Role of the epithelium in human papillomavirus and human immunodeficiency virus infections in the female genital tract.

Background: Two-thirds of people living with human immunodeficiency virus type 1 (HIV-1) infection reside in Sub-Saharan Africa, where there are the highest prevalence and incidence rates of human papillomavirus (HPV) infection. Both infections are sexually transmitted and enter the body via the epithelium. This review describes the extent of involvement of the epithelium in each infection in the female genital tract. Methods: A narrative review was conducted on the role of the epithelium in HPV and HIV-1 infections. Results: An intact epithelial barrier is the predominant form of protection against viral entry and infection, including from HIV-1 and HPV. HPV is an intraepithelial pathogen, and thus, its growth and amplification, which are dependent on squamous cell differentiation, occur in the epithelium. It gains entry to the basal cells of the stratified squamous epithelium via micro-abrasions or other epithelial injuries that expose the basement membrane. HIV-1, conversely, passes through the epithelium to infect subepithelial tissues. Following deposition of the HIV-1-containing inoculum into the lumen, the virus enters the mucosa through breaks in the epithelial barrier within hours of infection. Further, HIV-1 penetrates the epithelium via various mechanisms, including paracellular passage or across epithelial cells through transcytosis. The capture of the virus from the mucosal surface by intraepithelial and/or subepithelial target cells has also been documented. Conclusions: Epithelial disruption is the major pathogenetic pathway in HIV-1 and HPV infections. Therefore, biochemical compounds that strengthen the epithelial barrier must be prioritized to prevent these infections.

Recent Advances and New Challenges in Cisgender Women's Gynecologic and Obstetric Health in the Context of HIV.

Although rates of human immunodeficiency virus (HIV) have declined globally over the past 10 years, United Nations Programme on HIV/AIDS estimates 1.7 million new infections occurred in 2019, with cisgender women (cis women) and girls accounting for 48%. Acquired immune deficiency syndrome-related illnesses are the leading global cause of mortality in cis women aged 15 to 49, and in many sub-Saharan Africa countries, young women face substantially higher HIV risk than their male counterparts. Drivers of this increased risk include sexual and reproductive health characteristics unique to cis women. This review discusses the role of sexually transmitted infections, contraception and pregnancy in HIV risk, and biomedical HIV prevention technologies available and in development.

Localized cyclical variations in immunoproteins in the female genital tract and the implications on the design and assessment of mucosal infection and therapies.

PROBLEM: Fluctuating hormones regulate reproductive processes in the female genital tract. Consequent changes in the local immunological environment are likely to affect cellular interaction with infectious agents and the assessment of therapies that target mucosal infections. METHOD OF STUDY: We compared Softcup and Weck-Cel sampling protocols and assessed the changes in the concentrations of 39 soluble proteins with menstrual cycle progression in the mucosal and peripheral compartments. RESULTS: We demonstrate that the mucosal immunological profile is distinct from serum with inflammatory and migratory signatures that are localized throughout the cycle. The analytes highlighted in the mucosal compartment were generally highest at the follicular phase with a tendency to fall as the cycle progressed through ovulation to the luteal phase. CONCLUSION: Our results underscore the need to consider these localized cyclical differences in studies aimed at assessing the outcome of disease and the efficacy of mucosal vaccines and other therapies.

Lower concentrations of chemotactic cytokines and soluble innate factors in the lower female genital tract associated with the use of injectable hormonal contraceptive.

