Cervicovaginal Lavage Research Articles

Increased levels of immune activation in the genital tract of healthy young women from sub-Saharan Africa

To determine whether healthy, young women in sub-Saharan Africa have a more activated immune milieu in the genital tract (i.e. activated CD4 T cells) than a similar population in the United States. A cross-sectional study nested in a phase 1 microbicide trial. Cervical cytobrushes were collected from 18 to 24-year-old women in San Francisco, California, USA (n = 18) and Kisumu, Kenya (n = 36) at enrollment into a phase 1 microbicide trial. All participants tested negative for HIV, herpes simplex virus 2, gonorrhea, chlamydia, and trichomonas, and had abstained from sex for at least 7 days prior to enrollment. Cryopreserved T-cell populations were assayed by flow cytometry in a central laboratory. Secretory leukocyte protease inhibitor levels were assayed in cervicovaginal lavage samples. The Wilcoxon rank-sum test was used to compare immune parameters between sites. The total number of endocervical CD4(+) T cells was slightly higher in participants from San Francisco, but participants from Kisumu had a substantially higher number and proportion of CD4(+) T cells expressing the early activation marker CD69, with and without the HIV coreceptor C-C chemokine receptor type 5, and a greater proportion of activated CD8(+) T cells. Median (interquartile range) genital levels of secretory leukocyte protease inhibitor were lower in participants from Kisumu compared with those from San Francisco [190 (96-519) vs. 474 (206 817) pg/ml, P < 0.03]. Activated mucosal T cells were increased in the genital tract of young, sexually transmitted infection/HIV-free Kenyan women, independent of common genital coinfections, and secretory leukocyte protease inhibitor levels were reduced. The cause of these mucosal immune differences is not known, but could partly explain the high HIV incidence in young women from sub-Saharan Africa.

Mannose-binding lectin is produced by vaginal epithelial cells and its level in the vaginal fluid is influenced by progesterone

Mannose-binding lectin (MBL) is a recognition molecule of the complement (C) system and binds to carbohydrate ligands present on a wide range of pathogenic bacteria, viruses, fungi, and parasites. MBL has been detected in the cervico-vaginal cavity where it can provide a first-line defence against infectious agents colonizing the lower tract of the reproductive system. Analysis of the cervico-vaginal lavage (CVL) obtained from 11 normal cycling women at different phases of the menstrual cycle revealed increased levels of MBL in the secretive phase. Part of this MBL derives from the circulation as indicated by the presence of transferrin in CVL tested as a marker of vascular and tissue permeability. The local synthesis of MBL is suggested by the finding that its level is substantially higher than that of transferrin in the secretive phase. The contribution of endometrium is negligible since the MBL level did not change before and after hysterectomy. RT-PCR and in situ RT-PCR analysis showed that the vaginal tissue, and in particular the basal layer of the epithelium, is a source of MBL which binds to the basal membrane and to cells of the outer layers of the epithelium. In conclusion, we have shown that MBL detected in CVL derives both from plasma as result of transudation and from local synthesis and its level is progesterone dependent increasing in the secretive phase of the menstrual cycle.

Prevalence of human papillomavirus genotypes in HIV-1-infected women in Seattle, USA and Nairobi, Kenya: results from the Women's HIV Interdisciplinary Network (WHIN)

HIV-infected women have a high prevalence of human papillomavirus (HPV) infection and are more likely to be infected with HPV genotypes that are considered high-risk and have the potential for progressing to cervical cancer. The currently available HPV vaccines protect against specific HPV genotypes that may not be the most important causes of dysplasia and potentially of cervical cancer in HIV-1-infected women. African women have been underrepresented in the studies of global prevalence of HPV genotypes. We compared the HPV genotype distribution in HIV-1-infected women from Seattle, Washington, USA and Nairobi, Kenya. The reverse line blot assay and DNA sequencing on cervicovaginal lavage (CVL) specimens were carried out. The most commonly detected HPV types among the women from Seattle were HPV 56, 66, MM8, and 81; in contrast HPV 53, 33, and 58 were the most common HPV genotypes detected in the CVL specimens from the women in the Nairobi cohort. The HPV types associated with low-grade squamous intraepithelial lesions (LSIL) were HPV 53 and HPV 56. HPV types 58, 52, and 16 were associated with high-grade squamous intraepithelial lesions (HSIL). A better understanding of HPV genotype distribution in the most affected regions of the world is essential to planning effective vaccine strategies if we are unable to demonstrate cross-protection between HPV genotypes included in the present vaccines and those prevalent in the different populations.

