Clinical Practice Guidelines for Managing Genitourinary Symptoms Associated With Menopause.

Genitourinary syndrome of menopause encompasses the group of urogenital signs and symptoms resultant from hypoestrogenism, including genital dryness, burning or irritation, sexual discomfort, pain or dysfunction, and urinary urgency, dysuria, and recurrent urinary tract infections. Genitourinary syndrome of menopause can have a profound impact on well-being, functioning, and quality of life in postmenopausal women. Treatment includes vaginal moisturizers and lubricants geared towards providing symptomatic relief; hormonal treatments which promote epithelial thickening and production of vaginal secretions; and pelvic floor physical therapy along with behavioral therapies that address pelvic floor hypertonicity and psychosocial factors.

Methenamine hippurate compared with trimethoprim for the prevention of recurrent urinary tract infections: a randomized clinical trial.

INTRODUCTION AND HYPOTHESIS: The objective was to find an alternative treatment to a low-dose antibiotic for the prevention of recurrent urinary tract infections (UTI) and to evaluate the difference in rates of reinfection within 1 year when treated with methenamine hippurate for prophylaxis compared with trimethoprim. METHODS: We present a non-blinded randomized trial comparing methenamine hippurate with trimethoprim for the prevention of recurrent UTI at 12 months after starting treatment. Women over 18 who had at least two culture-positive UTI in the prior 6 months or three in the prior year were included. Ninety-two patients met enrollment criteria and were randomized to receive daily prophylaxis with methenamine hippurate or trimethoprim for a minimum of 6 months. Both intent-to-treat and per-protocol analyses if patients received the alternative drug after randomization were analyzed using Student's t test, Mann-Whitney U test, Kaplan-Meier curves, log-rank test, and a logistic and multivariate regression model. The primary outcome of this study was culture-proven UTI recurrence by 12 months after initiating prophylaxis. RESULTS: In the intent-to-treat analysis, we found no difference between groups in recurrent UTI, with a 65% (28 out of 43) recurrence in the trimethoprim group versus 65% (28 out of 43) in the methenamine hippurate group (p = 1.00). In the per-protocol analysis, 65% (26 out of 40) versus 65% (30 out of 46) of patients had UTI recurrences in the trimethoprim group versus the methenamine hippurate group (p = 0.98). CONCLUSIONS: Methenamine hippurate may be an alternative for the prevention of recurrent UTI, with similar rates of recurrence and adverse effects to trimethoprim.

MitoCellPhe reveals mitochondrial morphologies in single fibroblasts and clustered stem cells.

Mitochondria are dynamic organelles that differ significantly in their morphologies across cell types, reflecting specific cellular needs and stages in development. Despite the wide biological significance in disease and in health, delineating mitochondrial morphologies in complex systems remains challenging. Here, we present the Mitochondrial Cellular Phenotype (MitoCellPhe) tool developed for quantifying mitochondrial morphologies and demonstrate its utility in delineating differences in mitochondrial morphologies in a human fibroblast and human induced pluripotent stem cell (hiPSC) line. MitoCellPhe generates 24 parameters, allowing for a comprehensive analysis of mitochondrial structures and importantly allows for quantification to be performed on mitochondria in images containing single cells or clusters of cells. With this tool, we were able to validate previous findings that show networks of mitochondria in healthy fibroblast cell lines and a more fragmented morphology in hiPSCs. Using images generated from control and diseased fibroblasts and hiPSCs, we also demonstrate the efficacy of the toolset in delineating differences in morphologies between healthy and the diseased state in both stem cell (hiPSC) and differentiated fibroblast cells. Our results demonstrate that MitoCellPhe enables high-throughput, sensitive, detailed, and quantitative mitochondrial morphological assessment and thus enables better biological insights into mitochondrial dynamics in health and disease.

Patient Perceptions Impact Progression to Third-Line Therapy for Treatment of Overactive Bladder.

