γδ T cells are implicated to play crucial roles during early immune responses to pathogens. A subset of human γδ T cells carrying the Vγ9Vδ2 TCR recognize small, phosphorylated nonpeptidic Ags. However, the precise role of these cells and the ligands recognized in human immune responses against pathogens remains unclear because of the lack of suitable animal models. We have analyzed the reactivity of spleen cells of the New World monkey Aotus nancymaae against isopentenyl pyrophosphate (IPP), a phosphorylated microbial metabolite selectively activating Vγ9Vδ2 T cells. Spleen cells were stimulated by IPP and the expanding cell population expressed the Vγ9 TCR. TRGV-J and TRDV-D-J rearrangements expressed by IPP-stimulated cells of Aotus were analyzed by RT-PCR and DNA sequencing. The TRGV-J and TRDV-D-J rearrangements expressed by IPP-stimulated Aotus and human γδ T cells were similar with respect to 1) TCR gene segment usage, 2) a high degree of germline sequence homology of the TCR gene segments used, and 3) the diversity of the CDR3 regions. Phylogenetic analysis of human, Pan troglodytes, and A. nancymaae TRGV gene segments showed that the interspecies differences are smaller than the intraspecies differences with TRGV9 gene segments located on a distinct clade of the phylogenetic tree. The structural and functional conservation of Vγ9Vδ2 T cells in A. nancymaae and humans implicates a functionally important and evolutionary conserved mechanism of recognition of phosphorylated microbial metabolites.

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The New World primate Aotus nancymaae is susceptible to infection with the human malaria parasite Plasmodium falciparum and has therefore been recommended by the World Health Organization as a model for the evaluation of malaria vaccine candidates. Recently, we have shown that Aotus TCRVA genes and TCRJA segments exhibit a high degree of similarity to human counterparts. In the present report we used reverse transcription polymerase chain reaction to analyze the sequences of A. nancymaae TCR beta-chain gene rearrangements. Alignment with human sequences and phylogenetic comparison identified 18 distinct Aotus TCRBV genes homologous to the human TCRBV gene families 2, 4, 5, 6, 7, 9, 12, 15, 24, and 28. Multiple Aotus genes were found in the TCRBV4, 5, 6, and 7 families. Some of these TCRBV genes aligned best to the same human gene and thus do not seem to have separate human homologues. Amino acid sequences of the Aotus TCRBV genes were 77 to 90% identical to their closest human counterparts. Ten distinct Aotus TCRBJ segments homologous to the human segments J1-1, J1-2, J1-4, J1-5, J1-6, J2-1, J2-2, J2-3, J2-4, J2-5 were found. In some cases the amino acid sequences of Aotus and human TCRBJ segments were completely identical. A comparison of the proportion of synonymous and non-synonymous substitutions in Aotus vs human beta-chain-encoding genes revealed a dominance of synonymous substitutions in TCRBJ segments and of nonsynonymous substitutions in TCRBV segments. Dominance of nonsynonymous substitutions was more pronounced in TCRBV CDR1 and CDR2 regions than in the framework regions. No evidence for the emergence of new TCRBJ segments or TCRBV families was found. These results confirm that the TCR repertoire in primates is remarkably stable and support the concept of using Aotus monkeys as an infection model for the evaluation of future subunit vaccine candidates.

Mycobacteria target and persist within phagocytic monocytes and are strong adjuvants, making them attractive candidate vectors for DNA vaccines. We characterized the ability of mycobacteria to deliver transgenes to mammalian cells and the effects of various bacterial chromosomal mutations on the efficiency of transfer in vivo and in vitro. First, we observed green fluorescent protein expression via microscopy and fluorescence-activated cell sorting analysis after infection of phagocytic and nonphagocytic cell lines by Mycobacterium smegmatis or M. bovis BCG harboring a plasmid encoding the fluorescence gene under the control of a eukaryotic promoter. Next, we compared the efficiencies of gene transfer using M. smegmatis or BCG containing chromosomal insertions or deletions that cause early lysis, hyperconjugation, or an increased plasmid copy number. We observed a significant-albeit only 1.7-fold-increase in the level of plasmid transfer to eukaryotic cells infected with M. smegmatis hyperconjugation mutants. M. smegmatis strains that overexpressed replication proteins (Rep) of pAL5000, a plasmid whose replicon is incorporated in many mycobacterial constructs, generated a 10-fold increase in plasmid copy number and 3.5-fold and 3-fold increases in gene transfer efficiency to HeLa cells and J774 cells, respectively. Although BCG strains overexpressing Rep could not be recovered, BCG harboring a plasmid with a copy-up mutation in oriM resulted in a threefold increase in gene transfer to J774 cells. Moreover, M. smegmatis strains overexpressing Rep enhanced gene transfer in vivo compared with a wild-type control. Immunization of mice with mycobacteria harboring a plasmid (pgp120 h E ) encoding human immunodeficiency virus gp120 elicited gp120-specific CD8 T-cell responses among splenocytes and peripheral blood mononuclear cells that were up to twofold (P < 0.05) and threefold (P < 0.001) higher, respectively, in strains supporting higher copy numbers. The magnitude of these responses was approximately one-half of that observed after intramuscular immunization with pgp120 h E . M. smegmatis and other nonpathogenic mycobacteria are promising candidate vectors for DNA vaccine delivery.Injection of plasmid DNA vaccines encoding protective antigens under the control of a eukaryotic promoter induces protective T-and B-cell responses in mice (43, 45) and subhuman primates (32) and is being studied in phase I trials in humans (42). Despite the promise of DNA vaccination, the widespread use of DNA as an inexpensive and effective immunogen in humans may be limited by requirements for large inocula of highly purified DNA and/or the coadministration of expensive adjuvants. In an attempt to improve the efficiency of DNA vaccination, bacteria have recently been studied as gene delivery vectors through a process called "bactofection" (29), in which bacteria harboring antigen-encoding plasmids enter a mammalian cell and release the plasmids for uptake into the nucleus. Consequently, plasmid-encoded genes are expressed endogenously...