Anti-HIV Activity in Cervical-Vaginal Secretions from HIV-Positive and -Negative Women Correlate with Innate Antimicrobial Levels and IgG Antibodies

BackgroundWe investigated the impact of antimicrobials in cervicovaginal lavage (CVL) from HIV(+) and HIV(−) women on target cell infection with HIV. Since female reproductive tract (FRT) secretions contain a spectrum of antimicrobials, we hypothesized that CVL from healthy HIV(+) and (−) women inhibit HIV infection.Methodology/Principal FindingsCVL from 32 HIV(+) healthy women with high CD4 counts and 15 healthy HIV(−) women were collected by gently washing the cervicovaginal area with 10 ml of sterile normal saline. Following centrifugation, anti-HIV activity in CVL was determined by incubating CVL with HIV prior to addition to TZM-bl cells. Antimicrobials and anti-gp160 HIV IgG antibodies were measured by ELISA. When CXCR4 and CCR5 tropic HIV-1 were incubated with CVL from HIV(+) women prior to addition to TZM-bl cells, anti-HIV activity in CVL ranged from none to 100% inhibition depending on the viral strains used. CVL from HIV(−) controls showed comparable anti-HIV activity. Analysis of CH077.c (clone of an R5-tropic, mucosally-transmitted founder virus) viral inhibition by CVL was comparable to laboratory strains. Measurement of CVL for antimicrobials HBD2, trappin-2/elafin, SLPI and MIP3α indicated that each was present in CVL from HIV(+) and HIV(−) women. HBD2 and MIP3α correlated with anti-HIV activity as did anti-gp160 HIV IgG antibodies in CVL from HIV(+) women.Conclusions/SignificanceThese findings indicate that CVL from healthy HIV(+) and HIV(−) women contain innate and adaptive defense mechanisms that inhibit HIV infection. Our data suggest that innate endogenous antimicrobials and HIV-specific IgG in the FRT can act in concert to contribute toward the anti-HIV activity of the CVL and may play a role in inhibition of HIV transmission to women.

Effect of Non-Nucleoside Reverse Transcriptase Inhibitors on Cytokine, Chemokine, and Immunoglobulin Profiles in Serum and Genital Secretions of HIV-Infected Women

Non-protease inhibitor-based antiretroviral therapy (ART) is widely accepted as first-line ART in developing countries. Although reverse transcriptase inhibitor-based regimens have been studied in the peripheral blood, no studies have analyzed alterations in cytokine and chemokine levels, together in peripheral blood and genital secretions. Forty HIV-infected women with CD4 cell counts <200 cells/mm(3), asymptomatic, with no genital tract infection, willing to participate in the study, and adhere to ART were enrolled. Cervicovaginal lavage (CVL) was collected in the mid-cycle phase of menstrual cycle. Patients were initiated with reverse transcriptase-based antiretrovirals. Repeat sampling was performed at 24 weeks. Cytokines and chemokines were measured using ultrasensitive ELISA kits. Viral load declined to undetectable levels in 29 patients in the blood and in 33 cases in the CVL. Proinflammatory cytokines (tumor necrosis factor-alpha [TNF-alpha, interleukin-6 [IL-6], IL-1beta) in the serum and CVL showed a significant decrease in mean levels after therapy. IL-2 levels increased significantly whereas IL-12 and (IFN-gamma decreased in both compartments. Mean levels of IL-4 and IL-10 decreased significantly in the serum. There was direct correlation between serum and CVL levels of IL-2 and IL-10. IL-10 had a negative correlation with CD4% at baseline and 6 months of therapy. Mean levels of all alpha- and beta-chemokines decreased in serum after therapy. In CVL, mean levels of MIP-1alpha, RANTES, and IL-8 reduced and SDF-1alpha increased significantly (P value <0.001). Serum levels of all the cytokines, except IL-2, and all chemokines prior to therapy, were significantly higher than healthy controls. In CVL, mean levels of TNF-alpha, IL-6, IL-1beta, IL-12, IFN-gamma, IL-10, RANTES, and IL-8 were significantly higher, whereas IL-2, MIP-1alpha, and MIP-1beta were significantly lower than healthy controls. The mean levels of proinflammatory cytokines and chemokines significantly decreased in serum and CVL after therapy, possibly due to reduced viral load.

