HIV-1 Populations in Semen Arise through Multiple Mechanisms

Abstract

HIV-1 is present in anatomical compartments and bodily fluids. Most transmissions occur through sexual acts, making virus in semen the proximal source in male donors. We find three distinct relationships in comparing viral RNA populations between blood and semen in men with chronic HIV-1 infection, and we propose that the viral populations in semen arise by multiple mechanisms including: direct import of virus, oligoclonal amplification within the seminal tract, or compartmentalization. In addition, we find significant enrichment of six out of nineteen cytokines and chemokines in semen of both HIV-infected and uninfected men, and another seven further enriched in infected individuals. The enrichment of cytokines involved in innate immunity in the seminal tract, complemented with chemokines in infected men, creates an environment conducive to T cell activation and viral replication. These studies define different relationships between virus in blood and semen that can significantly alter the composition of the viral population at the source that is most proximal to the transmitted virus.

Figure 1. Neighbor-joining tree of SGA-derived env amplicons for two patients with HIV-1 subtype C demonstrating equilibration between blood and semen.

Blood SGA-env sequences (red circles) and semen SGA-env sequences (blue triangles). Bootstrap values ≥70 are shown. Genetic distance is indicated at the bottom of the figure, and represents the number of nucleotide substitutions per site. An outgroup was included to root the tree but is not shown.

Figure 2. Neighbor-joining tree of SGA-derived env amplicons for three patients with HIV-1 subtype C demonstrating clonal amplification of identical or nearly identical sequences within semen.

Blood SGA-env sequences (red circles) and semen SGA-env sequences (blue triangles). Vertical blue bars highlight clonal amplification of specific variants. Bootstrap values ≥70 are shown. An outgroup was included to root the tree but is not shown.

Figure 3. Neighbor-joining tree of SGA-derived env amplicons for two patients with HIV-1 subtype C demonstrating compartmentalization between blood and semen.

Blood SGA-env sequences (red circles) and semen SGA-env sequences (blue triangles). Ovals highlight clades of compartmentalized sequences in the semen or blood. Vertical blue bars highlight clonal amplification of specific variants. Bootstrap values ≥70 are shown. An outgroup was included to root the tree but is not shown.

Figure 4. Neighbor-joining tree of SGA-derived env amplicons for two patients with HIV-1 subtype B demonstrating amplification and equilibration in the left panel, and amplification and compartmentalization of seminal sequences in the right panel.

Blood SGA-env sequences (red circles) and semen SGA-env sequences (blue triangles). The oval highlights clades of compartmentalized sequences in the semen. Vertical blue bars highlight clonal amplification of specific variants. Bootstrap values ≥70 are shown. An outgroup was included to root the tree but is not shown.

Figure 5. Scatter plot of semen:blood analyte ratios of 17 cytokines and chemokines in HIV-1 infected (red circles) (n = 12), uninfected Malawi men (black circles) (n = 6), and uninfected US men (black triangles) (n = 12).

Values that were below the lower limit of detection were reported as the mid-point between the lower level of detection and zero. Semen:blood ratios were excluded if both compartments were below the level of detection; thus, IL12 and IL13 were not included in the analysis. In the infected men, there were 6 to 11 subjects with semen:blood analyte ratios for IL-1b, IL-2, IL-4, and IFN-g, whereas the remaining cytokines and chemokines had data from all 12 infected men. In addition, after exclusion of values below the limit of detection in both semen and blood compartments, we quantified the semen:blood ratio for each cytokine and chemokine from a range of 10 to 17 HIV-1 uninfected men. The dashed line illustrates an equivalent semen:blood analyte ratio. Horizontal lines represent median values. Blue asterisks denote significant differences between infected and uninfected men, P value from Mann-Whitney test <0.003 (to correct for multiple comparisons). Boxed cytokines/chemokines have median semen:blood analyte ratios >5 in both HIV-1 infected and uninfected men.

Figure 6. Model demonstrating HIV-1 populations in the blood and male genital tract.

Target cells traffic from blood to the male genital tract. A semi-permeable barrier separates blood from the genital tract that allows passage of some free virus and uninfected and infected cells. Equilibration of blood and seminal plasma sequences occurs through direct import of sequences from blood to semen. Clonal amplification results from establishment of a local focus of infection of anatomically isolated uninfected cells in the genital tract. Compartmentalization occurs when resident cell populations in the male genital tract become infected and there is persistent local replication. Gray circle, HIV-1 infected target cell. White circle, HIV-1 uninfected target cell. Red circle, blood-derived virus. Blue circle, male genital tract-derived virus.