PURPOSE: We aimed to understand the reasons patients choose to pursue third-line overactive bladder (OAB) therapy. MATERIALS AND METHODS: We conducted a mixed methods study that included patient interviews and survey data. Eligible patients were diagnosed by symptoms, had tried behavioral modifications, and OAB medications enrolled from October 2018 to August 2019. In addition to interviews, patients completed 4 surveys: the Pelvic Floor Distress Inventory, Overactive Bladder Questionnaire Short Form, Life Orientation Test-Revised, and a patient confidence in the health care system survey. Qualitative interview data were analyzed thematically. Logistic regression and chi-square analysis was used to analyze survey data. RESULTS: A total of 69 patients were consented, 4 withdrew, and 51 completed both interview and survey data. Overall 55% of patients were Caucasian, 45% were African American, and their average age was 71 (SD=10.4); 75% intended to pursue third-line OAB therapy and 31 (61%) expressed interest in a specific third-line therapy. Major interview themes included a desire for a better quality of life, embarrassment with accidents, and problems with medication. Themes leading patients away from third-line OAB treatment included concern about invasiveness and side effects of treatments, and restrictions to accessing care. CONCLUSIONS: Most patients desired to progress to third-line OAB therapy, were motivated by embarrassment, but were concerned about treatment side effects. We found that economic burden of OAB treatment is associated with patient interest in and decision to receive third-line therapies to include onabotulinumtoxinA and percutaneous tibial nerve stimulation. Improved quality of life, medication frustration, and concerns about side effects of further therapy are themes patients identified when patients considered third-line overactive bladder therapy.

Can patients independently identify their urinary incontinence symptoms?

INTRODUCTION AND HYPOTHESIS: The objective of our study is to compare patient self-reported urinary incontinence symptoms based on the International Consultation on Incontinence Questionnaire- Short Form (ICIQ-SF) question number 6 (When does urine leak?) with physician-assessed interpretation of the patient's urinary incontinence symptoms. METHODS: This trial is a cross-sectional study of patients who presented to a tertiary urogynecology center with symptoms of urinary incontinence between January 2014 and August 2016. We compared patient-reported symptoms on the ICIQ-SF with physician interpretation of urinary complaints during their initial visit. The urinary incontinence symptoms included stress urinary incontinence (SUI), urgency urinary incontinence (UUI), insensible urine loss, nocturnal enuresis, and post-micturition dribbling. RESULTS: A total of 432 patients with a mean age of 61 were included in this evaluation. The most common urinary incontinence symptoms according to the physician were UUI (n = 357, 83%), followed by SUI (n = 308, 71%). Of the patients who were diagnosed by a physician with the symptom of UUI, only 61% self-identified as having this symptom based on the ICIQ-SF, and for SUI, only 66% self-identified as having SUI symptoms based on the ICIQ-SF. Overall UUI (κ = 0.30) appears to have poor agreement, as does nocturnal enuresis (κ = 0.39), when compared with physician historical assessment. CONCLUSION: There is a discrepancy between patient-reported urinary incontinence symptoms on the ICIQ-SF and physician-assessed symptoms. Symptomatology entered into electronic medical records by patients is often inaccurate. Physician validation is essential in understanding the underlying the precise symptomatology.

Quantifying Mitochondrial Dynamics in Patient Fibroblasts with Multiple Developmental Defects and Mitochondrial Disorders.

Mitochondria are dynamic organelles that undergo rounds of fission and fusion and exhibit a wide range of morphologies that contribute to the regulation of different signaling pathways and various cellular functions. It is important to understand the differences between mitochondrial structure in health and disease so that therapies can be developed to maintain the homeostatic balance of mitochondrial dynamics. Mitochondrial disorders are multisystemic and characterized by complex and variable clinical pathologies. The dynamics of mitochondria in mitochondrial disorders is thus worthy of investigation. Therefore, in this study, we performed a comprehensive analysis of mitochondrial dynamics in ten patient-derived fibroblasts containing different mutations and deletions associated with various mitochondrial disorders. Our results suggest that the most predominant morphological signature for mitochondria in the diseased state is fragmentation, with eight out of the ten cell lines exhibiting characteristics consistent with fragmented mitochondria. To our knowledge, this is the first comprehensive study that quantifies mitochondrial dynamics in cell lines with a wide array of developmental and mitochondrial disorders. A more thorough analysis of the correlations between mitochondrial dynamics, mitochondrial genome perturbations, and bioenergetic dysfunction will aid in identifying unique morphological signatures of various mitochondrial disorders in the future.

