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.

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An apparently rare Neisseria meningitidis isolate containing one copy of a Neisseria gonorrhoeae 16S rRNA gene is described herein. This isolate was identified as N. meningitidis by biochemical identification methods but generated a positive signal with Gen-Probe Aptima assays for the detection of Neisseria gonorrhoeae. Direct 16S rRNA gene sequencing of the purified isolate revealed mixed bases in signature regions that allow for discrimination between N. meningitidis and N. gonorrhoeae. The mixed bases were resolved by sequencing individually PCR-amplified single copies of the genomic 16S rRNA gene. A total of 121 discrete sequences were obtained; 92 (76%) were N. meningitidis sequences, and 29 (24%) were N. gonorrhoeae sequences. Based on the ratio of species-specific sequences, the N. meningitidis strain seems to have replaced one of its four intrinsic 16S rRNA genes with the gonococcal gene. Fluorescence in situ hybridization (FISH) probes specific for meningococcal and gonococcal rRNA were used to demonstrate the expression of the rRNA genes. Interestingly, the clinical isolate described here expresses both N. meningitidis and N. gonorrhoeae 16S rRNA genes, as shown by positive FISH signals with both probes. This explains why the probes for N. gonorrhoeae in the Gen-Probe Aptima assays cross-react with this N. meningitidis isolate. The N. meningitidis isolate described must have obtained N. gonorrhoeae-specific DNA through interspecies recombination.

This study evaluated the performance characteristics of a new research use transcription-mediated amplification (TMA) assay for detection of ribosomal RNA from Treponema pallidum (Tp). Analytical sensitivity determined using dark field quantitated Tp was 1.4 organisms/ml (95% CI: 0.7-6.33). Dilution of Tp IVT RNA in STM yielded 100% positivity (n=3) at 10 copies/ml (4 copies/reaction). Analytical specificity testing of non-target microorganisms (N=59), including the closely related non-syphilis treponemes T. denticola and T. phagedenis, yielded 0% positivity. TMA testing of mucosal swab specimens collected from men who have sex with men (MSM) attending a sexually transmitted disease clinic yielded 1.8% (17/944) TMA positive results. A collection of 56 serum specimens obtained from a separate cohort of patients with known rapid plasma reagin (RPR) status and syphilis clinical diagnosis were 19.6% (11/56) TMA positive overall, 29.7% (11/37) positive among subjects with syphilis diagnosis, including 8 (36.3%) of 22 persons with primary or secondary syphilis, 2 (20%) of 10 persons with early latent syphilis and 1 (20%) of 5 persons with late latent or unstaged syphilis. None (0%) of 18 RPR positive sera from patients with a history of treated syphilis was TMA positive. These results show TMA is an analytically sensitive and specific method for detection of Tp rRNA and is compatible with serum specimens in addition to pharyngeal and rectal mucocutaneous swab specimens. Automated real time TMA testing for T. pallidum may be useful as an adjunctive method with serology for screening and diagnostic testing of selected patient populations for syphilis.