Long‐Term Impact of Acyclovir Suppressive Therapy on Genital and Plasma HIV RNA in Tanzanian Women: A Randomized Controlled Trial

Herpes simplex virus (HSV) suppressive therapy reduces genital and plasma human immunodeficiency virus type 1 (HIV-1) RNA over periods up to 3 months, but the long-term effect is unknown. A total of 484 HIV-1 and HSV type 2 seropositive Tanzanian women aged 16-35 years were enrolled in a randomized placebo-controlled trial of acyclovir administered at a dosage of 400 mg twice daily. Cervico-vaginal lavage and blood samples were collected at 6 months, 12 months, and 24 months for quantification of genital and plasma HIV-1 RNA and genital HSV DNA. Primary outcomes were detection and quantity of cervico-vaginal HIV-1 RNA at 6 months. At 6 months, there was little difference between the acyclovir and placebo arms for cervico-vaginal HIV-1 RNA detection (88 [ 41 .3%] of 213 vs 84 [ 44 .0%] of 191; odds ratio [OR], 0.90; 95% confidence interval [CI], 0.60-1.33), HSV DNA detection (20 [ 9 .4%] of 213 vs 22 [ 11 .5%] of 191; OR, 0.80; 95% CI, 0.42-1.51), genital HIV or HSV loads, or plasma HIV-1 RNA load. Estimated median adherence was 91%. There was a suggestion of an impact on cervico-vaginal HIV-1 RNA detection among women with estimated adherence 90% (OR, 0.74; 95% CI, 0.50-1.09) when data from all 3 visits were included. Acyclovir administered at a dosage of 400 mg twice daily is unlikely to be a useful long-term intervention to reduce HIV transmission. The lack of effect on HIV may be attributable to suboptimal adherence or treatment regimen.

HIV genital shedding and safety of Carraguard use by HIV-infected women: a crossover trial in Thailand

To evaluate the safety, including impact on genital HIV RNA shedding, of Carraguard vaginal gel in HIV-infected women. This is a randomized, controlled, crossover study of Carraguard in HIV-infected women in Thailand. Each woman (CD4 cell count 51-500 cells/microl and not on antiretroviral therapy) used each treatment (Carraguard, methylcellulose placebo, and no-product) once daily for 7 days during each 1-month period (3-week wash-out). Women were randomized to one of the six possible treatment sequences. Safety assessments were conducted at baseline (pregel), 15 min postgel, day 7, and day 14, and included HIV RNA measurements in cervicovaginal lavage (CVL) specimens. Sixty women were enrolled, and 99% of scheduled study visits were completed. At baseline, median age (34 years), CD4 lymphocyte count (296 cells/microl), plasma HIV viral load (4.6 log10 copies/ml), CVL HIV viral load (3.1 log10 total copies per CVL), and sexual behaviors were similar among randomization groups. HIV viral load, leukocyte and hemoglobin levels, and epithelial cell counts in CVLs were lower 15 min after application of Carraguard or placebo compared with no product; CVL HIV viral load was still lower at day 7 but returned to baseline by day 14. Carraguard use was not associated with prevalent or incident genital findings or abnormal vaginal flora. Carraguard appears to be well tolerated for once-daily vaginal use by HIV-infected women. The observed reduction in CVL HIV viral load in the gel months may be clinically relevant but could have resulted from interference with sample collection by study gels.

Human Immunodeficiency Virus Infection in Alabama Women: Sociodemographic, Behavioral, and Reproductive Health Characteristics and Factors Associated With Lack of Human Immunodeficiency Virus-1 Viral Control