Evaluating the Bioenergetics Health Index Ratio in Leigh Syndrome Fibroblasts to Understand Disease Severity.

Several pediatric mitochondrial disorders, including Leigh syndrome (LS), impact mitochondrial (mt) genetics, development, and metabolism, leading to complex pathologies and energy failure. The extent to which pathogenic mtDNA variants regulate disease severity in LS is currently not well understood. To better understand this relationship, we computed a glycolytic bioenergetics health index (BHI) for measuring mitochondrial dysfunction in LS patient fibroblast cells harboring varying percentages of pathogenic mutant mtDNA (T8993G, T9185C) exhibiting deficiency in complex V or complex I (T10158C, T12706C). A high percentage (>90%) of pathogenic mtDNA in cells affecting complex V and a low percentage (<39%) of pathogenic mtDNA in cells affecting complex I was quantified. Levels of defective enzyme activities of the electron transport chain correlated with the percentage of pathogenic mtDNA. Subsequent bioenergetics assays showed cell lines relied on both OXPHOS and glycolysis for meeting energy requirements. Results suggest that whereas the precise mechanism of LS has not been elucidated, a multi-pronged approach taking into consideration the specific pathogenic mtDNA variant, glycolytic BHI, and the composite BHI (average ratio of oxphos to glycolysis) can aid in better understanding the factors influencing disease severity in LS.

Cognitive changes in women starting anticholinergic medications for overactive bladder: a prospective study.

INTRODUCTION AND HYPOTHESIS: To assess cognitive changes in women 12 months after starting anticholinergic medications for overactive bladder syndrome (OAB). METHODS: We present a prospective cohort study assessing changes in cognition in women seen in a referral urogynecology practice. We compared women who started anticholinergic OAB medications with women not on anticholinergic OAB medications. The primary outcome was change over time on the Montreal Cognitive Assessment (MOCA) screening score. At enrollment, women completed a baseline MOCA screening, a Geriatric Depression Screen (GDS), and an assessment of medications to create an anticholinergic burden score (ACB). At 3, 6, 9, and 12 months after enrollment women were administered the MOCA, GDS, and a review of their medications and medical problems. Statistical analysis was performed using a linear mixed effects model taking into account correlated error terms given multiple MOCA assessments at various time points per patient. RESULTS: A total of 106 women were enrolled, 60 in the OAB medication group and 46 in the control (non-OAB medication) group. The mean age was 77 years, 93% of women were Caucasian, and 98% completed high school, with no difference between groups. Over time there was no difference in change of MOCA score between the OAB and control groups when controlling for age, GDS score, and ACB score (p = 0.78). This association did not change when women with a neurological diagnosis were excluded (n = 6). On average MOCA scores for the OAB group increased by 0.76 over 12 months and the control group increased 0.39, with no difference between the groups (p = 0.53). CONCLUSIONS: We found no changes in MOCA scores between OAB medication and control groups after controlling for age, depression, and polypharmacy after 12 months of follow-up.

Resistance to type 1 interferons is a major determinant of HIV-1 transmission fitness.

Sexual transmission of HIV-1 is an inefficient process, with only one or few variants of the donor quasispecies establishing the new infection. A critical, and as yet unresolved, question is whether the mucosal bottleneck selects for viruses with increased transmission fitness. Here, we characterized 300 limiting dilution-derived virus isolates from the plasma, and in some instances genital secretions, of eight HIV-1 donor and recipient pairs. Although there were no differences in the amount of virion-associated envelope glycoprotein, recipient isolates were on average threefold more infectious (P = 0.0001), replicated to 1.4-fold higher titers (P = 0.004), were released from infected cells 4.2-fold more efficiently (P < 0.00001), and were significantly more resistant to type I IFNs than the corresponding donor isolates. Remarkably, transmitted viruses exhibited 7.8-fold higher IFNα2 (P < 0.00001) and 39-fold higher IFNß (P < 0.00001) half-maximal inhibitory concentrations (IC50) than did donor isolates, and their odds of replicating in CD4+ T cells at the highest IFNα2 and IFNß doses were 35-fold (P < 0.00001) and 250-fold (P < 0.00001) greater, respectively. Interestingly, pretreatment of CD4+ T cells with IFNß, but not IFNα2, selected donor plasma isolates that exhibited a transmitted virus-like phenotype, and such viruses were also detected in the donor genital tract. These data indicate that transmitted viruses are phenotypically distinct, and that increased IFN resistance represents their most distinguishing property. Thus, the mucosal bottleneck selects for viruses that are able to replicate and spread efficiently in the face of a potent innate immune response.

