) activated PBMCs treated with a citrate-phosphate buffer at pH 3 to release endogenous peptides bound to surface MHC. This treatment resulted in transient expression of empty class I molecules which could be subsequently stabilized with peptide and beta 2-microglobulin (beta 2m). SAC-I activated PBMCs from HLA-A2.1 normal donors loaded with HBV core 18-27 peptide following acid treatment were used to stimulate PBMCs depleted of CD4+ T cells, in the presence of recombinant interleukin-7 (rIL-7). After 12 days, cells were restimulated with autologous, peptide-pulsed, adherent cells and tested for CTL activity 7 days later. In 23 independent experiments from 13 different HLA-A2.1 donors, this protocol resulted in induction of primary CTL more than 90% of the time. As indicated by both the frequency and magnitude of the response against peptide-sensitized target cells, SAC-I activated PBMCs treated with acid were the most efficient stimulator APC. Thirteen per cent of the cultures generated were capable of lysing target cells transfected with the HBV core antigen and, in general, these CTL cultures exhibited high avidity for the HBV core peptide. This protocol is generally applicable to different antigens and class I alleles, and thus, may be utilized to screen large numbers of peptides to identify human CTL epitopes.

Immunization with the HBV core 18-27 epitope elicits CTL responses in humans expressing different HLA-A2 supertype molecules.

The human leukocyte antigen (HLA)-A2 restricted HBV core 18-27 epitope is immunodominant in the context of HLA-A2.1 and subdominant in the context of the other HLA-A2 supertype molecules, as defined by frequency of recognition by memory cytotoxic T lymphocyte (CTL) responses from acute hepatitis B virus (HBV) patients, and on the basis of its binding affinity to purified HLA molecules in vitro. Herein, we show that immunization with a lipopeptide containing HBV core 18-27 epitope induces CTL responses in patients expressing different HLA-A2 supertype molecules, with indistinguishable frequency and magnitude. No difference in responses was noted between patients expressing either one or two different HLA-A2 supertype molecules. Thus, complexes of HBV core 18-27 bound to different HLA-A2 supertype alleles do not appear to act as altered peptide ligands, and do not cross antagonize CTL responses. These results substantiate the immunological relevance of the HLA supertypes concept, and illustrate its potential usefulness for the development of vaccines.

Universal influenza DNA vaccine encoding conserved CD4+ T cell epitopes protects against lethal viral challenge in HLA-DR transgenic mice.

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.

Class I molecules with similar peptide-binding specificities are the result of both common ancestry and convergent evolution

Abstract.HLA class I molecules can be classified into supertypes associated with overlapping peptide-binding motifs and repertoires. Herein, overlaps in peptide-binding and T-cell recognition repertoires were demonstrated between mouse and human molecules. Since rodent and primate lineages separated before the current allelic variation of mouse and human class I molecules, these data demonstrate that supertypic specificities originated by convergent evolution. Phylogenetic and structural analyses demonstrated that convergent evolution also occurs amongst primates and within the human species, resulting from the selection of different pocket structures having similar specificity or independent repeated selection of the same pocket structure.

Towards prediction of degenerate CTL epitope recognition

The cellular immune system is characterized by flexibility with respect to epitope recognition at the level of peptide binding to HLA molecules and HLA-peptide complexes to T-cell receptors (TCRs). For epitopes recognized by cytotoxic T-lymphocytes (CTLs), amino acid substitutions at different positions have varying impact on recognition. By analyzing the frequencies of specific amino acid substitutions at each position in conjunction with HLA-peptide binding and immune-response data, we have developed new methods to predict cross-reactive recognition of epitope variants by CTLs. We derived position-specific substitution matrices (EPSSMs) through the analysis of known HLA ligands and achieved relatively accurate prediction of detrimental and tolerated amino acid substitutions. Initial analysis of amino acid substitutions in CTL epitopes with degenerate recognition showed strong position-specific preferences. This first systematic analysis further suggested that spatial constraint may be the major molecular factor determining the degenerate epitope recognition. As the data cumulates, we anticipate that eventually EPSSMs will be available for prediction of degenerate T-cell epitope recognition.