In this study, we examined the genetics of breakthrough HIV-1 in an ES subject (LSC63) who had been seronegative for more than 2 years despite repeated unprotected sexual contacts with an HIV-1-positive partner (P63), as well as the HIV-1-specific CTL responses before and after LSC63's seroconversion. Genetic analyses of HIV-1 sequences of LSC63, P63, and P63's infecting source (PP63) indicate that subject LSC63 was not infected by P63 viruses. Low viral load (<50 copies/ml) in P63 during the sexual relationship with LSC63 may have prevented the transmission of P63's HIV-1 to LSC63. However, broad CTL responses were detected in LSC63 before seroconversion. Compared to those detected after seroconversion, these CTL responses were of low magnitude and half of them targeted different regions of the viral proteome. Furthermore, for the majority of the coding regions of HIV-1 (with the possible exceptions of polB, gp41, and nef), the genetic distances between the infecting and the exposed viruses of LSC63 were comparable to the distances between random subtype B HIV-1 sequences and the exposed viruses. These results suggest that repeated sexual exposure to low levels of HIV-1 from a seropositive partner was able to elicit HIV-1-specific CTL responses in LSC63. However, these responses were not able to prevent HIV infection in the subject, even though the infecting viral strain was not significantly dissimilar from random HIV-1 strains.
Viral loads in HIV-1-positive partners are a strong predictor of heterosexual transmission (51
). It has also been reported that the HIV-1-positive partners in monogamous homosexual relationships between whom HIV-1 transmission had not occurred had significantly lower cellular infectious viral loads than HIV-1-transmitting donors in monogamous homosexual relationships (5
). In addition, heterozygous CCR5-Δ32 and CCR5 promoter mutations associated with resistance to HIV-1 infection (18
) were identified in LSC63 and may have also contributed to the delay in HIV-1 infection in LSC63. LSC63 was HSV-2 positive 1.6 years before his HIV-1 seroconversion. Thus, HSV-2 infection might have played a minor role in his acquisition of HIV-1.
Consistent with findings for the late seroconverters from the Nairobi commercial sex worker cohort (24
), we found that CTL responses in LSC63 before seroconversion were of low magnitudes and that CTL targets before and after seroconversion were different. Of the 13 peptides recognized after seroconversion, 6 were located in pools recognized before seroconversion. In only 3/13 peptides were the sequences of the exposed and the infecting viruses identical over both the targeted peptides and the immediate 5-amino-acid flanking regions, and two of them were located in pools recognized before seroconversion. It has been reported that mutations flanking CTL epitopes at least as far as the fifth position downstream can prevent appropriate epitope processing and presentation (67
) and lead to escape (10
). Although our in silico analysis did not predict alteration of epitope processing by sequence differences at flanking regions of the epitopes between the exposed and the infecting viruses, in vitro digestion analyses of peptides containing those epitopes and flanking regions will be required (34
) to further determine the impact of the sequence variation observed in the flanking regions. Therefore, discordant CTL responses before and after seroconversion might reflect the sequence differences between the exposed and the infecting viruses.
HIV-1-specific CTL activities have been associated with resistance to HIV-1 infection (1
). However, in the study of HIV-1-resistant female commercial sex workers in Nairobi (27
), most of the peptides recognized before seroconversion were present in the later-infecting viruses, indicating a minor role of viral escape in late seroconversion in these individuals. We were not able to identify the individual CTL peptides recognized before seroconversion in LSC63 or examine the functionality of CTL responses before seroconversion due to a lack of specimens. However, our genetic analyses suggested that, when compared to random HIV-1 subtype B sequences, the viruses establishing infection in LSC63 had distances similar to those of the exposed viruses over most of the coding regions of HIV-1. Only for Nef and the 3′ half of Pol were CTL responses detected before seroconversion and significantly greater genetic distances observed between the exposed and the infecting viruses and random strain sequences. We note that the choice of random control sequences might affect these interpretations (D. C. Nickle, Y. Liu, K. Davis, C. Celum, G. H. Learn, and J. I. Mullins, unpublished data). The ideal control sequences for this study would be random sequences from subjects that had been infected for a period of time comparable to P63 and had effective HAART shortly after HIV-1 infection. Nevertheless, our results indicate that although the viruses infecting LSC63 might contain sequences that could have escaped some specific CTL responses elicited before seroconversion, the majority of the protein-coding regions of HIV-1 variants in LSC63 (except gp41
, and the 3′ half of pol
) were not unusually distant from the viruses to which LSC63 had been previously regularly exposed.
Without antiviral treatment, LSC63 maintained a viral load of ~103
copies/ml and a CD4+
T-cell count of over 500 cells/μl during the 2.5 years of study after seroconversion. LSC63 had the protective B27 allele (23
); developed strong CTL responses after seroconversion, especially B27-restricted responses; and was CCR5-Δ32 and CCR5 promoter heterozygous. All of these factors might have contributed to the initial relatively benign clinical outcome. Before seroconversion, however, in spite of broad CTL responses, no B27-restricted CTL responses were detected.
In summary, we found that repeated exposure to low-level HIV-1 was able to elicit multiple, although low-magnitude, HIV-1-specific CTL responses in LSC63. However, only marginal evidence was obtained for these responses being able to restrict infection with related viruses. Further study of ES individuals who seroconvert and those who fail to seroconvert may assist in the identification of protective CTL responses.