Human immunodeficiency virus‐1 reverse transcriptase immunodominant CD4⁺ T cell epitopes: A peptide‐based multiparametric assessment in the mouse

Abstract

We previously identified an immunodominant CD4+ T cell determinant in the carboxy-terminal region of HIV-1 reverse transcriptase (RT528-543). The present study aimed at enumerating all the potential sites of HIV-1 RT recognized by Th cells in the BALB/c (H-2d) mouse model. To achieve this we used a panel of 62 overlapping 15-mer synthetic peptides covering the whole RT sequence to assay the following parameters: (i) immunogenicity in naive BALB/c mice injected either with peptides pools or individual peptides; (ii) antigenicity, as detected by their ability to restimulate in vitro T cells from BALB/c mice primed with native RT; (iii) MHC class II (Ad)-binding capacity as measured by the inhibition of the antigen-specific, Ad-restricted presentation of unfolded apamin (4-Acm) by fixed antigen-presenting cells to Ad/4-Acm-specific, interleukin-2-producing T hybridoma cells; and (iv) the presence of typical or degenerate consensus Ad-binding motifs. The results in this study permitted identification of three novel immunodominant RT mouse CD4+ T cell sites (RT276-290, RT375-389 and RT411-425) located in regions of limited polymorphism among RT from several HIV isolates. Some of these RT segments were found to be in the vicinity of B cell or H-2Kk- or HLA-A2-restricted cytotoxic T lymphocyte epitopes. Finally, the approach used in this study was found to be very efficient for enumerating most T cell recognition sites in a complex protein, a result that would have not been achieved by a single parameter-based analysis.

Cellular Immunogenicity of Novel Gene Immunogens in Mice Monitored by in Vivo Imaging

“…Tests assessed IFN-c, IL-2, and IL-4 production in response to stimulation with recombinant RT and peptides representing amino acids 375-389 and 528-543 of RT, both characterized as promiscuous T-cell epitopes in BALB/c mice. [50][51][52] In the RT-immunized mice, the number of IL-2-secreting splenocytes did not differ from that in the vector-immunized group. Low levels of IL-2 secretion were detected in the RT-Ii and RT-Cgar gene recipients.…”

“…Regions between RT amino acids 375 to 389 and 528 to 543 contain known T-helper cell epitopes in mice and CTL epitopes in humans. [50][51][52] The pattern of cytokine secretion in response to these peptides would thus reflect the mode of action of each of the RT gene chimeras. A weak to moderate antipeptide IFN-c response was detected in all groups.…”

Fusion to the Lysosome Targeting Signal of the Invariant Chain Alters the Processing and Enhances the Immunogenicity of HIV-1 Reverse Transcriptase

“…The control mice received either a plasmid encoding the parental RT gene, or an empty vector. The immune response was assessed by the capacity of murine splenocytes to produce IFN-γ and IL-2 after in vitro stimulation with the protein RT or RT -derived peptide representing its immunodominant epitopes at amino acid residues 375–389 and 528–543 [31, 32]. IFN-γ, IL-2, or IFN-γ / IL-2 producing cells were identified with the Fluorospot assay ( Fig.…”

Lack of patent liver autoimmunity after breakage of tolerance in a mouse model

“…pep23 (KDSWTVNDIQKLVGK), corresponding to the amino acid sequence 248±262 of the HIV-1 reverse transcriptase. This peptide carries a T h epitope recognized in a MHC A d -restricted fashion in BALB/c mice (17). A double-stranded oligonucleotide codifying the pep23 was cloned in-frame upstream or downstream to the mGST cDNA in pTRC99a as described in Methods.…”