More on Viral Reservoirs and Transmission
Session 54 Poster Presentations
Below are the abstracts from this session on HIV reservoirs and their relationship to transmission of HIV. The potential significance of reservoirs investigated in these studies include the prostate, the cervix, semen cells, seminal plasma, the genital tract, and genital epithelial cells. These reports discuss transmission of HIV potential as it relates to the reservoirs.
The Prostate as a Reservoir for HIV-1
HIV in the genital tract is often genetically distinct from that in the bloodstream. This variation results from differing host cell selection, local immune responses, and varying penetration of drugs into the genital tract. Male genital secretions are a complex mixture of cells and secretions from the testes, epidydimis, vas deferens, seminal vesicles, prostate, urethral and paraurethral glands. Since the prostate can harbor bacterial and fungal infections, it was investigated as a reservoir for HIV.
Seven male participants submitted genital secretion samples weekly for 11 weeks in this self-controlled trial. Samples from weeks 1-4 and 11 were collected without prior prostate massage (PM), and samples from weeks 5-10 were submitted after PM. 4/7 subjects were on stable antiretroviral regimens (>3months) before starting the trial; 3/7 were on no antiretrovirals. There were no medication changes during the trial. HIV RNA was extracted from 1 ml of seminal plasma (SP) from each sample via the Boom method (Nuclisens, Biomerieux) and then quantitated (Amplicor, Roche). At study enrollment subjects tested negative for active sexually transmitted infections, and they remained asymptomatic throughout.
4/7 participants had undetectable SP HIV RNA in all samples without prior PM but then became detectable in 1-3 samples collected after prior PM (mean 473 cop/ml, range 4-3488). No correlation was discerned between PM and the amount of SP collected. The remaining 3/7 participants had SP samples with detectable HIV RNA at all collections, which showed no correlation with PM. Blood plasma HIV RNA levels measured at the start and the end of the study were unchanged in all subjects except one who showed a steep decrease in blood plasma HIV RNA levels from 42,102 cop/ml to <50 cop/ml. The SP HIV RNA levels from this subject varied greatly (3,041 to 285,732 cop/ml) but did not correlate to prior PM.
These data suggest that the prostate is a reservoir for HIV. This has significant transmission consequences, as PM resembles receptive anal intercourse in men who have sex with men. Prostate stimulation via anal intercourse could therefore increase the amount of HIV RNA in the receptive partner's genital secretions, which could then be transmitted when he became the insertive partner. These data could, in part, explain the high HIV infection rates historically seen in men who have sex with men.
Abst. 459a. D. M. Smith*1, J. D. Kingery2, C. C. Ignacio1, J. K. Wong1, D. D. Richman1, S. J. Little1. 1Univ of California, San Diego and 2Loma Linda Univ, CA
Seminal Super Shedding of HIV: Implications for Sexual Transmission
HIV-1 is usually detected in seminal plasma (SP) less frequently than the corresponding blood plasma (BP) and usually at lower levels. However, in most cohorts there are a significant minority of men in which SP levels are equal to, or higher than BP levels. We have identified these individuals as "seminal super shedders" (SSS) of HIV-1 and postulate that they may represent a group of individuals at increased risk of transmitting HIV-1.
Seventy-three (73) HIV+ men not on therapy were enrolled. They produced matched blood and semen samples at the same time as undergoing tests for sexually transmitted infections. Viral load was determined by NASBA. Variables considered were; age, CDC status, CD4 count, BPVL > 100,000 c/ml, and the presence or absence of urethritis. SSS were defined as individuals with a SPVL/BPVL ratio > 1.
Overall BPVL was significantly correlated with SPVL, Spearman's ? 0.53 p < 0.0001. No men had BPVL values < 400 c/ml. In contrast 22/72 (30%) had SPVL < 400 c/ml despite detectable BPVL (e.g., non-shedders). Men who shed virus into semen had significantly higher BPVL than non shedders; 5.01 log10 c/ml (range 3.4-6.3) vs 4.2 log10 c/ml (3.0-5.5) p < 0.0001, Mann-Whitney U. Nine (9) men met the SSS criteria. These men had significantly higher SPVL compared with the main seminal shedding (SS) group; 5.6 log10 c/ml (4.1-6.5) vs 3.9 log10 c/ml (2.6-6.2) p < 0.001. In contrast, BPVL was not significantly different between SSS and SS (4.8 log10 c/ml vs 5.02 log10 c/ml, p = 0.9). SSS were generally older; 48 yrs vs 35 yrs p < 0.02 and the presence of urethritis was significantly over represented in the SSS group compared with the other groups, 3/9 (33%) vs 3/63 (4.8%) of other groups (Fishers Exact test p = 0.02).