Subjective and objective responses to PTNS and predictors for success: a retrospective cohort study of percutaneous tibial nerve stimulation for overactive bladder.

INTRODUCTION AND HYPOTHESIS: We investigated the objective improvements in overactive bladder (OAB) symptoms in patients undergoing percutaneous tibial nerve stimulation (PTNS) and predictive factors of patient satisfaction. METHODS: In this single-center retrospective cohort study at a tertiary urogynecology center, we identified all female patients who underwent PTNS therapy from 1 October 2007 - 1 January 2016 and followed them from their initial visit through medication therapy and PTNS treatments. Patients who tried at least one medication prior to starting PTNS therapy and completed at least one PTNS visit were included. Baseline demographic data, urinary data, and details of medication and PTNS therapy sessions were collected from records through chart review. Paired or two-sample t-tests were used to compare changes over time or groups. Bivariate and multivariable logistic regression were performed. RESULTS: Two hundred thirteen patients underwent PTNS therapy and 183 patients met the criteria. Overall patients were able to decrease voiding frequency by 1 h, decrease nocturia episodes by 0.8, and decrease urge incontinence episodes with PTNS therapy by ten episodes per week (p = 0.02). Patients who continued OAB medications did not have additional improvements compared with patients who did not continue OAB medications during PTNS. Overall, 25.4% (43/169) patients reported ≥ 75% improvement during PTNS therapy, and 61.5% (104/169) reported ≥ 50% improvement. When evaluating predictive factors of ≥ 50% overall improvement, the number of PTNS sessions increased odds of subjective success (OR = 1.8, p = 0.004). Other factors were not significant predictors of subjective PTNS success. CONCLUSIONS: PTNS can provide both objective and subjective improvements for patients who do not respond to OAB medication therapy.

CD4 mimetics sensitize HIV-1-infected cells to ADCC.

HIV-1-infected cells presenting envelope glycoproteins (Env) in the CD4-bound conformation on their surface are preferentially targeted by antibody-dependent cell-mediated cytotoxicity (ADCC). HIV-1 has evolved a sophisticated mechanism to avoid exposure of ADCC-mediating Env epitopes by down-regulating CD4 and by limiting the overall amount of Env at the cell surface. Here we report that small-molecule CD4-mimetic compounds induce the CD4-bound conformation of Env, and thereby sensitize cells infected with primary HIV-1 isolates to ADCC mediated by antibodies present in sera, cervicovaginal lavages, and breast milk from HIV-1-infected individuals. Importantly, we identified one CD4 mimetic with the capacity to sensitize endogenously infected ex vivo-amplified primary CD4 T cells to ADCC killing mediated by autologous sera and effector cells. Thus, CD4 mimetics hold the promise of therapeutic utility in preventing and controlling HIV-1 infection.

Transvaginal mesh: a historical review and update of the current state of affairs in the United States.

INTRODUCTION: Transvaginal mesh usage has been at the forefront of popular media and academic debate for the past 10 years. Several US Food and Drug Administration (FDA) communications, society statements, and research articles have been written in an attempt to define and articulate the classification system, safety data, and efficacy of this approach to transvaginal surgery. In this review, we explore the history of transvaginal mesh surgery for pelvic organ prolapse (POP), review FDA and society statements, and research current practice in the United States. METHODS: We searched the English language literature using PubMed for articles related to safety and monitoring of transvaginal mesh and reviewed all FDA publication and notices and gynecology and urogynecology society statements on its use in the United States. We then reviewed 22 articles and grouped them into several sections. RESULTS: Mesh used to augment transvaginal repair of POP was introduced in the United States in 2005 without clinical safety and efficacy data. In the subsequent years of use, both major and minor complications were increasingly reported, leading to several FDA notifications and warnings. The type of mesh used, reporting and classifications systems, and provider usage has varied widely over time. CONCLUSION: We present a historical review of transvaginal mesh use for pelvic organ prolapse in the United States from 2005 to 2016. There continues to be heated debate among practitioners about balancing the efficacy of mesh use to decrease recurrent prolapse and complications. Research into safety and efficacy, along with tighter FDA regulations, is ongoing.

