Engineered T-cell receptor tetramers bind MHC-peptide complexes with high affinity.

In this study we extend tetramerization technology to T-cell receptors (TCRs). We identified TCR alpha beta pairs in the absence of accessory molecules, ensuring isolation of high-affinity TCRs that maintain stable binding characteristics after tetramerization. Subtle changes in cognate peptide levels bound to the class I molecule were accurately reflected by parallel changes in the mean fluorescence intensity of cells that bound TCR tetramers, allowing us to accurately assess the binding affinity of a panel of peptides to major histocompatibility complex (MHC) class I. Using a TCR tetramer specific for the Mamu-A(*)01 allele, we identified animals expressing this restricting class I allele from a large cohort of outbred rhesus macaques. TCR tetramers should facilitate analysis of the MHC-peptide interface and, more generally, the design of immunotherapeutics and vaccines.

Multiclade human immunodeficiency virus type 1 envelope immunogens elicit broad cellular and humoral immunity in rhesus monkeys.

The development of a human immunodeficiency virus type 1 (HIV-1) vaccine that elicits potent cellular and humoral immune responses recognizing divergent strains of HIV-1 will be critical for combating the global AIDS epidemic. The present studies were initiated to examine the magnitude and breadth of envelope (Env)-specific T-lymphocyte and antibody responses generated by vaccines containing either a single or multiple genetically distant HIV-1 Env immunogens. Rhesus monkeys were immunized with DNA prime-recombinant adenovirus boost vaccines encoding a Gag-Pol-Nef polyprotein in combination with either a single Env or a mixture of clade-A, clade-B, and clade-C Envs. Monkeys receiving the multiclade Env immunization developed robust immune responses to all vaccine antigens and, importantly, a greater breadth of Env recognition than monkeys immunized with vaccines including a single Env immunogen. All groups of vaccinated monkeys demonstrated equivalent immune protection following challenge with the pathogenic simian-human immunodeficiency virus 89.6P. These data suggest that a multicomponent vaccine encoding Env proteins from multiple clades of HIV-1 can generate broad Env-specific T-lymphocyte and antibody responses without antigenic interference. This study demonstrates that it is possible to generate protective immune responses by vaccination with genetically diverse isolates of HIV-1.

Evaluation of CD62L expression as a marker for vaccine-elicited memory cytotoxic T lymphocytes.

The development of successful vaccination strategies for eliciting cytotoxic T lymphocytes (CTLs) will be facilitated by the definition of strategies for subdividing CTLs into functionally distinct subpopulations. We assessed whether surface expression of a number of cell-surface proteins could be used to define functionally distinct subpopulations of memory CTLs in mice immunized with a recombinant vaccinia virus expressing human immunodeficiency virus (HIV)-1 envelope (Env). We found changes in cell-surface expression of CD11a, CD44, CD45RB, CD49d, CD54 and CD62L on Env-specific CD8(+) T cells that appeared to differentiate them from other CD8(+) T cells within 1 week to 1 month following immunization. Further, we saw an up-regulation of CD62L surface expression on Env-specific CD8(+) memory T cells several months after immunization. However, CD62L expression did not correlate with differences in the abilities of CTLs to proliferate or produce interferon gamma (IFN-gamma) and tumour necrosis factor alpha (TNF-alpha) in vitro in response to Env peptide stimulation. Moreover, the expression of CD62L did not allow differentiation of CTLs into subpopulations with distinct expansion kinetics in vivo after adoptive transfer into naïve mice and subsequent boosting of these mice with a recombinant adenovirus expressing HIV-1 Env. Therefore, the definition of memory CD8(+) T-cell subpopulations on the basis of CD62L expression in mice does not allow the delineation of functionally distinct CTL subpopulations.

Magnitude and diversity of cytotoxic-T-lymphocyte responses elicited by multiepitope DNA vaccination in rhesus monkeys.

In an effort to develop an AIDS vaccine that elicits high-frequency cytotoxic-T-lymphocyte (CTL) responses with specificity for a diversity of viral epitopes, we explored two prototype multiepitope plasmid DNA vaccines in the simian-human immunodeficiency virus/rhesus monkey model to determine their efficiency in priming for such immune responses. While a simple multiepitope vaccine construct demonstrated limited immunogenicity in monkeys, this same multiepitope genetic sequence inserted into an immunogenic simian immunodeficiency virus gag DNA vaccine elicited high-frequency CTL responses specific for all of the epitopes included in the vaccine. Both multiepitope vaccine prototypes primed for robust epitope-specific CTL responses that developed following boosting with recombinant modified vaccinia virus Ankara vaccines expressing complete viral proteins. The natural hierarchy of immunodominance for these epitopes was clearly evident in the boosted monkeys. These studies suggest that multiepitope plasmid DNA vaccine-based prime-boost regimens can efficiently prime for CTL responses of increased breadth and magnitude, although they do not overcome predicted hierarchies of immunodominance.

Heterologous envelope immunogens contribute to AIDS vaccine protection in rhesus monkeys.

Because a strategy to elicit broadly neutralizing anti-human immunodeficiency virus type 1 (HIV-1) antibodies has not yet been found, the role of an Env immunogen in HIV-1 vaccine candidates remains undefined. We sought to determine whether an HIV-1 Env immunogen genetically disparate from the Env of the challenge virus can contribute to protective immunity. We vaccinated Indian-origin rhesus monkeys with Gag-Pol-Nef immunogens, alone or in combination with Env immunogens that were either matched or mismatched with the challenge virus. These animals were then challenged with a pathogenic simian-human immunodeficiency virus. The vaccine regimen included a plasmid DNA prime and replication-defective adenoviral vector boost. Vaccine regimens that included the matched or mismatched Env immunogens conferred better protection against CD4(+) T-lymphocyte loss than that seen with comparable regimens that did not include Env immunogens. This increment in protective immunity was associated with anamnestic Env-specific cellular immunity that developed in the early days following viral challenge. These data suggest that T-lymphocyte immunity to Env can broaden the protective cellular immune response to HIV despite significant sequence diversity of the strains of the Env immunogens and can contribute to immune protection in this AIDS vaccine model.