Importantly SSS was not simply explained by high BPVL as patients (pts) with BPVL > 100,000 c/ml were equally represented in both the SSS group 4/9 (44%) vs SS 25/63 (39%), p > 0.9. CD4 counts and CDC status were not significantly different between the SSS and SS.
In HIV+1 men with detectable BPVL not on therapy, 30% had undetectable SPVL, 58% had SPVL detectable at concentrations below their BPVL. Thus 12% of this cohort was classified as SSS. Although urethritis accounted for 3/9 cases of SSS, studies are required to elucidate possible reasons for the excess shedding in those pts without urethritis. This important group of pts may pose a higher risk of transmitting HIV to their sexual partners.
Abst. 454. S. Taylor *1, T. Sadiq 2, C. Sabin 2, D.White 3, P.Cane 4, S.Drake 3, D.Pillay 4. 1Univ of Birmingham and Birmingham Heartlands Hosp, UK; 2Royal Free and Univ Coll Med Sch, London, UK; 3Birmingham Heartlands Hosp, UK; and 4Univ of Birmingham, UK
Seminal Reservoirs During an HIV-1 Eradication Approach
Despite of the reduction of the levels of HIV-1 virions in blood and seminal plasma of HIV-1-infected patients (pts), HAART does not eradicate HIV-1 disease. Low-levels of viral replication can be shown in pts with undetectable plasma HIV-1 RNA load, and viral rebound occurs after interruption of therapy. Residual HIV-1 disease was approached in vivo with a rationally designed combination therapeutic eradication protocol, and the role
of the seminal compartment in the phenomenon of viral rebound was investigated.
Three (3) HIV-1 infected pts, with < 50 copies/ml of plasma viral RNA for > 1 yr, were enrolled in this trial. DDI and hydroxyurea (HU) were added to their baseline HAART to attack cryptic viral replication. After a month of therapy, low dose OKT3, followed by a 2-wk course of IL-2 were administrated to stimulate latent provirus. Plasma viral RNA and 2-LTR DNA circles were analyzed. CD8-depleted PBMCs and semen cells were studied in co-culture to detect replication-competent virus. The sequences of the V3 loop of the gp120 region were aligned for cell-free RNA and proviral DNA in blood and seminal compartments at different time points.
HU and DDI were continued with HAART after OKT3 and IL-2 stimulation. Replication-competent virus was undetectable after treatment and
plasma viral RNA was either undetectable or very low in each of the pts. When the pts were given trials off all antiretroviral therapy, they developed blood, but not semen, viral rebound. Based on viral sequences and tropism, we found that the two major viral isolates, in semen cells, were macrophage-tropic and dual-tropic, and these were also present in PBMCs. Interestingly, in one pt, a third major isolate was identified after immuno-stimulation, but never afterwards. Six (6) months after the viral rebound, we found a shift toward dual tropism in semen cell-associated HIV-1 proviral DNA, with the first appearance of a T-lymphotropic virus solely in this compartment. The virus responsible for the blood plasma viral rebound was not found in the semen compartment, neither before nor at completion of the protocol.
This study suggests viral compartmentalization of the semen micro-environment after an intensification and stimulatory protocol, with evidence of viral evolution. Whether the latter will play an active role, as a viral reservoir, along the course of HIV-1 disease will be determined by ongoing
Abst. 455. G Nunnari*, J Kulkosky, D Leto, J Sullivan, Y Xu, RJ Pomerantz. Thomas Jefferson Univ, Philadelphia, PA
Differences in HIV-1 Replication Dynamics between Peripheral Blood and Vagina Suggest a Compartmentalized Viral Production in the Female Genital Tract
The female genital tract is suspected to be an independent compartment of HIV-1 replication, which would have significant implications for HIV-1 transmission and for the design of systemic and mucosal vaccination against HIV.
