Impact of Short-Term HAART Initiated during the Chronic Stage or Shortly Post-Exposure on SIV Infection of Male Genital Organs

International audienceBACKGROUND: The male genital tract is suspected to constitute a viral sanctuary as persistent HIV shedding is found in the semen of a subset of HIV-infected men receiving effective antiretroviral therapy (HAART). The origin of this persistent shedding is currently unknown. Phylogenetic studies indicated that HIV in semen from untreated men arises from local sources and/or passive diffusion from the blood. We previously demonstrated in human and macaque low levels and localized infection of several semen-producing organs by HIV/SIV. Using a macaque model, this study investigates the impact of short term HAART (2-4 weeks) initiated either during the asymptomatic chronic stage or 4 h post-intravenous inoculation of SIVmac251 on the infection of male genital organs. METHODOLOGY/PRINCIPAL FINDINGS: Short term HAART during the chronic stage decreased blood viral load. No major impact of HAART was observed on SIV DNA levels in male genital organs using a sensitive nested PCR assay. Using in situ hybridization, SIV RNA+ cells were detected in all male genital tract organs from untreated and treated animals with undetectable blood viral load following HAART. Infected CD68+ myeloid cells and CD3+ T lymphocytes were detected pre- and post-HAART. In contrast, short term HAART initiated 4 h post-SIV exposure led to a drastic decrease of the male genital tissues infection, although it failed to prevent systemic infection. In both cases, HAART tended to decrease the number of CD3+ T cells in the male organs. CONCLUSIONS: Our results indicate that the established infection of male genital organs is not greatly impacted by short term HAART, whereas the same treatment during pre-acute phase of the infection efficiently impairs viral dissemination to the male genital tract. Further investigations are now needed to determine whether infection of male genital organs is responsible for long term persistent HIV shedding in semen despite HAART

SIV RNA+ cells detection and identification within MGT of acutely infected macaques treated or not.

<p>(A) Non radioactive ISH for SIV viral RNA in the MGT organs of untreated versus treated animals. Arrows indicate SIV RNA+ cells. Scale bar = 50 µm. (B) Identification of SIV-expressing cells using non radioactive ISH for SIV (green) combined with immunofluorescence for cell marker CD3 (a, red) or CD68 (b, red). Nuclei labeled with DAPI are shown in blue. Side panels represent individual channels. Large panel represents a merged image combining all channels. Arrows show co-labeled cells. Scale bars = 20 µm.</p

Quantitative analysis of CD3+ T lymphocytes in MGT organs of untreated and treated animals.

<p>Stars indicate statistical differences (p<0.05) using a Mann-Whitney test.</p

Viral dynamics in chronically-infected macaques receiving HAART and SIV nucleic acids detection in MGT organs.

<p>6 animals were untreated, 3 received a 2 weeks long AZT/3TC/IDV treatment and 3 received 4 weeks of the same treatment. (A) Plasma viral load and (B) total viral DNA in PBMCs were measured before and after treatment. Median is represented by a line. The grey area indicates the quantitative threshold of the qRT-PCR and qPCR assays. Star indicates statistical differences (p<0.05) using a Wilcoxon test. (C) Frequency of detection of SIV DNA in the seminal vesicle, prostate, epididymis and testis of untreated, 2 or 4 week treated macaques, using nested SIV gag PCR. Two independent fragments of each tissue were assayed. Median of frequency is represented by a line. (D) Tat/rev spliced RNA detection in MGT organs (SV: seminal vesicle; P: prostate; E: epididymis, T: testis) by RT nested PCR. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037348#s3" target="_blank">Results</a> are shown for one representative treated animal (20475) with an undetectable PVL after treatment. C− and C+ represent negative and positive controls respectively.</p

SIV RNA+ cells detection and identification within MGT of chronically infected macaques treated or not.

<p>(A) Detection of SIV positive cells in the seminal vesicle (a, a′), prostate (b, b′), epididymis (c, c′) and testis (d, d′) of macaques, treated or not, using radioactive (a, b, c, d) or non-radioactive (a′, b′, c′, d′) ISH for SIV gag RNA. Scale bars = 20 µm. (B) Due to the low and localized nature of the infection, an extensive screening for SIV RNA+ cells was performed in a minimum of 30 tissues section/experiment in 2 independent radioactive ISH experiments on the seminal vesicle, prostate, epididymis and testis of 2 untreated and 2 treated macaques with undetectable PVL following HAART. Median is represented by a line. (C) Identification of SIV-expressing cells using either non-radioactive (a, b) or radioactive (c) ISH for SIV RNA combined with immunostaining for cell markers in treated versus untreated animals. The pictures presented show the detection of CD3+ (a, red) or CD68 (b, red) SIV+ (green) co-labeled cell in the prostate (a) and epididymis (b) of one treated animal. Nuclei labeled with DAPI are shown in blue. Side panels represent individual channels. Large panel represents a merged image combining all channels. Arrows show co-labeled cells. (c) Detection of CD68+ (brown) SIV+ (black dots) co-labeled cell in the epididymis of one treated animal with undetectable PVL following HAART. Insert show enlargement of co-labeled cell. Scale bars = 20 µm.</p

Viral dynamics in acutely-infected macaques receiving HAART and SIV nucleic acids detection in MGT organs.

<p>4 macaques received a AZT/3TC/IDV combination (HAART 4 h-2 weeks) while 4 others were untreated. (A) Plasma viral load and (B) total viral DNA in PBMCs were measured during the 2 week protocol. Median is represented by a line. The grey area indicates the quantitative threshold of the qRT-PCR and qPCR assays. (C) Frequency of detection of SIV DNA in the seminal vesicle, prostate, epididymis and testis of untreated and 2 week treated macaques, using nested SIV gag PCR. Two independent fragments of each tissue were assayed. Median of frequency is represented by a line.</p