Vaginal vault prolapse in a patient with Mayer-Rokitansky-Küster-Hauser syndrome: a video case presentation.

INTRODUCTION AND HYPOTHESIS: Several reports have described vaginal prolapse in Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome patients after creation of a neovagina. To our knowledge, no reports of primary vaginal prolapse of a blind pouch without previous intervention, or surgery for this condition, have been described. METHODS: In this case report, we describe a 19-year-old woman with MRKH and complete prolapse of her shortened vaginal pouch. Surgical correction utilizing permanent suture-based sacrospinous ligament fixation was performed. RESULTS: The patient had a successful outcome. CONCLUSIONS: Sacrospinous ligament fixation provided a safe and effective method for the management of vaginal pouch prolapse. Long-term follow-up is planned. To our knowledge, this is the first report describing surgical repair of primary prolapse of a blind vaginal pouch in the setting of MRKH.

Which bladder instillations are more effective? DMSO vs. bupivacaine/heparin/triamcinolone: a retrospective study.

INTRODUCTION AND HYPOTHESIS: Bladder pain syndrome/interstitial cystitis (BPS/IC) is a chronic and debilitating condition. Our objective was to compare two different bladder instillation treatments in patients with BPS/IC: dimethyl sulfoxide with triamcinolone (DMSO) vs. bupivacaine with heparin and triamcinolone (B/H/T). Our hypothesis was that both treatments are equally effective. METHODS: A retrospective cohort study of instillation-naïve patients was conducted comparing responses to either DMSO or B/H/T at our tertiary urogynecology center from 2012 to 2014. The primary outcome was patient-reported percent of overall improvement from baseline. Secondary outcomes were change in patient-reported daytime voiding frequency (hours) and change in number of nighttime voiding episodes. Variables analyzed as potential confounders included pelvic pain, cystoscopy findings, levator spasm, and fibromyalgia. The two-sided Student's t test, chi-squared test, Poisson regression, and repeated-measure analysis of variance (ANOVA) were used for analyses. RESULTS: One hundred and ninety-three eligible patients were identified (45 receiving DMSO, 146 receiving B/H/T). Compared with baseline, DMSO patients reported 63% improvement (p < 0.0001), increased time between daytime voids by 1.5 h (p < 0.00), and a 40% reduction in nocturia episodes (p < 0.00). B/H/T patients reported 51% improvement (p < 0.00), increased time between daytime voids by 1.4 h (p < 0.00), and an 8% reduction in nocturia episodes (p = 0.26). When comparing the two treatments, DMSO resulted in a greater percentage of overall improvement (p = 0.02) and a significant decrease in nocturia episodes when compared with B/H/T (p = 0.02). There was no significant difference between treatments for daytime voiding frequency (p = 0.50). CONCLUSION: Bladder instillations with DMSO or B/H/T provide overall symptomatic improvement and improved frequency and nocturia. DMSO appears to provide greater improvement in nocturia and overall.

The effect of HIV-1 Vif polymorphisms on A3G anti-viral activity in an in vivo mouse model.

Humans encode seven APOBEC3 proteins (A-H), with A3G, 3F and 3H as the major factors restricting HIV-1 replication. HIV-1, however, encodes Vif, which counteracts A3 proteins by chaperoning them to the proteasome where they are degraded. Vif polymorphisms found in HIV-1s isolated from infected patients have varying anti-A3G potency when assayed in vitro, but there are few studies demonstrating this in in vivo models. Here, we created Friend murine leukemia viruses encoding vif alleles that were previously shown to differentially neutralize A3G in cell culture or that were originally found in primary HIV-1 isolates. Infection of transgenic mice expressing different levels of human A3G showed that these naturally occurring Vif variants differed in their ability to counteract A3G during in vivo infection, although the effects on viral replication were not identical to those seen in cultured cells. We also found that the polymorphic Vifs that attenuated viral replication reverted to wild type only in A3G transgenic mice. Finally, we found that the level of A3G-mediated deamination was inversely correlated with the level of viral replication. This animal model should be useful for studying the functional significance of naturally occurring vif polymorphisms, as well as viral evolution in the presence of A3G.