In the present study, the levels of cell-free HIV-1 RNA and proviral DNA were determined in genital and systemic compartments of 30 clinically asymptomatic and treatment-naive African women by means of ultra-sensitive PCR-based techniques. Patients (pts) were selected from a cohort of 213 HIV-1 infected women (Bangui, Central African Republic) provided they were
non-pregnant and free of cervicitis, sexual transmitted diseases, genital bleeding, and seminal pollution in the genital tract.
Five (5) women (17%) only showed proviral DNA in vaginal secretions. HIV-1 RNA levels in plasma ranged from < 20 to 199,000 copies per ml (mean: 42,296 copies per ml), whereas vaginal amounts of HIV-1 RNA ranged from < 50 to 110,830 copies per ml (mean: 9,891 copies per ml). Amounts of HIV-1 DNA in blood ranged from < 5-943 copies per µg of cellular DNA (mean: 63 copies per µg), while genital proviral levels ranged from < 5-925 copies of HIV-1 DNA per µg of cellular DNA (mean: 91 copies per µg). Levels of HIV-1 RNA and HIV-1 proviral DNA in vaginal lavage samples were weakly correlated with those in paired blood samples (r = 0.45, p = 0.014; r= 0.37, p = 0.047, respectively). Levels of HIV-1 DNA in peripheral blood were strongly correlated with levels of plasma HIV-1 RNA (r = 0.76, p = 0.0001), whereas no such correlation was evidenced in vaginal secretions. Total HIV RNA loads in cervical mucus were
positively correlated with cell-free HIV RNA in vaginal lavage samples (r = 0.61, p = 0.005), but not with those in plasma.
Our results suggest that the basal replication dynamic of HIV in the female genital tract is relatively independent from that of peripheral blood, and that genital production of cell-free and cell-associated viruses are largely unrelated in treatment-naive women. These findings further document that the female genital tract is an independent compartment of HIV-1 replication, which may result in viral or proviral populations with important genetic differences from those found in peripheral blood at the chronic phase of the infection.
Abst. 456. L. Andreoletti, N. Chomont, G. Gresenguet, M. Matta M. Matta, P. Coudol, L. Belec. 1Fac de Med de Reims, France; INSERM U430, Paris, France; CNR des MST Bangui, Ctr Afrique; and HEGP/AP-HP, Paris, France
Determination of Viral DNA in T-cells and Monocytes/Macrophages in the Cervix and Blood of HIV+ Women
Heterosexual transmission is the predominant route of HIV infection. To further understand the dynamics of sexual transmission and levels of virus persistence following the initiation of HAART, CD4+ bearing cells capable of harbouring virus were isolated and the level of viral DNA quantitated.
Twenty-four (24) women without evidence of cervical inflammation or sexually transmitted infection were investigated in this prospective study.
Cervical intraepithelial cells were obtained using a cytobrush. Matched cervical and blood samples were obtained from HIV+ women who were therapy naive, receiving HAART with a low viral load (< 200 copies/mm3) and those women on HAART with a virus load > 200 copies/mm3. DNA was extracted from purified populations of T-cells and monocytes/macrophages, and the provirus load calculated using nested PCR. In addition, 4-colour flow cytometry was used to determine the relative proportion of leukocyte subpopulations. Significance was determined using a students t-test.
In the cervical epithelium, macrophages constituted the predominant leukocyte subset, whereas in blood T-cells were prevalent. Viral DNA in
blood resided mostly in T-cells; however, comparable levels of viral DNA was identified in T-cells and macrophages in the cervix irrespective of HAART
and viral load. The level of viral DNA in cervical preparations exceeded the levels observed in blood per 10,000 purified cells. This increase was evident in women not on therapy (p < 0.05), and those on therapy with a high viral load (p < 0.05).
These data suggest differences exist in the immunophentypic profile of leukocytes that reside within the cervical mucosa and blood. The higher
levels of viral DNA observed in the cervix indicates an important site for the transfer of virus from women to men irrespective of systemic viral load and
CD4 count. Thus, despite successful HAART the cervical mucosa remains a plausible reservoir of HIV infection driving local immunodeficiency and
potentially a site for the emergence of drug-resistant variants.
