Bacteria associated with BV increase genital-tract HIV RNA levels. Quantitative bacterial counts for lactobacilli and M. hominis are better correlates of CVL HIV RNA than are Nugent score or Amsel criteria. Since plasma virus and CD4 cell levels did not differ between the BV and no-BV groups, these data suggest that the bacterial flora associated with BV influence genital-tract HIV shedding.

Objective Lactobacillus dominates the lower genital tract microbiota of many women, producing a low vaginal pH, and is important for healthy pregnancy outcomes and protection against several sexually transmitted pathogens. Yet, factors that promote Lactobacillus remain poorly understood. We hypothesized that the amount of free glycogen in the lumen of the lower genital tract is an important determinant of Lactobacillus colonization and a low vaginal pH.MethodsFree glycogen in lavage samples was quantified. Pyrosequencing of the 16S rRNA gene was used to identify microbiota from 21 African American women collected over 8–11 years.ResultsFree glycogen levels varied greatly between women and even in the same woman. Samples with the highest free glycogen had a corresponding median genital pH that was significantly lower (pH 4.4) than those with low glycogen (pH 5.8; p<0.001). The fraction of the microbiota consisting of Lactobacillus was highest in samples with high glycogen versus those with low glycogen (median = 0.97 vs. 0.05, p<0.001). In multivariable analysis, having 1 vs. 0 male sexual partner in the past 6 months was negatively associated, while BMI ≥30 was positively associated with glycogen. High concentrations of glycogen corresponded to higher levels of L. crispatus and L. jensenii, but not L. iners.ConclusionThese findings show that free glycogen in genital fluid is associated with a genital microbiota dominated by Lactobacillus, suggesting glycogen is important for maintaining genital health. Treatments aimed at increasing genital free glycogen might impact Lactobacillus colonization.

SummaryThis study investigates whether cell-derived glycosylphosphatidylinositol-linked complement control proteins CD55 and CD59 can be incorporated into HIVol virions and contribute to complement resistance. Virus was prepared by transfection of cell lines with pNL4-3, and primary isolates of HIV-1 were derived from patients' PBMCs. Virus was tested for sensitivity to complementmediated virolysis in the presence of anti-gp160 antibody. Viral preparations from JY33 cells, which lack CD55 and CD59, were highly sensitive to complement. HIV-1 preparations from H9 and U937 cells, which express low levels of CD55 and CD59, had intermediate to high sensitivity while other cell line-derived viruses and primary isolates of HIV-1 were resistant to complement-mediated virolysis. Although the primary isolates were not lysed, they activated complement as measured by binding to a complement receptor positive cell line. While the primary isolates were resistant to lysis in the presence of HIV-specific antibody, antibody to CD59 induced lysis. Likewise, antibody to CD55 and CD59 induced lysis of cell line-derived virus. Western blot analysis of purified virus showed bands corresponding to CD55 and CD59. Phosphatidylinositol-specific phospholipase C treatment of either cell line-derived or primary isolates of HIV-1 increased sensitivity to complement while incubation of sensitive virus with purified CD55 and CD59 increased resistance to complement. These results show that CD55 and CD59 are incorporated into HIV-1 particles and function to protect virions from complementmediated destruction, and they are the first report of host cell proteins functioning in protection of HIV-1 from immune effector mechanisms.

Lactobacillus colonization of the lower female genital tract provides protection from the acquisition of sexually transmitted diseases, including human immunodeficiency virus, and from adverse pregnancy outcomes. While glycogen in vaginal epithelium is thought to support Lactobacillus colonization in vivo, many Lactobacillus isolates cannot utilize glycogen in vitro. This study investigated how glycogen could be utilized by vaginal lactobacilli in the genital tract. Several Lactobacillus isolates were confirmed to not grow in glycogen, but did grow in glycogen-breakdown products, including maltose, maltotriose, maltopentaose, maltodextrins, and glycogen treated with salivary α-amylase. A temperature-dependent glycogen-degrading activity was detected in genital fluids that correlated with levels of α-amylase. Treatment of glycogen with genital fluids resulted in production of maltose, maltotriose, and maltotetraose, the major products of α-amylase digestion. These studies show that human α-amylase is present in the female lower genital tract and elucidates how epithelial glycogen can support Lactobacillus colonization in the genital tract.

Human immunodeficiency virus type 1 (HIV-1) can be either resistant or sensitive to complementmediated destruction depending on the host cells. Incorporation of different levels of host cell CD46, CD55 and CD59 may account for this differential sensitivity to complement. However, it has not been determined whether CD46, CD55 and CD59 can all be incorporated at levels which protect virions. To determine whether each of these proteins can protect HIV-1, virions were derived from CHO cells expressing either human CD46, CD55 or CD59. Virions were shown to incorporate both glycosyl phosphatidylinositol (GPI)-anchored CD55 and CD59 as well as transmembrane CD46. Importantly, all three virus preparations were significantly more resistant to complement lysis than control virus. This study demonstrates that HIV-1 incorporates both transmembrane and GPI-anchored complement control proteins from host cells and that both types of protein increase complement resistance of virus.