Phenotypic properties of transmitted founder HIV-1.

Defining the virus-host interactions responsible for HIV-1 transmission, including the phenotypic requirements of viruses capable of establishing de novo infections, could be important for AIDS vaccine development. Previous analyses have failed to identify phenotypic properties other than chemokine receptor 5 (CCR5) and CD4+ T-cell tropism that are preferentially associated with viral transmission. However, most of these studies were limited to examining envelope (Env) function in the context of pseudoviruses. Here, we generated infectious molecular clones of transmitted founder (TF; n = 27) and chronic control (CC; n = 14) viruses of subtypes B (n = 18) and C (n = 23) and compared their phenotypic properties in assays specifically designed to probe the earliest stages of HIV-1 infection. We found that TF virions were 1.7-fold more infectious (P = 0.049) and contained 1.9-fold more Env per particle (P = 0.048) compared with CC viruses. TF viruses were also captured by monocyte-derived dendritic cells 1.7-fold more efficiently (P = 0.035) and more readily transferred to CD4+ T cells (P = 0.025). In primary CD4+ T cells, TF and CC viruses replicated with comparable kinetics; however, when propagated in the presence of IFN-α, TF viruses replicated to higher titers than CC viruses. This difference was significant for subtype B (P = 0.000013) but not subtype C (P = 0.53) viruses, possibly reflecting demographic differences of the respective patient cohorts. Together, these data indicate that TF viruses are enriched for higher Env content, enhanced cell-free infectivity, improved dendritic cell interaction, and relative IFN-α resistance. These viral properties, which likely act in concert, should be considered in the development and testing of AIDS vaccines.

Transmitted/founder and chronic HIV-1 envelope proteins are distinguished by differential utilization of CCR5.

Infection by HIV-1 most often results from the successful transmission and propagation of a single virus variant, termed the transmitted/founder (T/F) virus. Here, we compared the attachment and entry properties of envelope (Env) glycoproteins from T/F and chronic control (CC) viruses. Using a panel of 40 T/F and 47 CC Envs, all derived by single genome amplification, we found that 52% of clade C and B CC Envs exhibited partial resistance to the CCR5 antagonist maraviroc (MVC) on cells expressing high levels of CCR5, while only 15% of T/F Envs exhibited this same property. Moreover, subtle differences in the magnitude with which MVC inhibited infection on cells expressing low levels of CCR5, including primary CD4(+) T cells, were highly predictive of MVC resistance when CCR5 expression levels were high. These results are consistent with previous observations showing a greater sensitivity of T/F Envs to MVC inhibition on cells expressing very high levels of CCR5 and indicate that CC Envs are often capable of recognizing MVC-bound CCR5, albeit inefficiently on cells expressing physiologic levels of CCR5. When CCR5 expression levels are high, this phenotype becomes readily detectable. The utilization of drug-bound CCR5 conformations by many CC Envs was seen with other CCR5 antagonists, with replication-competent viruses, and did not obviously correlate with other phenotypic traits. The striking ability of clade C and B CC Envs to use MVC-bound CCR5 relative to T/F Envs argues that the more promiscuous use of CCR5 by these Env proteins is selected against at the level of virus transmission and is selected for during chronic infection.

Comparison of viral Env proteins from acute and chronic infections with subtype C human immunodeficiency virus type 1 identifies differences in glycosylation and CCR5 utilization and suggests a new strategy for immunogen design.

Understanding human immunodeficiency virus type 1 (HIV-1) transmission is central to developing effective prevention strategies, including a vaccine. We compared phenotypic and genetic variation in HIV-1 env genes from subjects in acute/early infection and subjects with chronic infections in the context of subtype C heterosexual transmission. We found that the transmitted viruses all used CCR5 and required high levels of CD4 to infect target cells, suggesting selection for replication in T cells and not macrophages after transmission. In addition, the transmitted viruses were more likely to use a maraviroc-sensitive conformation of CCR5, perhaps identifying a feature of the target T cell. We confirmed an earlier observation that the transmitted viruses were, on average, modestly underglycosylated relative to the viruses from chronically infected subjects. This difference was most pronounced in comparing the viruses in acutely infected men to those in chronically infected women. These features of the transmitted virus point to selective pressures during the transmission event. We did not observe a consistent difference either in heterologous neutralization sensitivity or in sensitivity to soluble CD4 between the two groups, suggesting similar conformations between viruses from acute and chronic infection. However, the presence or absence of glycosylation sites had differential effects on neutralization sensitivity for different antibodies. We suggest that the occasional absence of glycosylation sites encoded in the conserved regions of env, further reduced in transmitted viruses, could expose specific surface structures on the protein as antibody targets.