Progesterone-based injectable hormonal contraceptives (HCs) potentially modulate genital barrier integrity and regulate the innate immune environment in the female genital tract, thereby enhancing the risk of STIs or HIV infection. We investigated the effects of injectable HC use on concentrations of inflammatory cytokines and other soluble factors associated with genital epithelial repair and integrity. The concentrations of 42 inflammatory, regulatory, adaptive growth factors and hematopoietic cytokines, five matrix metalloproteinases (MMPs), and four tissue inhibitors of metalloproteinases (TIMPs) were measured in cervicovaginal lavages (CVLs) from 64 HIV-negative women using injectable HCs and 64 control women not using any HCs, in a matched case-control study. There were no differences between groups in the prevalence of bacterial vaginosis (BV; Nugent score ≥7), or common sexually transmitted infections (STIs). In multivariate analyses adjusting for condom use, sex work status, marital status, BV and STIs, median concentrations of chemokines (eotaxin, MCP-1, MDC), adaptive cytokines (IL-15), growth factors (PDGF-AA) and a metalloproteinase (TIMP-2) were significantly lower in CVLs from women using injectable HCs than controls. In addition, the pro-inflammatory cytokine IL-12p40 and the chemokine fractalkine were less likely to have detectable levels in women using injectable HCs compared with those not using HCs. We conclude that injectable HC use was broadly associated with an immunosuppressive female genital tract innate immune profile. While the relationship between injectable HC use and STI or HIV risk is yet to be resolved, our data suggest that the effects of injectable HCs were similar in STI-positive and STI-negative participants.

HIV disease progression in seroconvertors from the CAPRISA 004 tenofovir gel pre-exposure prophylaxis trial.

BACKGROUND: Although antiretroviral pre-exposure prophylaxis prevents HIV acquisition, it is not known if it alters HIV disease progression. This study assesses whether tenofovir gel impacted on disease progression among CAPRISA 004 microbicide trial seroconvertors. METHODS: Eighty-three seroconvertors from the tenofovir and placebo gel arms of the CAPRISA 004 trial were monitored prospectively for a minimum of 2 years by CD4 count and viral load (VL). Linear mixed models were fitted to HIV VL, and log rank test was used to compare time to reach CD4 counts of <350 cells per microliter. RESULTS: Median 2-week postinfection VL was 4.74 and 4.45 log copies per milliliter in women assigned to tenofovir gel (n = 32) and placebo gel (n = 51) (P = 0.189). Corresponding 12-month postinfection VLs were 4.24 and 3.70 log copies per milliliter (P = 0.016). After adjusting for clinical and behavioral characteristics and protective HLA alleles, mean VLs within the first 2 years were 4.51 and 4.02 log copies per milliliter in women from the tenofovir and placebo arms (P = 0.013). Among women with vaginal tenofovir measurements, mean VLs were 4.53 and 4.60 log copies per milliliter in those with detectable versus undetectable levels (P = 0.840). Overall mean CD4 counts were 463 and 514 cells per microliter in women assigned to tenofovir and placebo (P = 0.290). Thirty-two women (38.6%) reached CD4 counts of <350 cells per microliter at median 9.4 months postinfection, 13 (40.6%) from the tenofovir and 19 (37.3%) from the placebo arms (P = 0.786). CONCLUSIONS: Tenofovir gel had no impact on postinfection CD4 counts or the rate of CD4 decline. Although seroconvertors from the tenofovir arm experienced higher VLs, this did not result in a need for earlier antiretroviral therapy.

How can we design better vaccines to prevent HIV infection in women?

The human immunodeficiency virus (HIV) burden in women continues to increase, and heterosexual contact is now the most common route of infection worldwide. Effective protection of women against HIV-1 infection may require a vaccine specifically targeting mucosal immune responses in the female genital tract (FGT). To achieve this goal, a much better understanding of the immunology of the FGT is needed. Here we review the architecture of the immune system of the FGT, recent studies of potential methods to achieve the goal of mucosal protection in women, including systemic-prime, mucosal-boost, FGT-tropic vectors and immune response altering adjuvants. Advances in other fields that enhance our understanding of female genital immune correlates and the interplay between hormonal and immunological systems may also help to achieve protection of women from HIV infection.

Sensitive tenofovir resistance screening of HIV-1 from the genital and blood compartments of women with breakthrough infections in the CAPRISA 004 tenofovir gel trial.