Background This study investigated whether HIV-infection is associated with a change in diversity of genital microbiota in women. Methods Amplicon length heterogeneity PCR (LH-PCR) and pyrosequencing of the 16S rRNA gene were used to analyze diversity of the microbiota from HIV-positive (HIV+) and HIV-negative (HIV-) women with or without bacterial vaginosis (BV). Results LH-PCR analysis showed significantly more diversity in BV-positive (BV+) women than in BV-negative (BV-) women, but no significant difference between HIV+ women and HIV- women. Pyrosequencing revealed that Lactobacillus constituted a median of 96% of the bacteria in BV- women. BV+ women had a significantly higher number of taxa found at ≥ 1% of the microbiota (median of 11). Common taxa in BV were Prevotella, Megasphaera, Gardnerella, Coriobacterineae, Lachnospira, and Sneathia. There was a trend (p=0.07) toward a higher number of taxa in HIV+BV+ compared to HIV-BV+ women. Propionibacterineae, Citrobacter and Anaerococcus were found only in HIV+ women (p<0.05). Conclusions This study showed that both LH-PCR and pyrosequencing differentiated BV+ from BV- microbiota and that pyrosequencing indicated a trend toward increased diversity in BV+HIV+ suggesting that HIV-infection is associated with changes in diversity of genital microbiota.

Bacterial vaginosis (BV) is a clinical syndrome presenting with a malodorous vaginal discharge and increased vaginal pH. Diagnosis has been based on clinical Amsel criteria and direct Gram stain of vaginal secretions. Human immunodeficiency virus (HIV)-infected participants in the WomenOf the group with Nugent scores of 7 to 10, 83% and 81% had log 10 G. vaginalis counts and log 10 M. hominis counts greater than 6.81 and 4.82, respectively, while only 30% and 31% of the group with Nugent scores of 0 to 3 were above these thresholds, respectively. There was significant overlap in the log 10 lactobacillus counts between the two groups. Utilizing all three log 10 bacterial counts (G. vaginalis, M. hominis, and lactobacilli) in our model improved the sensitivity and specificity to 83% and 78%, respectively, in comparison with Nugent score. In this cohort, Amsel criteria were poorly predictive of BV. PCR quantification of G. vaginalis and M. hominis from CVL is significantly more sensitive than Amsel criteria for diagnosing BV.In women of childbearing age, bacterial vaginosis (BV) is the most common cause of vaginitis. In BV, normal vaginal flora, consisting of hydrogen peroxide-producing lactobacilli, are replaced by Gardnerella vaginalis, Mycoplasma hominis, Mobiluncus species, and anaerobic gram-negative rods (14). BV has been associated with preterm delivery, chorioamnionitis, postabortion infection, and pelvic inflammatory disease. In 1983, Amsel et al. established clinical criteria for diagnosing BV. The presence of three of the following four criteria is considered to be consistent with the presence of BV: vaginal pH of Ͼ4.5, clue cells on saline wet mount, release of a fish amine odor on addition of 10% KOH to a drop of vaginal discharge, and a characteristic thin, homogenous vaginal discharge (1). In 1991, Nugent et al. described a Gram stain scoring system of vaginal smears to diagnose BV (10). The Nugent score is calculated by assessing for the presence of large gram-positive rods (Lactobacillus morphotypes; decrease in Lactobacillus scored as 0 to 4), small gram-variable rods (G. vaginalis morphotypes; scored as 0 to 4), and curved gram-variable rods (Mobiluncus spp. morphotypes; scored as 0 to 2) and can range from 0 to 10. A score of 7 to 10 is consistent with BV.Compared to the Amsel criteria, the Nugent score allows for assessment of alteration in vaginal flora as a continuum rather than a dichotomy. Vaginal cultures for G. vaginalis are sensitive but not specific, as 50 to 60% of healthy asymptomatic women will be culture positive (14). Because Amsel criteria are dependent on the acumen of the clinician, the Nugent score has been favored for diagnosing BV due to superior reproducibility and sensitivity (9). Nevertheless evaluation of smears is also subjective and therefore requires an experienced slide reader (10).Recently we reported on a PCR method to quantify M. hominis, G. vaginalis, and lactobacilli in cervicovaginal lavage (CVL) samples obtained from human immunodeficiency virus (HIV)-infected ...

Mannose-binding lectin (MBL), a serum lectin that mediates innate immune functions including activation of the lectin complement pathway, binds to carbohydrates expressed on some viral glycoproteins. In this study, the ability of MBL to bind to virus particles pseudotyped with Ebola and Marburg envelope glycoproteins was evaluated. Virus particles bearing either Ebola (Zaire strain) or Marburg (Musoke strain) envelope glycoproteins bound at significantly higher levels to immobilized MBL compared with virus particles pseudotyped with vesicular stomatitis virus glycoprotein or with no virus glycoprotein. As observed in previous studies, Ebola-pseudotyped virus bound to cells expressing the lectin DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin). However, pre-incubation of virus with MBL blocked DC-SIGN-mediated binding to cells, suggesting that the two lectins bind at the same or overlapping sites on the Ebola glycoprotein. Neutralization experiments showed that virus pseudotyped with Ebola or Marburg (Musoke) glycoprotein was neutralized by complement, while the Marburg (Ravn strain) glycoprotein-pseudotyped virus was less sensitive to neutralization. Neutralization was partially mediated through the lectin complement pathway, since a complement source deficient in MBL was significantly less effective at neutralizing viruses pseudotyped with filovirus glycoproteins and addition of purified MBL to the MBL-deficient complement increased neutralization. These experiments demonstrated that MBL binds to filovirus envelope glycoproteins resulting in important biological effects and suggest that MBL can interact with filoviruses during infection in humans.