BackgroundHuman immunodeficiency virus (HIV) infection among women in the southern United States is on the rise. This study examined sociodemographic profile and behavioral risk factors for HIV and sexually transmitted infections and assessed factors associated with HIV-1 viral control in a cohort of 280 HIV-infected Alabama women aged 17 to 66years. MethodsWomen receiving care for HIV infection at a university outpatient HIV clinic were enrolled in the study. Women completed a self-administered questionnaire on demographics and behavioral risk factors at enrollment. They were followed up with appointments at least every 6months with Papanicolaou smears, cervicovaginal lavages, cervical and vaginal swabs, and blood specimens collected at each visit. ResultsOf the women in the study, 69% were black, had mean age of 36years, and~three fourths were mothers with annual household income<$20,000. White women were likely to have been HIV infected for a longer period (50.2 versus 36.3months; P=0.02) and had significantly lower viral loads at enrollment (P=0.04) than black women. Factors associated with lack of HIV-1 control (≥10,000 RNA copies/mL) at enrollment included black race/ethnicity (odds ratio [OR]: 2.8; 95% confidence interval [CI]: 1.2−6.8), CD4+ T-cell count <200 cells/μL (OR: 20.1; CI: 8.6−47.0), being diagnosed with HIV <6months (OR: 3.5; CI: 1.4−8.9) and not being on any antiretroviral therapy (OR: 2.5; CI: 1.1−5.7). ConclusionsPoorer HIV-1 viral control in black women at enrollment may indicate suboptimal access to HIV testing, delays in receipt of medical care after HIV-1 diagnosis, and/or some underlying biologic or social race-related influence.

Postcoital Bioavailability and Antiviral Activity of 0.5% PRO 2000 Gel: Implications for Future Microbicide Clinical Trials

BackgroundThe pharmacokinetics and pharmacodynamics of vaginal microbicides are typically assessed among sexually abstinent women. However, the physical act of sex may modulate gel distribution, and preclinical studies demonstrate seminal plasma interferes with the antiviral activity of several microbicides. This study compared the biological activity and concentration of PRO 2000 in cervicovaginal lavage (CVL) collected in the absence or following coitus.MethodsCVL samples were collected from ten heterosexual couples at baseline, after sex, after a single dose of 0.5% PRO 2000 gel and sex, and after gel application without sex. The impact of CVL on HIV-1 infection of TZM-bl cells and HSV-2 infection of CaSki cells was monitored by luciferase and plaque assay, respectively. PRO 2000 concentrations were measured by fluorescence.ResultsCVL collected after PRO 2000 application significantly inhibited HIV-1 and HSV-2 (p = 0.01). However, the antiviral activity was reduced following sex and no significant protective effect was observed in postcoital CVL obtained in the presence compared to the absence of PRO 2000 for HIV (p = 0.45) or HSV-2 (p = 0.56). Less PRO 2000 was recovered in postcoital CVL, which, in conjunction with interference by seminal plasma, may have contributed to lower antiviral activity.ConclusionsPostcoital responses to PRO 2000 differ from precoital measures and the results obtained may provide insights into the clinical trial findings in which there was no significant protection against HIV-1 or HSV-2. Postcoital studies should be incorporated into clinical studies before embarking on large-scale efficacy trials.

Trappin‐2/Elafin: a novel innate anti‐human immunodeficiency virus‐1 molecule of the human female reproductive tract

Trappin-2/Elafin is a serine protease inhibitor that plays a major role as an anti-inflammatory mediator at mucosal surfaces. In addition, Trappin-2/Elafin has antibacterial activity against Gram-positive and Gram-negative bacterial and fungal pathogens. In this study we examined the production of Trappin-2/Elafin by epithelial cells from the human upper and lower female reproductive tract as well as its activity as an anti-human immunodeficiency virus (HIV)-1 molecule. We found that primary uterine, Fallopian tube, cervical and ectocervical epithelial cells produce Trappin-2/Elafin constitutively and that production of Trappin-2/Elafin is enhanced following stimulation with Poly(I:C), especially by the uterine cells. Given the presence of Trappin-2/Elafin in the reproductive tract, we tested the ability of recombinant Trappin-2/Elafin to inhibit HIV-1, an important sexually transmitted pathogen. We found that recombinant Trappin-2/Elafin was able to inhibit both T-cell-tropic X4/IIIB and macrophage-tropic R5/BaL HIV-1 in a dose-dependent manner. The inhibitory activity was observed when virus was incubated with Trappin-2/Elafin but not when Trappin-2/Elafin was added to cells either before infection or after infection. This suggests that the mechanism of inhibition is likely to be a direct interaction between HIV-1 and Trappin-2/Elafin. Additionally, we measured the levels of secreted Trappin-2/Elafin in cervico-vaginal lavages (CVL) from both HIV-positive and HIV-negative women and found that average levels of secreted Trappin-2/Elafin were higher in the CVL from HIV-negative women, although the values did not reach statistical significance. We also found that women at the secretory phase of the menstrual cycle produced more Trappin-2/Elafin in CVL relative to women at the proliferative phase of the menstrual cycle. Our data suggest that Trappin-2/Elafin might be an important endogenous microbicide of the female reproductive tract that is protective against HIV-1.