The Centre for the AIDS Programme of Research in South Africa 004 (CAPRISA 004) study demonstrated that vaginally applied tenofovir gel is a promising intervention for protecting women from sexually acquiring human immunodeficiency virus (HIV). However, the potential for emergence of tenofovir resistance remains a concern in women who seroconvert while using the gel despite the lack of plasma virus resistance as assessed by population sequencing during the trial. We applied highly sensitive polymerase chain reaction-based assays to screen for tenofovir resistance in plasma and vaginal swab specimens. The absence of mutation detection suggested little immediate risk of tenofovir-resistant HIV-1 emergence and forward transmission in settings in which gel users are closely monitored for HIV seroconversion.

Women with pregnancies had lower adherence to 1% tenofovir vaginal gel as HIV preexposure prophylaxis in CAPRISA 004, a phase IIB randomized-controlled trial.

BACKGROUND: Antiretroviral prophylaxis may be a critical strategy to reduce periconception HIV transmission. Maximizing the benefit of periconception pharmacologic HIV risk-reduction requires an understanding of the links between pregnancy and adherence to this prevention strategy. METHODS: We assessed study gel adherence among women with pregnancies compared to women without pregnancies enrolled in the CAPRISA 004 phase IIB trial of 1% vaginal tenofovir gel. Pregnancy was assessed with monthly urine tests. Adherence was measured monthly and defined as proportion of sex acts covered by two returned, used applicators based on pre- and post-coital dosing. High adherence was defined as a median adherence score of >80%, that is, more than 80% of sex acts were covered by two applications of study gel. A multivariate generalized estimating equations (GEE) model with a binomial distribution was used to assess covariates associated with high adherence (>80%) over time. Median adherence before and after pregnancy was compared using Wilcoxon signed rank test. RESULTS: Among 868 women, 53 had at least 1 pregnancy (4.06 per 100 woman years, 95% CI: 3.04, 5.31). Women with pregnancies had lower median adherence compared to women without pregnancies (50% [IQR: 45-83] vs. 60% [IQR: 50-100], p = 0.02). Women with pregnancies also had a 48% lower odds of high adherence compared to women without pregnancies when adjusting for confounders (aOR 0.52, 95%CI: 0.41-0.66, p<0.0001). Among women with pregnancies, adherence before and after pregnancy was not different (50% [IQR: 46-83] vs. 55% [IQR: 20-100], p = 0.68). CONCLUSIONS: Women with pregnancies were less likely to have high adherence to study gel compared to women without pregnancies. Understanding these differences may inform findings from HIV prevention trials and future implementation of antiretroviral prophylaxis for at-risk women who choose to conceive. The protocol for the parent trial is registered on ClinicalTrials.gov, NCT00441298, http://www.clinicaltrials.gov/ct2/show/NCT00441298.

Recommendations for the follow-up of study participants with breakthrough HIV infections during HIV/AIDS biomedical prevention studies.

OBJECTIVE: To facilitate collection of cumulative data on longitudinal HIV disease outcomes during HIV prevention studies by developing recommendations for follow-up of the relatively few study participants with breakthrough infections. DESIGN: We formed a working group to compare and contrast the various approaches taken in recent HIV prevention trials, to summarize the advantages and disadvantages associated with each, and to explore the feasibility of developing protocols for the long-term follow-up of seroconverters. METHODS: We reviewed study designs, objectives, and assessments in 15 interventional studies that followed HIV seroconverters. Protocol team members joined discussions of the various approaches and developed recommendations. RESULTS: Most HIV prevention clinical trials share a core set of objectives, including the description/comparison of virological, immunological, and clinical course of HIV, and sometimes a comparison of preseroconversion and postseroconversion behavior. Long-term follow-up of seroconverters can be conducted in separate studies if the transition from parent protocol is effectively managed. CONCLUSION: We recommend the development of harmonized seroconverter protocols. Although specific research questions in the postseroconversion period may differ depending on prevention modality, harmonizing key evaluations would create an opportunity to ask overarching questions that inform the prevention field with respect to design and implementation of future combination prevention studies. Follow-up immediately postseroconversion should be conducted in the parent protocol before roll over into a follow-up protocol. Development of specimen repositories with ample volumes for future assays, standardized definitions of infection, diagnosis and seroconversion dates, and harmonization of study objectives and sample collections at key time points are important.

