Amino acid sequences from chronic infection plasma viral RNA from SIVΔ<i>vpx</i>-infected monkeys.

<p><b>(A).</b> Vpx N-terminal sequences accumulated debilitating mutations in two animals (cases 4 and 5) (*). <b>(B).</b> Novel amino acid sequence changes of Vif were present in case 4 (with highest viral loads) with leucine to proline (L->P) and alanine to threonine (A->T). <b>(C).</b> Analysis of Vpr revealed changes in amino acid sequences detected at the C-terminal region in cases 3 and 4 (I94T, P95L, S99N or G). <b>(D).</b> Alignments of Tat demonstrated multiple non-conservative changes, such as I22T, S32L, L35P.</p

Survival and viral load data.

<p>(<b>A)</b> Survival days post-inoculation (dpi) for rhesus macaques inoculated with SIVΔ<i>vpx</i> compared to animals inoculated with SIVmac239 (SIV239E with encephalitis or SIV239noE without encephalitis) or other mutant viruses (SIVΔ<i>nef</i> or SIVΔ3. SIVΔ<i>vpx</i>-infected macaques survived significantly longer with a slower disease progression compared to SIV239E or SIV239noE animals, but did not differ in survival length compared to SIVΔ<i>nef</i> or SIVΔ3. <b>(B)</b> Plasma viral RNA from SIVΔ<i>vpx</i>-infected rhesus macaques expressed as RNA copy equivalents per ml plasma from chronic disease or near-terminal collections (range 329–1140 dpi, median 730 dpi).</p

Double-label immunohistochemistry and <i>in situ</i> hybridization of colon.

<p><b>(A).</b> Representative images of SIV ISH (blue) with double-label immunohistochemistry for macrophage marker Ham56 (DAB, brown) in rhesus macaques in groups SIV239E (left), SIV239noE (middle), and SIVΔ<i>vpx</i> (right). SIV+ Ham56+ macrophages were frequent in SIV239E monkeys (left), but absent in the SIVΔ<i>vpx</i> group (right). <b>(B).</b> SIVΔ<i>vpx</i>-infected monkeys had significantly less virus in the colon (mean 10.5 SIV+ cells) compared to monkeys infected with SIV239 (SIV239E mean 105.3 SIV+ cells, SIV239noE mean 33.5 SIV+ cells), but no difference with animals in the SIVΔ<i>nef</i>/SIVΔ3 group. <b>(C).</b> SIVΔ<i>vpx</i> monkeys (mean 0 cells) had significantly fewer SIV+ Ham56+ macrophages compared to SIV239E (mean 66.7 cells), SIV239noE (mean 9.8 cells), and SIVΔ<i>nef</i>/SIVΔ3 groups (mean 3.3 cells) groups. <b>(D).</b> There was a significantly lower percentage of SIV+ cells that were macrophages in SIVΔ<i>vpx</i>-infected rhesus (mean 0%) compared to both the SIV239E (mean 63.3%) and SIV239noE (mean 22.4%) groups and a trend in the SIVΔ<i>nef</i>/SIVΔ3 group (mean 25.6%).</p

HSV-2 impacts the expression of integrins and co-stimulatory molecules on CD3⁻ HLA-DR⁺ cells.

<p>Cells were gated on singlets, live, CD3⁻ HLA-DR⁺. A) The fold increases in the frequencies of α₄β₇^high CD80⁺, CD80⁺ and CD103⁺ DCs in HSV-2 infected vaginal tissues are shown compared to the tissues treated with HSV-2 in presence of Acyclovir (uninfected control; ACV; set as 1) 3 days after HSV-2 infection. Means ± SEM are shown from n = 21 independent experiments; each experiment = 1 RM; each condition run in duplicate. Paired Wilcoxon signed rank p values are shown (p<0.05 is considered significant). B–C) The frequencies of α₄β₇^high CD80⁺, CD80⁺, CD103⁺, CD62L⁺ and CCR7⁺ DCs in the mucosa at day 3 are plotted against the HSV-2 copies/ml (each dot represents 1 explant; n = 27–29). Fitting linear regression lines, Spearman rank correlation p values and correlation coefficients r are shown. p<0.05 was considered significant.</p

Summaryoflentiviral infection of macrophages in spleen, lymph node, anin macaques inoculated intravenously with SIVΔ<i>vpx</i> compared to SIVmac239 (with or without SIV encephalitis) and SIVΔ<i>nef</i> or Δ3 assessed in spleen, lymph node, and colon.

<p>*p<0.05 difference between SIVΔ<i>vpx</i> and both SIVmac239 and SIVΔ<i>nef</i>/Δ3cases.</p

HSV-2 infection impacts the expression of α₄β₇, CD103 and CD11c on vaginal DCs.