ORIGINAL ARTICLE: Female Genital Tract Secretions Inhibit Herpes Simplex Virus Infection: Correlation with Soluble Mucosal Immune Mediators and Impact of Hormonal Contraception

Female genital tract secretions inhibit herpes simplex virus (HSV) infection, however, the intra- and inter-subject variability, contribution of specific mediators, and impact of reproductive hormones have not been defined. of study Cervicovaginal lavage (CVL) (n = 89) obtained from nine cyclers and seven women on hormonal contraception (HC), who completed between three and eight weekly visits, were examined for anti-herpes simplex virus activity and concentrations of mediators. The CVL inhibited HSV infection by a mean value of approximately 57% during the follicular or luteal phase, but only by 36% in hormonal contraceptive users. Human neutrophil peptides 1-3 (HNP1-3) (P = 0.03), IL-8 (P = 0.003), lactoferrin (P = 0.005), lysozyme (P = 0.003), IgA (P = 0.002), and IgG (P = 0.02) correlated with antiviral activity. Intra-subject and inter-subject variability was observed, suggesting that factors other than hormones contribute to innate defense. Endogenous antimicrobial activity may provide a biomarker of healthy mucosal immunity and may be reduced in the setting of HC. However, larger prospective studies are needed.

HPV testing on self collected cervicovaginal lavage specimens as screening method for women who do not attend cervical screening: cohort study

Objective To determine whether offering self sampling of cervicovaginal material for high risk human papillomavirus (HPV) testing is an effective screening method for women who do not attend regular cervical...

Targeting Trojan Horse leukocytes for HIV prevention

…. is sure design'd, by fraud or force: trust not their presents, nor admit the horse. Virgil, Aeneid Human immunodeficiency virus type 1 (HIV-1), the lymphotropic virus that causes AIDS, has infected more than 60 million people worldwide since its clinical appearance in 1981. Despite intensive prevention efforts, the HIV/AIDS epidemic continues to spread, particularly in developing countries in sub-Saharan Africa, southeast Asia and the Caribbean, as well as the developed world [1]. Although HIV can be transmitted very efficiently parenterally, the advent of routine blood screening prior to transfusion and harm reduction programs for injection drug users, have made this mode of transmission much less common than mucosal transmission. Most new HIV infections are attributable to mucosal transmission: through genital and rectal mucosae in the case of sexual transmission and through oral or gastrointestinal mucosae in the case of mother-to-child transmission [2]. Much has been learned about HIV pathogenesis and infection mechanisms at the molecular level, but the scientific community has yet to develop an effective vaccine or microbicide for HIV prevention. Many unanswered questions remain concerning HIV-1 sexual transmission. In 1983, barely 2 years into the AIDS epidemic, we hypothesized that the agent that was subsequently identified as HIV-1 may be sexually transmitted by infected ‘Trojan Horse’ leukocytes in semen [3]. This hypothesis was based on our knowledge at the time that human semen contains substantial numbers of T lymphocytes and macrophages, which could host a T-cell tropic virus, and the following assumptions: intracellular virus would be better protected than free virus from adverse effects of antiviral factors in the genital environment such as antiviral antibodies likely to be present in genital secretions of the virus-infected transmitter, as well as antimicrobial peptides that play an important role in genital innate immune defense; and virus-infected allogeneic cells could also escape early detection by major histocompatibility complex (MHC)-restricted cytotoxic T cells in a new host. Over the intervening 25+ years, others have also championed this cause [4,5], and convincing evidence has emerged from clinical research as well as in-vitro and animal studies that infected leukocytes indeed play a role in HIV transmission. Yet, most recent research on sexual HIV transmission has focused on cell-free HIV in genital secretions because of the wide availability of HIV RNA quantification assays. Furthermore, the majority of HIV vaccines and microbicides have been designed to block transmission of cell-free virus and have been tested in animal and in-vitro models that use cell-free virus as the only infectious inoculum. As the molecular events underlying cell-associated HIV transmission differ from those underlying cell-free virus transmission, many of the current vaccine and microbicide candidates might not be expected to protect against cell-associated HIV transmission. The failure of several recent vaccine and microbicide clinical trials may be due in part to this oversight. It should be possible to design strategies that block cell-associated HIV transmission as well as cell-free HIV transmission. In this article, we present an overview of research that has been conducted on cell-associated HIV mucosal transmission and recommendations for future research. We focus on sexual HIV transmission, but this review also has relevance for mother-to-child HIV transmission, which may occur through mucosal transmission of cell-associated HIV from maternal genital or mammary gland secretions [6–8]. We review published reports that describe and enumerate HIV-infected cells in genital secretions, and compelling evidence from clinical, animal and in-vitro studies demonstrating that such cells can transmit HIV across genital tract epithelial surfaces; potential molecular mechanisms underlying cell-associated HIV transmission that could be specifically targeted by future HIV prevention strategies; and in-vitro and animal cell-associated HIV transmission models currently used for studies on cell-associated HIV transmission mechanisms and for testing vaccine and microbicide candidates. Using this information as a foundation, we discuss the evidence and probability that various current microbicide and vaccine approaches prevent cell-associated HIV transmission, and suggest additional microbicide and vaccine concepts and experiments that will move this field forward.

