HIV viral shedding in semen

Semen is a major transmission vector for HIV. Virus-specific CD8 T cells are critical in HIV control, but their relationship with semen viral load is unknown. We therefore examined the association between systemic HIV-specific IFN-γ CD8 responses and viral load in the semen and blood of HIV-infected men. No correlation was observed between viral load in either semen or blood and systemic CD8 T-cell responses. Further studies of immune correlates of semen HIV shedding are needed. HIV-specific CD8 T lymphocytes are critical in the host control of HIV replication. There is a clear temporal relationship between the development of HIV-specific CD8 T cells and viral control during acute HIV infection [1], and between experimental CD8 T-cell depletion and increases in the plasma viral load [2]. In addition, epitope-specific CD8 T cells place considerable evolutionary pressure on the virus in both primates and humans [3–8]. The magnitude of the systemic HIV-specific CD8 T-cell response has been inversely correlated with rates of disease progression [9] and plasma viral load in some studies [10–12], but not in others [13–15]. This may be because the inverse relationship only applies during early HIV infection [16–18], or to CD8 T cells recognizing specific HIV gene products, such as Gag [10]. The HIV plasma viral load is strongly associated with the probability of sexual HIV transmission [19], probably because the plasma viral load correlates with the amount of viral shedding in both semen [20–23] and female genital tract secretions [24–26]. HIV-specific CD8 T cells can be detected in the semen [27] and the female cervix [28–30] of HIV-infected individuals. Furthermore, the specificity, function and ontogeny of HIV-specific CD8 T cells in the genital tract of infected individuals tend to reflect that found in the blood [31]. We therefore studied the relationship between systemic CD8 T cell responses and HIV shedding in the male urogenital tract. Simultaneous blood and semen samples were collected from 20 treatment-naive, chronically HIV-infected men enrolled through the Canadian Immunodeficiency Research Collaborative (CIRC), Toronto, Canada. Semen samples were collected by masturbation into a dry sterile container, and were immediately transported to the clinic at room temperature; blood was collected into acid citrate dextran. A first-void urine sample was screened for the presence of leukocytes using a dipstick (Bayer Diagnostics, Puteaux Cedex, France), and for infection by either Neisseria gonorrhoeae (NG) or Chlamydia trachomatis (CT) (Amplicor CT/NG assay, Roche Diagnostic, Quebec, Canada). Any participant with a positive result was excluded from analysis. Seminal plasma was isolated by centrifugation (∼850 g/10 min), and peripheral blood mononuclear cells (PBMC) were obtained through Ficoll–Hypaque density centrifugation. The viral load in blood and seminal plasma was measured using the Versant HIV-1 RNA 3.0 assay (branched DNA; Bayer Diagnostics), as reverse transcriptase–polymerase chain reaction-based assays may be inhibited by semen constituents [32]. IFN-γ enzyme-linked immunospot assays were set up using PBMC at 1 × 105 per well, as previously described [33,34]. A matrix of 756 15-mer peptides overlapping by 11 amino acids, spanning the clade B HIV-1 genome (AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH) was established, with each peptide appearing uniquely in two separate pools at a final working concentration of 2 μM. Responses were counted using an automated enzyme-linked immunospot counter (Cellular Technology Ltd., Cleveland, OH, USA). A positive HIV-specific CD8 response was defined as an HIV-specific response at least twofold higher than background (PBMC + 2 μm DMSO), and 100 or more spot-forming units (SFU)/106 cells. All positive responses were confirmed using individual 15-mer peptides. No PCR inhibition was evident when assaying the seminal plasma viral load. There was a trend towards an inverse relationship between the peripheral blood CD4 T-cell count (mean 574 cells/mm3, range 350–850 cells/mm3) and the HIV viral load in both semen and plasma (P = 0.2 for both). Viral loads in seminal and blood plasma were closely correlated (r = 