Transmitted/founder and chronic subtype C HIV-1 use CD4 and CCR5 receptors with equal efficiency and are not inhibited by blocking the integrin α4ß7.

Sexual transmission of human immunodeficiency virus type 1 (HIV-1) most often results from productive infection by a single transmitted/founder (T/F) virus, indicating a stringent mucosal bottleneck. Understanding the viral traits that overcome this bottleneck could have important implications for HIV-1 vaccine design and other prevention strategies. Most T/F viruses use CCR5 to infect target cells and some encode envelope glycoproteins (Envs) that contain fewer potential N-linked glycosylation sites and shorter V1/V2 variable loops than Envs from chronic viruses. Moreover, it has been reported that the gp120 subunits of certain transmitted Envs bind to the gut-homing integrin α4ß7, possibly enhancing virus entry and cell-to-cell spread. Here we sought to determine whether subtype C T/F viruses, which are responsible for the majority of new HIV-1 infections worldwide, share biological properties that increase their transmission fitness, including preferential α4ß7 engagement. Using single genome amplification, we generated panels of both T/F (n = 20) and chronic (n = 20) Env constructs as well as full-length T/F (n = 6) and chronic (n = 4) infectious molecular clones (IMCs). We found that T/F and chronic control Envs were indistinguishable in the efficiency with which they used CD4 and CCR5. Both groups of Envs also exhibited the same CD4+ T cell subset tropism and showed similar sensitivity to neutralization by CD4 binding site (CD4bs) antibodies. Finally, saturating concentrations of anti-α4ß7 antibodies failed to inhibit infection and replication of T/F as well as chronic control viruses, although the growth of the tissue culture-adapted strain SF162 was modestly impaired. These results indicate that the population bottleneck associated with mucosal HIV-1 acquisition is not due to the selection of T/F viruses that use α4ß7, CD4 or CCR5 more efficiently.

Early low-titer neutralizing antibodies impede HIV-1 replication and select for virus escape.

Single genome sequencing of early HIV-1 genomes provides a sensitive, dynamic assessment of virus evolution and insight into the earliest anti-viral immune responses in vivo. By using this approach, together with deep sequencing, site-directed mutagenesis, antibody adsorptions and virus-entry assays, we found evidence in three subjects of neutralizing antibody (Nab) responses as early as 2 weeks post-seroconversion, with Nab titers as low as 1∶20 to 1∶50 (IC(50)) selecting for virus escape. In each of the subjects, Nabs targeted different regions of the HIV-1 envelope (Env) in a strain-specific, conformationally sensitive manner. In subject CH40, virus escape was first mediated by mutations in the V1 region of the Env, followed by V3. HIV-1 specific monoclonal antibodies from this subject mapped to an immunodominant region at the base of V3 and exhibited neutralizing patterns indistinguishable from polyclonal antibody responses, indicating V1-V3 interactions within the Env trimer. In subject CH77, escape mutations mapped to the V2 region of Env, several of which selected for alterations of glycosylation. And in subject CH58, escape mutations mapped to the Env outer domain. In all three subjects, initial Nab recognition was followed by sequential rounds of virus escape and Nab elicitation, with Nab escape variants exhibiting variable costs to replication fitness. Although delayed in comparison with autologous CD8 T-cell responses, our findings show that Nabs appear earlier in HIV-1 infection than previously recognized, target diverse sites on HIV-1 Env, and impede virus replication at surprisingly low titers. The unexpected in vivo sensitivity of early transmitted/founder virus to Nabs raises the possibility that similarly low concentrations of vaccine-induced Nabs could impair virus acquisition in natural HIV-1 transmission, where the risk of infection is low and the number of viruses responsible for transmission and productive clinical infection is typically one.