T-lymphocyte epitope identification and their use in vaccine development for HIV-1.

Cellular immune responses mediated by CD8+ cytotoxic T-lymphocytes (CTL) and CD4+ helper T-lymphocytes (HTL) are needed to effectively control and clear many viral pathogens, including HIV-1. Thus, vaccines for HIV-1 capable of inducing CTL and HTL responses are now the focus of multiple academic and industry-based research and development programs. The use of defined, minimal CTL and HTL epitopes in vaccines has several potential advantages. Firstly, it is possible to use epitopes that are conserved thus targeting the majority of viral variants within a given clade or across clades. Secondly, epitopes from multiple viral structural or accessory gene products can be included in vaccines, which supports the induction cellular immune responses with significant breadth. Finally, dominance relationships between epitopes can be altered to increase immune recognition of subdominant epitopes. HTL and CTL epitopes from HIV-1 have recently been identified and characterized in numbers that are large enough to support their use in experimental vaccines. Initial studies with prototype DNA vaccines encoding epitopes indicate the need to include intracellular targeting sequences, to direct the encoded gene products to different cellular compartments, and amino acid spacer sequences between epitopes to optimize the processing, and subsequent presentation, of individual epitopes. Vaccines composed of CTL or HTL epitopes are now being developed for clinical testing.

Triaryl-substituted Schiff bases are high-affinity subtype-selective ligands for the estrogen receptor.

We have explored the isoelectronic replacement of the C═C double bond found at the core of many nonsteroidal estrogen ligands with a simple Schiff base (C═N). Di- and triaryl-substituted imine derivatives were conveniently prepared by the condensation of benzophenones with various anilines without the need for phenolic hydroxy protection. Most of these imines demonstrated high affinity for the estrogen receptors, which, in some cases exceeded that of estradiol. In cell-based assays, these imines profiled as ERα agonists but as ERß antagonists, showing preferential reliance on the N-terminal activation function (AF1), which is more active in ERα. X-ray analysis revealed that the triaryl-imines distort the ligand-binding pocket in a new way: by controlling the separation of helices 3 and 11, which appears to alter the C-terminal AF2 surface that binds transcriptional coactivators. This work suggests that C═N for C═C substitution might be more widely considered as a general strategy for preparing drug analogues.

Recognition of variant HIV-1 epitopes from diverse viral subtypes by vaccine-induced CTL.

Recognition by CD8(+) T lymphocytes (CTL) of epitopes that are derived from conserved gene products, such as Gag and Pol, is well documented and conceptually supports the development of epitope-based vaccines for use against diverse HIV-1 subtypes. However, many CTL epitopes from highly conserved regions within the HIV-1 genome are highly variable, when assessed by comparison of amino acid sequences. The TCR is somewhat promiscuous with respect to peptide binding, and, as such, CTL can often recognize related epitopes. In these studies, we evaluated CTL recognition of five sets of variant HIV-1 epitopes restricted to HLA-A*0201 and HLA-A*1101 using HLA transgenic mice. We found that numerous different amino acid substitutions can be introduced into epitopes without abrogating their recognition by CTL. Based on our findings, we constructed an algorithm to predict those CTL epitopes capable of inducing responses in the HLA transgenic mice to the greatest numbers of variant epitopes. Similarity of CTL specificity for variant epitopes was demonstrated for humans using PBMC from HIV-1-infected individuals and CTL lines produced in vitro using PBMC from HIV-1-uninfected donors. We believe the ability to predict CTL epitope immunogenicity and recognition patterns of variant epitopes can be useful for designing vaccines against multiple subtypes and circulating recombinant forms of HIV-1.

A decaepitope polypeptide primes for multiple CD8+ IFN-gamma and Th lymphocyte responses: evaluation of multiepitope polypeptides as a mode for vaccine delivery.

Proteins are generally regarded as ineffective immunogens for CTL responses. We synthesized a 100-mer decaepitope polypeptide and tested its capacity to induce multiple CD8(+) IFN-gamma and Th lymphocyte (HTL) responses in HLA transgenic mice. Following a single immunization in the absence of adjuvant, significant IFN-gamma in vitro recall responses were detected for all epitopes included in the construct (six A2.1-, three A11-restricted CTL epitopes, and one universal HTL epitope). Immunization with truncated forms of the decaepitope polypeptide was used to demonstrate that optimal immunogenicity was associated with a size of at least 30-40 residues (3-4 epitopes). Solubility analyses of the truncated constructs were used to identify a correlation between immunogenicity for IFN-gamma responses and the propensity of these constructs to form particulate aggregates. Although the decaepitope polypeptide and a pool of epitopes emulsified in IFA elicited similar levels of CD8(+) responses using fresh splenocytes, we found that the decaepitope polypeptide more effectively primed for in vitro recall CD8(+) T cell responses. Finally, immunogenicity comparisons were also made between the decaepitope polypeptide and a corresponding gene encoding the same polypeptide delivered by naked DNA immunization. Although naked DNA immunization induced somewhat greater direct ex vivo and in vitro recall responses 2 wk after a single immunization, only the polypeptide induced significant in vitro recall responses 6 wk following the priming immunization. These studies support further evaluation of multiepitope polypeptide vaccines for induction of CD8(+) IFN-gamma and HTL responses.