Exploring the acceptability of two self-sampling devices for human papillomavirus testing in the cervical screening context: a qualitative study of Muslim women in London

We explored Muslim women's attitudes to self-sampling for human papillomavirus (HPV) in the context of cervical cancer screening and their responses to two self-sampling devices. A Muslim community centre in north-east London. Following a talk given on the subject of cervical cancer and HPV at the community centre, 28 women were recruited to take part in three focus group discussions. The discussion covered cervical screening, self-sampling and HPV testing. Women were also asked for their responses to a swab self-sampling kit and a cervico-vaginal lavage device. Discussions were recorded and transcribed verbatim and the qualitative data were analysed using Framework Analysis. Participants were generally positive about cervical screening but acknowledged that some women in their community were reluctant to attend because of embarrassment, language difficulties, fear or because they were unmarried and did not want to communicate implicit messages about being sexually active. Self-sampling met a mixed response - women were concerned about not doing the test correctly, but thought that it might overcome barriers to screening for some women. HPV testing itself was thought to raise potentially difficult issues relating to trust and fidelity within marriages. Although most women said they would prefer to continue to have screening by a health professional, if they were to perform self-sampling, there was overwhelming preference for the swab over the lavage kit. There was limited enthusiasm for self-sampling in this group of Muslim women who had mostly attended for cervical screening, but a clear preference for a swab rather than a cervico-vaginal lavage.

Importance of specimen type in detecting human papillomavirus DNA from the female genital tract

HPV testing is a valuable tool in cervical cancer screening and efficacy assessment of HPV vaccines. Concordance of specimens from three sites for detection of HPV DNA in the female genital tract was evaluated. At a single visit, the following specimens were collected: an endo-ecto-cervical swab (EEC), labial/vulvar/perineal/perianal swab (LVPP) and cervicovaginal lavage (CVL). Specimens were evaluated with HPV6, HPV11, HPV16, and HPV18 type- and gene-specific PCR assays. Of the 898 women evaluated at baseline, 232 were HPV PCR positive in at least one specimen. Of these, for HPV6, HPV11, HPV16, and HPV18, respectively, throughout: (a) 70.4%, 40.0%, 65.3%, and 64.1% tested three-site positive; (b) 13.6%, 30.0%, 19.7%, and 18.8% tested two-site positive; and (c) 16.4%, 30.0%, 15.0%, and 17.2% tested single-site positive. For patients who tested single-site positive for HPV6, HPV11, HPV16, or HPV18, respectively, the specimen was: LVPP in 92.3%, 33.3%, 68.2%, and 72.7%; EEC in 0.0%, 33.3%, 18.2%, and 9.1%; and CVL in 7.7%, 33.3%, 13.6%, and 18.2%. Combining results of swab specimens together increases detection of HPV6, HPV11, HPV16, and HPV 18, respectively, to 98.7%, 90.0%, 97.9%, and 96.9%. HPV DNA is detectable from all three sites using type-specific PCR assays; most women who tested positive for a given HPV type were positive for that type in all three specimens.