<p>Cells from vaginal tissue and blood were gated on live, singlets and lin⁻ HLA-DR⁺. The frequencies of different subsets are shown for HSV-2 positive and negative RMs. Bars represent mean±SEM. Unpaired Mann-Whitney test p values are shown when ≤0.125 to indicate a tendency toward a significant difference. p<0.05 was considered significant.</p

HSV-2-Driven Increase in the Expression of α₄β₇ Correlates with Increased Susceptibility to Vaginal SHIV_SF162P3 Infection

<div><p>The availability of highly susceptible HIV target cells that can rapidly reach the mucosal lymphoid tissues may increase the chances of an otherwise rare transmission event to occur. Expression of α₄β₇ is required for trafficking of immune cells to gut inductive sites where HIV can expand and it is expressed at high level on cells particularly susceptible to HIV infection. We hypothesized that HSV-2 modulates the expression of α₄β₇ and other homing receptors in the vaginal tissue and that this correlates with the increased risk of HIV acquisition in HSV-2 positive individuals. To test this hypothesis we used an in vivo rhesus macaque (RM) model of HSV-2 vaginal infection and a new ex vivo model of macaque vaginal explants. In vivo we found that HSV-2 latently infected RMs appeared to be more susceptible to vaginal SHIV_SF162P3 infection, had higher frequency of α₄β₇^high CD4⁺ T cells in the vaginal tissue and higher expression of α₄β₇ and CD11c on vaginal DCs. Similarly, ex vivo HSV-2 infection increased the susceptibility of the vaginal tissue to SHIV_SF162P3. HSV-2 infection increased the frequencies of α₄β₇^high CD4⁺ T cells and this directly correlated with HSV-2 replication. A higher amount of inflammatory cytokines in vaginal fluids of the HSV-2 infected animals was similar to those found in the supernatants of the infected explants. Remarkably, the HSV-2-driven increase in the frequency of α₄β₇^high CD4⁺ T cells directly correlated with SHIV replication in the HSV-2 infected tissues. Our results suggest that the HSV-2-driven increase in availability of CD4⁺ T cells and DCs that express high levels of α₄β₇ is associated with the increase in susceptibility to SHIV due to HSV-2. This may persists in absence of HSV-2 shedding. Hence, higher availability of α₄β₇ positive HIV target cells in the vaginal tissue may constitute a risk factor for HIV transmission.</p></div

Vaginal tissues infected ex vivo with HSV-2 are more susceptible to SHIV infection.

<p>The fold increases in SHIV copies/ml of the vaginal explants co-infected with HSV-2 are shown compared to the SHIV copies/ml of vaginal explants infected with SHIV alone (set as 1). Mean ± SEM are shown from n = 11 independent experiments; each experiment = 1 RM; each condition run in duplicate. Paired Wilcoxon signed-rank test p values are shown. p<0.05 was considered significant.</p

HSV-2 modulates the phenotype of migratory CD4⁺ T cells and CD3⁻ HLA-DR⁺ cells.

<p>A) The fold increases in the MFI of the α₄β₇^high CD4⁺ T cells (left) and the frequencies of CD62L⁺ (center) and CCR6⁺ (right) CD4⁺ T cells that migrated out of the HSV-2 infected mucosa 18 hours after infection are shown compared to the Acyclovir control (+ACV, set as 1; n = 13–19). B) The fold increases in the frequencies of α₄β₇^high CD80⁺ (left) CCR6⁺ (center) and CD103⁺ (right) CD3⁻ HLA-DR⁺ cells that migrated out of the HSV-2 infected mucosa 3 days after infection are shown compared to the Acyclovir control (+ACV, set as 1; n = 6–13). Bars represent mean ± SEM. Wilcoxon signed-rank test p values are shown. p<0.05 was considered significant.</p

Double-label immunohistochemistry and <i>in situ</i> hybridization of lymphoid tissues.

<p><b>(A)</b> Representative images of SIV <i>in situ</i> hybridization (ISH, blue) with double-label immunohistochemistry for monocyte/macrophage lineage cell marker Ham56 (DAB, brown) in rhesus macaques in groups SIV239E (left), SIV239noE (middle), and SIVΔ<i>vpx</i> (right). The top images of lymphoid follicles depict Ham-56+ cells morphologically consistent with follicular DCs. Images in the second row depict HAM56+ cells in the red pulp of the spleen (left and center) or paracortex in lymph node (right). Frequent SIV+ HAM56+ macrophages/DCs (Mφ) were observed in tissues from the SIV239E and SIV239noE groups, while only rare SIV+ Ham56+ macrophages/DCs were observed in lymph node from a SIVΔ<i>vpx</i>-infected rhesus (arrow). <b>(B).</b> The overall numbers of infected cells in the spleen and lymph node from the 4 groups of animals were not significantly different. <b>(C)</b> However, there were significantly fewer SIV+ Mφ in SIVΔ<i>vpx</i> monkeys (mean 0.5 SIV+ Mφ) compared to the SIV239E (mean 13.64 SIV+ Mφ), SIV239noE (mean 4.5 SIV+ Mφ) and SIVΔ<i>nef</i>/SIVΔ3 monkeys (mean 6.25 SIV+ Mφ). <b>(D)</b> SIV infected macrophages made up a much lower percentage of all SIV+ cells in SPL and LN of SIVΔ<i>vpx</i>-infected rhesus (mean 2.2%) compared to SIV239E (mean 22.7%), SIV239noE (mean 8.3%), and SIVΔ<i>nef</i>/SIVΔ3 monkeys (10.1%) (p<0.05).</p