Background:   Some individuals remain HIV-1 seronegative despite repeated exposures to the virus, termed exposed seronegatives. In early 1998, we initiated a study to identify and characterize HIV-1 infection in long-term seronegative persons who reported repeated unprotected sexual activities with multiple HIV-1-infected partners.

Methods:  Limiting-dilution PCR were performed to amplify HIV-1 provirus sequences within env, gag, pol , and vpr from both late seroconverters and their long-term sexual partners. HIV-1 sequences were aligned using CLAUSTAL W, and analyzed with phylogenetic programs including PAUP* and MEGA. Statistic analyses were performed using PRISM version 3.0, InStat Version 3.0 and EXCEL.

Results:  We have identified HIV-1 sequences in 12  of 94 exposed seronegative individuals. Of these 12 infected exposed seronegatives, 2 have remained seronegative and healthy with persistent HIV-1 DNA at extraordinarily low levels. However, the other 10 HIV-1-infected exposed seronegatives seroconverted later. We first analyzed HIV-1 env sequences in 4 of the late seroconverters and 1 exposed seronegatives whose HIV-1-infected, long-term sexual partners were identified.  To our surprise, none of the 5 late seroconverters/exposed seronegatives were infected or superinfected by HIV-1 strains from their long-term sexual partners.  In fact, env genetic distances between the breakthrough virus in late seroconverters and their partners' virus were significantly higher than those of pairwise distances between each pair of HIV-1 control sequences randomly chosen from GenBank (p < 0.001). Interestingly, a higher rate of non-synonymous mutations (dN) (p < 0.05), but not synonymous mutations (dS) (p = 0.34), was found between 3 of the 4 late seroconverters who had HIV-1-specific CD8⁺ responses (CTL) before seroconversion and their long-term partners, as compared with random HIV-1 pairs. In contrast, the dS but not dN was higher in the late seroconverters who had no detectable CTL responses pre-seroconversion. Furthermore, significant difference in dN but not dS was found between late seroconverters and partners’ HIV-1 sequences corresponding to multiple CTL epitope pools (including Env, Pol, Gag, and Vpr) that were detected pre-seroconversion. 

Conclusions:  These findings indicate that breakthrough HIV-1 strains in exposed seronegatives/late seroconverters tends to be divergent from those of their long-term sexual partners, suggesting that continued virus exposures might protect partner-like HIV-1, but allow distinct viral strains to infect. Pre-infection CTL responses might play an important role in the positive selection of breakthrough HIV-1 infection in late seroconverters.

Keywords: HIV-1; infection; exposed-seronegative