Abst. 457. M. Prakash, S. Patterson, F. Gotch, M. Kapembwa. Imperial Coll, London, UK
Human Urogenital Epithelial Cells Capture Cell-free HIV-1 and Transmit the Virus to CD4+ Cells-Implications to Mechanisms of Sexual Transmission
Sexual transmission of HIV-1 has become the dominant route of the epidemic, especially in parts of the world where preventative resources
are limited, therapeutics are inaccessible to the majority of the population, and genital-related illnesses are prevalent. However, despite profound
understanding of the life cycle of the virus, little is known on how the virus breaks through the genital mucosal barrier, disseminates to peripheral lymphoid tissues, and establishes systemic infection.
We investigated whether HIV-1 infected genital epithelial cells and how, subsequently, the virus disseminated to CD4+ cells by developing a
genital epithelial cell-based co-culture system to understand the sexual transmission of the virus.
We found that 1) HIV does not productively infect genital epithelial cells, instead the virus was sequestered by the cells via a specific receptor,
similar to DC-SIGN on dendritic cells; 2) the sequestered virus remains infectious for a prolonged period; 3) the epithelial cell-attached virus could be efficiently transmitted to CD4+ cells through cell-cell contact; and 4) M-tropic isolates appeared to be transmitted significantly more efficiently than
HIV-1 does not productively infect genital epithelial cells; instead the virus becomes sequestered by the cells and remains infectious for a prolonged period. The sequestered virus can efficiently infect CD4+ cells via cell-cell contact and M-tropic isolates are more readily transmitted to these CD4+ cells. We will discuss the mechanism of sexual transmission of HIV and selective viral transmission, its significance and implication for vaccine
development, and other preventative measures.
Abst. 458. Z. Wu, Z. Chen, D. Phillips. Univ of Pennsylvania, Philadelphia; Aaron Diamond AIDS Res Ctr, New York, NY; and Pop Council, New York, NY
Mucosal CCR5 Expression is Down-regulated in HIV Infection
CCR5 is a key receptor for HIV infection and has been identified on CD4+ lymphocytes in both blood and intestinal mucosa. In seronegative controls, CCR5 expression is increased in mucosa compared to in blood. Following HIV infection, the predominant viral tropism is for the CCR5 receptor and CCR5 expression in blood is increased. The purpose of this study was to determine whether HIV infection results in altered CCR5 co-receptor expression in intestinal mucosa.
Mononuclear cells from blood and intestinal mucosa were obtained from HIV-infected (n = 20) and control subjects (n = 8) and were assessed by flow cytometry for 1) Percentage CD4+/ CD45+, CD8+/CD45+ and CD4+/CCR5+ phenotypes and 2) Percentage and intensity of CCR5 expression on CD4+ cells. Data for cell phenotype are expressed as percentages and the CCR5 receptor intensity. Data for CCR5 median fluorescent intensity (MFI) were log transformed for statistical analysis and reflect the number of CCR5 antibody
molecules bound per cell.
Of the 20 subjects with HIV infection, the mean plasma viral load was 33,766 copies/ml plasma (range < 50-293,864), mean CD4 count was 318 (range 153-612) and all subjects were receiving combination antiretroviral therapy. CD4 lymphopenia was seen in both blood and mucosal samples. An increase in CD8+ cells was seen in both compartments. As previously reported, an increase in CD4+/CCR5+ cells was seen in blood from HIV-infected subjects. However, a significant decrease in CCR5 frequency and MFI was seen
in intestinal mucosal samples from the HIV group. Mean values by group are listed below.
In contrast to blood, the frequency and intensity of CCR5 expression on mucosal CD4+ lymphocytes is reduced in HIV-infected subjects. This may occur as a response to the increased mucosal levels of RANTES (the natural ligand of CCR5), which is increased in this population. Alternatively, infection of CCR5+ cells with HIV may lead to a selective loss of this cell population. Whatever the mechanistic basis of this observation, sufficient CCR5+ cells remain to facilitate further cycles of mucosal infection.
Abst. 459. McGowan*, J Elliott, P Taing, M Fuerst, J Boscardin, P Anton. David Geffen Sch of Med at UCLA, Los Angeles, CA