0.5, P = 0.03; Fig. 1a), although viral levels in the blood were approximately 10-fold higher than in semen (mean 4.3 versus 3.3 log10 copies/ml; P < 0.001, paired t-test). Study participants could be divided into three subgroups, based on their semen viral load; (i) three out of 20 (15%) had no detectable virus in the semen (≤ 50 copies/ml); (ii) three out of 20 (15%) had high levels of HIV in the semen (≥ 60% blood viral load); and (iii) 14/20 individuals (70%) had detectable semen viral loads less than 60% of the blood viral load. In one individual, the semen viral load was 10-fold higher than in blood (5.3 versus 4.3 log10 copies/ml) in the absence of a detectable sexually transmitted infection or urethral inflammation.Fig. 1.: Lack of association between semen or blood HIV-RNA viral load and systemic HIV-specific CD8 T-cell responses. There was a significant correlation between semen HIV-RNA shedding and blood RNA viral load (r = 0.5, P = 0.03) (a). Semen HIV shedding did not correlate with the magnitude of overall or Gag-specific systemic HIV-1-specific IFN-γ CD8 responses (b, dotted and solid lines of best fit, respectively), or with the number of epitopes recognized overall or within HIV-1 Gag (c, dotted and solid lines of best fit, respectively). Likewise, the blood viral load did not correlate with the magnitude of overall or Gag-specific systemic HIV-1-specific IFN-γ CD8 responses (d, dotted and solid lines of best fit, respectively), or with the number of HIV-1 epitopes recognized overall or within HIV-1 Gag (e, dotted and solid lines of best fit, respectively). See text for statistical analysis.Systemic CD8 IFN-γ responses were detected in all participants. A total of 159 epitopes were recognized (mean eight epitopes per participant, range two to 20 epitopes), and all were confirmed to be CD8 T-cell mediated by flow cytometry with intracellular cytokine staining. Responses focused on Gag (37% of epitopes), Pol (20%), Nef (20%), Env (13%), and less commonly on Tat, Rev, Vpr, Vpu and Vif. The mean total magnitude of CD8 T-cell IFN-γ responses for each participant was 6776 SFU/106 PBMC (range 1135–25 955 SFU/106 PBMC). Neither semen nor blood viral loads correlated with the total HIV-specific CD8 cell response magnitude (semen r = 0.1, P = 0.6; blood r = 0.3, P = 0.3) or the response breadth, defined as the total number of HIV epitopes recognized in an individual (semen r = −0.03, P = 0.9; blood r = 1.0, P = 0.7; Fig. 1). Although there was an inverse association between both the magnitude and breadth of Tat-specific responses and semen HIV shedding (r = −0.5 for both, P = 0.02; P = 0.03, respectively), only three out of 20 participants responded to Tat, limiting our power to draw firm conclusions, and significance was lost when correcting for multiple comparisons. No association was seen between the magnitude or breadth of Gag-specific CD8 T-cell responses and the viral load at either site (semen r = 0.06, P = 0.8; blood r = −0.09, P = 0.7; Fig. 1). In conclusion, we have confirmed that there is a direct correlation between the HIV viral load in the semen and blood, that the viral load in semen tends to be approximately 10-fold lower than in blood, and that occasional ‘super-shedders’ [35] may pose a particular HIV-1 transmission risk [19]. Therefore, a better understanding of the immune correlates of viral shedding in semen will be important to inform the development of immunotherapeutics, topical microbicides and rational public health policy. However, we found no association between systemic HIV-specific CD8 T-cell responses and semen HIV shedding, or with the blood plasma viral load. In addition, no association was seen between Gag-specific systemic responses and viral levels in either semen or blood. These results strongly suggest that systemic HIV-specific CD8 T-cell IFN-γ responses are not directly associated with viral levels in the blood or semen compartments. Elucidating the immune associations of HIV semen shedding will be important in directing strategies to reduce sexual transmission. Acknowledgements The authors would like to thank Professor Mario Ostrowski for his invaluable help in reviewing this manuscript.

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