Human Papillomavirus Infection and Cervical Cytology in HIV‐Infected and HIV‐Uninfected Rwandan Women

Data on human papillomavirus (HPV) prevalence are essential for developing cost-effective cervical cancer prevention programs. In 2005, 710 human immunodeficiency virus (HIV)-positive and 226 HIV-negative Rwandan women enrolled in an observational prospective cohort study. Sociodemographic data, CD4+ cell counts, and cervical specimens were obtained. Cervicovaginal lavage specimens were collected from each woman and tested for >40 HPV types by a polymerase chain reaction assay; HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 were considered primary carcinogenic HPV types. The prevalence of HPV was higher in HIV-positive women than in HIV-negative women in all age groups. Among HIV-infected women, 69% were positive for >or=1 HPV type, 46% for a carcinogenic HPV type, and 10% for HPV-16. HPV prevalence peaked at 75% in the HIV-positive women aged 25-34 years and then declined with age to 37.5% in those >or=55 years old (Ptrend<.001). A significant trend of higher prevalence of HPV and carcinogenic HPV with lower CD4+ cell counts and increasing cytologic severity was seen among HIV-positive women. We found a higher prevalence of HPV infection in HIV-positive than in HIV-negative Rwandan women, and the prevalence of HPV and carcinogenic HPV infection decreased with age.

142 HIV Frequently Elicits Mucosal and Plasma Env-Specific IgA With a Rapid Initial Decline In Acute Infection

An optimal HIV vaccine would elicit protective immune responses, preferably neutralizing antibodies, at mucosal surfaces in order to prevent infection. One key to eliciting vaccine-induced antibody responses at mucosal surfaces is to understand what antibodies are present at these sites soon after transmission during acute HIV infection (AHI). However, past attempts to detect HIV-specific IgA in mucosal specimens have been largely unsuccessful. The goal of this project is to characterize the binding antibody reponses elicited during AHI in plasma and mucosal (semen and cervicovaginal lavage (CVL)) samples compared to chronic infection with respect to antibody isotype, specificity, and timing in the CHAVI 001 acute infection cohort. To determine the presence of binding antibodies to HIV envelope proteins (gp140 consensus oligomers and recombinant gp41) and p55 gag protein, a standardized colorimetric ELISA as well as a customized Luminex assay were performed. Luminex is a bead-based flow cytometric assay that we customized to create a highly sensitive multiplexed assay for determining HIV-specific isotypes in plasma and mucosal specimens. Our results are the first to demonstrate the presence of HIV-specific IgA in approximately 88% of mucosal samples obtained during AHI, whereas approximately 33% of samples from chronically infected patients were positive for HIV-specific IgA. Each of the AHI patients had env-specific mucosal IgA, some of which mapped to the immunodominant region of gp41. High levels of HIV-specific IgG were also observed in the acute and chronic mucosal samples while HIV-specific IgM was undetectable. Furthermore, HIV env-specific plasma and mucosal IgA levels rapidly declined over time. In addition, in approximately 40% of AHI patients, systemic levels of p55 gag-specific IgA rose over the course of the infection despite the decrease in env-specific IgA. The rapid decline in HIV env-specific IgA suggests mucosal B cells may lose the capacity for env-specific IgA production during the course of AHI. Furthermore, the concurrent rise in systemic gag-specific IgA levels over time may be a reflection of HIV env-mediated differential regulation of IgA production in activated mucosal B cells. Future work includes functional studies to determine the role of HIV-specific IgA in AHI versus chronic infection as well as more epitope mapping to determine the specificity of isotypes in mucosal samples. These results have important implications in our understanding of how HIV infection regulates mucosal B cell responses and the barriers that need to be overcome by an HIV vaccine.