Safety of tenofovir gel, a vaginal microbicide, in South African women: results of the CAPRISA 004 Trial.

BACKGROUND: Tenofovir gel, used vaginally before and after coitus, reduced women's acquisition of HIV by 39%. This is a safety assessment of tenofovir gel, including renal, bone, gastrointestinal, genital and haematological parameters. METHODS: In the Centre for the AIDS Programme of Research in South Africa (CAPRISA) 004, a double-blind, randomized placebo-controlled trial, 445 of the 889 eligibly enrolled women were assigned to tenofovir gel. All participants were advised to use the gel vaginally only, with one dose of gel within 12 h before and a second dose as soon as possible after sex, with no more than two doses in 24 h. Clinical and laboratory safety data were collected at monthly and quarterly visits, respectively. Genital assessments were undertaken at enrolment and quarterly thereafter, or as indicated. RESULTS: Women assigned to tenofovir gel were exposed to an average monthly vaginal dose of 240 mg of tenofovir (six applications). In total, six women, three in each group, had mild creatinine elevations, all of which occurred in July/August 2008. The incidence of anaemia was 3.5 and 3.8 per 100 women-years in tenofovir and placebo groups, respectively (P=0.80). Of the six women (four tenofovir and two placebo) experiencing bone fractures, none were associated with abnormal phosphate or calcium values. The proportion of women with diarrhoea was higher in the tenofovir gel group (17% versus 11%; P=0.026). There was no significant increase of any genital adverse event in the tenofovir group. CONCLUSIONS: No significant renal, haematological, genital or bone effects were associated with the use of tenofovir gel. Aside from a puzzling increase in diarrhoea, tenofovir gel has an excellent safety profile.

From the laboratory to clinical trials and back again: lessons learned from HIV prevention trials.

PROBLEM: Inadequate, irrelevant, or inappropriate timing of biological specimen collection during clinical trials is a cause for delay in understanding and explaining correlates of protection and/or effectiveness, particularly at the portal of entry in the context of sexual HIV transmission and its prevention. METHODS: We present examples of HIV prevention trials to illustrate the impact of preplanned versus unplanned laboratory science program on the interpretation of trial results and advancement of the field. RESULTS: Of the five completed pre-exposure prophylaxis trials, only two announced main outcome results simultaneously with data on correlates of drug-related effectiveness. In four of the vaccine trials completed, the only one that showed a protective effect presented data on protection correlates significantly later. CONCLUSION: Clinical trials must preplan collaborative immunophysiological research and prioritize biological specimen collection and storage for enhancement of research on correlates of protection. Similarly appropriate specimens should be prioritized for pathogenesis research.

Innate immune activation enhances hiv acquisition in women, diminishing the effectiveness of tenofovir microbicide gel.

The antiretroviral agent, tenofovir, formulated as a vaginal microbicide gel, reduces human immunodeficiency virus (HIV) acquisition by 39% in women. This study assessed the role of preexisting immune activation in HIV acquisition in women from the CAPRISA 004 trial, to identify potential strategies to increase the effectiveness of tenofovir gel. Systemic cytokine and cellular immune mediators (platelets and natural killer [NK] cells) were assessed in women at high risk for HIV assigned to either tenofovir or placebo gel in the CAPRISA 004 trial. Notwithstanding tenofovir gel use, women who acquired HIV had significantly higher systemic innate immune activation prior to infection than women who remained uninfected. Activation of both soluble (cytokine) and cellular (NK cells) immune mediators were associated with HIV acquisition, individually or in combination. Hence, an innate immune activation suppressant could be added to tenofovir gel as a potential combination gel strategy in developing the next generation of higher efficacy antiretroviral microbicides.

