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.

Simplified fluorescent multiplex PCR method for evaluation of the T-cell receptor V beta-chain repertoire.

RATIONALE: Evaluation of the T-cell receptor (TCR) V beta-chain repertoire by PCR-based CDR3 length analysis allows fine resolution of the usage of the TCR V beta repertoire and is a sensitive tool to monitor changes in the T-cell compartment. A multiplex PCR method employing 24 labeled upstream V beta primers instead of the conventionally labeled downstream C beta primer is described. METHOD: RNA was isolated from purified CD4 and CD8 T-cell subsets from umbilical cord blood and clinical samples using TRI reagent followed by reverse transcription using a C beta primer and an Omniscript RT kit. The 24 V beta primers were multiplexed based on compatibility and product sizes into seven reactions. cDNA was amplified using 24 V beta primers (labeled with tetrachloro-6-cardoxyfluorescein, 6-carboxyfluorescein, and hexachloro-6-carboxyfluorescein), an unlabeled C beta primer, and Taqgold polymerase. The fluorescent PCR products were resolved on an automated DNA sequencer and analyzed using the Genotyper 2.1 software. RESULTS: V beta spectratypes of excellent resolution were obtained with RNA amounts of 250 ng using the labeled V beta primers. The resolution was superior to that obtained with the labeled C beta primer assay. Also the numbers of PCRs were reduced to 7 from the 12 required in the C beta labeling method, and the sample processing time was reduced by half. CONCLUSION: The method described for T-cell receptor V beta-chain repertoire analysis eliminates tedious dilutions and results in superior resolution with small amounts of RNA. The fast throughput makes this method suitable for automation and offers the feasibility to perform TCR V beta repertoire analyses in clinical trials.

CD4+ and CD8+ T cell receptor repertoire perturbations with normal levels of T cell receptor excision circles in HIV-infected, therapy-naive adolescents.

Our objective was to determine whether treatment-naive HIV-infected adolescents manifest abnormalities in thymus function and peripheral T cell repertoire, and to assess relationships of these immunologic characteristics with each other, with plasma HIV virus load, and T cell surface markers. TCR Vbeta repertoire was determined by CDR3 length spectratyping in purified CD4(+) and CD8(+) T cells of high-risk, HIV-negative adolescents and of treatment-naive, HIV-infected adolescents. Thymus function was investigated by the simultaneous examination of T cell receptor excision circles (TRECs) in the CD4(+) and CD8(+) T cell subsets. HIV-infected adolescents exhibited significantly greater perturbations in their TCR Vbeta repertoire in comparison with HIV-negative subjects. Perturbations in the CD8(+) T cell compartment were more profound in comparison with CD4(+) T cells. The CD4(+) TCR Vbeta perturbations were negatively correlated with the total and phenotypically naive CD4(+) T cells, and with CD4(+) TRECs. CD8(+) TRECs, although not correlated with CD8(+) TCR Vbeta perturbations, showed negative correlation with memory and activated CD8(+) T cells. Interestingly, TRECs in CD4(+) and CD8(+) T cells were not significantly different between HIV-infected and uninfected adolescents. The TCR Vbeta repertoire in adolescents is profoundly perturbed even in early stages of HIV infection, when total CD4(+) cell counts in most subjects are within normal limits. The correlative analyses demonstrating negative association of CD4(+) cell TRECs with CD4(+) TCR Vbeta perturbations and of CD8(+) TRECs with CD8(+) cell activation markers provide evidence of the intense activation of the central and peripheral immune compartments in this study population.

Determinants of HIV-specific CD8 T-cell responses in HIV-infected pediatric patients and enhancement of HIV-gag-specific responses with exogenous IL-15.

Cellular immune responses play a central role in controlling HIV-1 infection. HIV-specific IFN-gamma production by CD8 T cells was evaluated in 17 HLA-A2+ HIV-infected pediatric patients (age range 1 month to 16 years) in an ELISPOT assay. Most patients (15/17) exhibited responses to HIV-gag, followed by responses to envelope gp120, gp41, and V3 loop. Only 7 patients responded to all four antigenic peptides. Treatment-related immune reconstitution of CD4 T cells was associated with increase in gag-specific responses, but these declined with prolonged viral suppression. Exogenous IL-15 resulted in augmentation of HIV-gag-specific response in 71% of patients, while IL-2 and IL-7 had variable effects, augmenting responses in 25% patients. Thus, HIV-specific CD8 T-cell responses are dependent on both CD4 T-cell help and antigenic stimulation. The cytokine IL-15 may be a useful modality as adjunctive therapy to augment HIV-specific memory CD8 T cells.