The Differential Binding and Activity of PRO 2000 Against Diverse HIV-1 Envelopes

PRO 2000 is a polyanionic microbicide that binds directly to the glycoprotein 120 (gp120) envelope protein to inhibit HIV-1 entry. We studied the breadth of PRO 2000 activity against HIV-1 derived from recently transmitted R5 viruses. We also investigated the interaction of this compound with X4 and R5 HIV-1 envelope glycoproteins using an epitope-mapping strategy. The anti-HIV activity of PRO 2000 against subtype B and C Env-pseudotyped viruses was assessed in saline and cervicovaginal lavage fluid. Competitive binding assays were performed with X4 and R5 monomeric and virus-associated gp120. PRO 2000 was found to be active against recently transmitted subtype B and C viruses tested in vitro, however, at 1 microg/mL in saline, activity against subtype C was decreased compared with subtype B. Epitope mapping using anti-V3 region antibodies showed that PRO 2000 binds to the V3 region of monomeric and virus-associated X4 gp120 with a higher affinity than to V3 of R5 gp120. In contrast, the interaction of PRO 2000 with the CD4-binding site was similar for both X4 and R5 monomeric and virus-associated gp120. PRO 2000 has significant activity against recently transmitted viruses, although some activity is lost at low concentrations. Epitope binding studies suggest that this broad activity is due to direct and indirect interactions with multiple gp120 sites rather than V3 binding alone.

Human Immunodeficiency Virus (HIV) Antigens and RNA in HIV-Seronegative Women with Cervical Intraepithelial Neoplasia

While investigating whether proteins retrieved by cervicovaginal lavages (CVL) from women with cervical intraepithelial neoplasia (CIN) might correlate with risk of progression to invasive cervical cancer, we unexpectedly identified HIV gag and env glycoprotein in CVL from women with HIV-negative serology. HIV antigens were consistently identified by mass spectrometry (MS) in CVL from 4 women but were absent in CVL from the remaining 16 women. HIV serologies of all 20 patients were negative for both HIV-1 and HIV-2 antibodies. To validate the unexpected MS findings we performed Western blot (WB) and immunoaffinity chromatography (IC) analysis of CVL for HIV proteins, viral load assays of paired CVL and blood samples, and immunohistochemical HIV p24 expression in cervical biopsy specimens. WB analysis of CVL for prostate-specific antigen (PSA) was performed to exclude semen contamination as the source of HIV proteins. WB and IC results demonstrated the presence of HIV-1 gp41 and p24 antigens in four CVL that were identified by MS to have the HIV proteins. Despite negative serology, HIV RNA in CVL and HIV p24 in cervix biopsies were detected in patients with HIV antigen-positive CVL. HIV p24-positive CVL were PSA negative. All 20 subjects remained HIV seronegative throughout the study. Women with HIV proteins and RNA were comparatively older. Our findings suggest that CVL HIV proteins in women with CIN could be markers for unrecognized HIV exposure or subclinical infection. Proteomic screening of cervical secretions may be useful in identifying seronegative women exposed to HIV and/or at risk for AIDS.

Genetic Variants in TAP Are Associated with High-Grade Cervical Neoplasia

The transporter associated with antigen processing (TAP) is essential in assembling MHC-I proteins. Human papillomavirus (HPV) evades immune recognition by decreasing class I MHC cell surface expression through down-regulation of TAP1 levels. Consistent with heterogeneity in MHC expression is the individual variability in clearing detectable HPV infections. Genetic polymorphisms in TAP genes may affect protein structure, function, and the ability to clear HPV infection. Case-control study of women with cervical intraepithelial neoplasia (CIN) II or III (n = 114) and women without high-grade CIN (n = 366). Five nonsynonymous single nucleotide polymorphisms (SNP) in TAP1 and TAP2 were genotyped using DNA collected in cervicovaginal lavage samples using microsphere array technology (Luminex xMAP). HPV typing was done using a PCR-based system with MY09/MY11 primers. TAP1 and TAP2 SNPs were validated by direct sequencing. Differences in allele distribution between women with high-grade cervical neoplasia and women without was seen for TAP1 I333V (P = 0.02) and TAP1 D637G (P = 0.01). The odds ratios (OR) for CIN III were significantly lower among carriers of the TAP1 I333V polymorphism (OR, 0.28; 95% confidence interval, 0.1-0.8), and TAP1 D637G polymorphism (OR, 0.27; 95% confidence interval, 0.1-0.7). These associations remained significant even after restricting the evaluation to women who were positive for high-risk HPV types. In addition to the down-regulation of MHC-1 by oncogenic HPV, HPV pathogenesis might be facilitated by polymorphisms in the TAP proteins. Identifying TAP polymorphisms may potentially be used to identify women less susceptible to progression to high-grade CIN and cervical cancer.