The development of CD4 binding site antibodies during HIV-1 infection.

Broadly neutralizing antibodies to the CD4 binding site (CD4bs) of gp120 are generated by some HIV-1-infected individuals, but little is known about the prevalence and evolution of this antibody response during the course of HIV-1 infection. We analyzed the sera of 113 HIV-1 seroconverters from three cohorts for binding to a panel of gp120 core proteins and their corresponding CD4bs knockout mutants. Among sera collected between 99 and 258 weeks post-HIV-1 infection, 88% contained antibodies to the CD4bs and 47% contained antibodies to resurfaced stabilized core (RSC) probes that react preferentially with broadly neutralizing CD4bs antibodies (BNCD4), such as monoclonal antibodies (MAbs) VRC01 and VRC-CH31. Analysis of longitudinal serum samples from a subset of 18 subjects revealed that CD4bs antibodies to gp120 arose within the first 4 to 16 weeks of infection, while the development of RSC-reactive antibodies was more varied, occurring between 10 and 152 weeks post-HIV-1 infection. Despite the presence of these antibodies, serum neutralization mediated by RSC-reactive antibodies was detected in sera from only a few donors infected for more than 3 years. Thus, CD4bs antibodies that bind a VRC01-like epitope are often induced during HIV-1 infection, but the level and potency required to mediate serum neutralization may take years to develop. An improved understanding of the immunological factors associated with the development and maturation of neutralizing CD4bs antibodies during HIV-1 infection may provide insights into the requirements for eliciting this response by vaccination.

CAPRISA 004 tenofovir microbicide trial: no impact of tenofovir gel on the HIV transmission bottleneck.

Alterations of the genital mucosal barrier may influence the number of viruses transmitted from a human immunodeficiency virus-infected source host to the newly infected individual. We used heteroduplex tracking assay and single-genome sequencing to investigate the effect of a tenofovir-based microbicide gel on the transmission bottleneck in women who seroconverted during the CAPRISA 004 microbicide trial. Seventy-seven percent (17 of 22; 95% confidence interval [CI], 56%-90%) of women in the tenofovir gel arm were infected with a single virus compared with 92% (13 of 14; 95% CI, 67%->99%) in the placebo arm (P = .37). Tenofovir gel had no discernable impact on the transmission bottleneck.

Contraception and pregnancy in microbicide trials.

The distinctive feature of the human immunodeficiency virus (HIV) epidemic in Sub-Saharan Africa is the burden on women, in particular young women of reproductive age. Consequently, most late-phase effectiveness microbicide clinical trials are conducted in sub-Saharan Africa where fertility rates are high. Because late-phase clinical trials are conducted over prolonged periods of time, women participating in these trials may fall pregnant during the trial. Their unborn babies may be exposed to a drug whose teratogenic potential is unknown if the investigational drug is not withdrawn. High pregnancy rates in such trials may compromise statistical integrity, as women will be withdrawn from the study drug for the duration of the pregnancy. It is therefore imperative for microbicide trials to implement effective contraceptive and pregnancy management programmes that maintain low pregnancy rates and the safety of unborn babies while not compromising the conduct and statistical integrity of the trial.

Preservation HIV-1-specific IFNγ+ CD4+ T-cell responses in breakthrough infections after exposure to tenofovir gel in the CAPRISA 004 microbicide trial.

The Centre for the AIDS Program of Research in South Africa 004 trial demonstrated reduction of sexual HIV-1 acquisition in women using a vaginal microbicide containing tenofovir. A better understanding of the consequences of antiretroviral-containing microbicides for immune responses in individuals with intercurrent HIV-1 infection is needed for future trials combining the use of microbicides with HIV-1 vaccines. Investigation of immune responses in women who acquired HIV-1 although using tenofovir gel showed significantly higher (P = 0.01) Gag-specific IFNγ+ CD4+ T-cell responses. The use of tenofovir-containing gel around the time of infection can modulate HIV-1 immunity, and these immunological changes need to be considered in future trials combining vaccines and microbicides.

Contraceptive choices, pregnancy rates, and outcomes in a microbicide trial.

OBJECTIVE: Women who become pregnant during the conduct of biomedical human immunodeficiency virus prevention trials are taken off the study product for safety reasons. High pregnancy rates can compromise statistical integrity in these trials. The comprehensive contraceptive curriculum developed for the Centre for the AIDS Programme of Research in South Africa (CAPRISA) 004 trial was evaluated for its ability to enhance contraceptive uptake, reduce pregnancy rates, and preserve statistical integrity. METHODS: Contraceptive- and pregnancy-related eligibility criteria were specified in the protocol. We enrolled women who opted for a nonbarrier method of contraceptive and provided hormonal contraceptives onsite at no cost. At each monthly study visit, we provided pregnancy prevention counseling and performed pregnancy testing. Study product was withheld on pregnancy diagnosis, but women continued with monthly follow-up. RESULTS: Contraceptive use was high throughout the study with 100% uptake at baseline and 94.71% use after a mean of 18 months follow-up at exit. Injectable progestins, particularly medroxyprogesterone acetate, remained the preferred choice of contraceptive. After 30 months of follow-up, 54 pregnancies were reported out of 889 participants, giving a pregnancy incidence rate of 3.95 per 100 woman-years (95% confidence interval 2.96-5.17). Of all pregnancies, two thirds (64.81%) resulted in a full-term live birth, whereas 18.52% and 11.11% pregnancies culminated as miscarriage and terminated pregnancies, respectively. There were no congenital anomalies in the early neonatal period. Pregnancies resulted in 1.56% of woman-years of study follow-up lost as a result of temporary product withdrawal. CONCLUSION: The CAPRISA 004 contraceptive curriculum was an effective strategy for maintaining low pregnancy rates, thereby minimizing product withdrawal and loss of follow-up time. LEVEL OF EVIDENCE: III.

Recruitment of high risk women for HIV prevention trials: baseline HIV prevalence and sexual behavior in the CAPRISA 004 tenofovir gel trial.

BACKGROUND: Young women in sub-Saharan Africa bear a disproportionate burden of HIV infection compared to men but have limited options to reduce their HIV risk. Microbicides could fill an important HIV prevention gap for sexually active women who are unable to successfully negotiate mutual monogamy or condom use. PURPOSE: This paper describes the baseline sample characteristics in the CAPRISA 004 trial which assessed the safety and effectiveness of the vaginal microbicide, 1% tenofovir gel for HIV prevention in South Africa. METHODS: This analysis assessed the baseline demographic, clinical and sexual behavior data of women screened and enrolled into the trial. The characteristics were summarized using descriptive summary measures; expressed as means and percent for categorical variables. RESULTS: HIV prevalence at screening was 25.8% [95% Confidence Interval (CI):23.9-27.7). Of the 889 eligibly enrolled women who contributed follow-up data, rural participants recruited from a family planning (FP) clinic were younger, more likely to be living apart from their regular partner, reported lower coital frequency, had lower condom use (p < 0.001). In contrast, urban participants recruited from a sexually transmitted disease (STD) clinic reported higher numbers of lifetime sexual partners, new partners in the last 30 days and receiving money in exchange for sex (p < 0.001). CONCLUSION: The populations selected provide suitable diverse target groups for HIV prevention intervention studies. TRIAL REGISTRATION: ClinicalTrials.gov: NCT 00441298.

The neutralization breadth of HIV-1 develops incrementally over four years and is associated with CD4+ T cell decline and high viral load during acute infection.

An understanding of how broadly neutralizing activity develops in HIV-1-infected individuals is needed to guide vaccine design and immunization strategies. Here we used a large panel of 44 HIV-1 envelope variants (subtypes A, B, and C) to evaluate the presence of broadly neutralizing antibodies in serum samples obtained 3 years after seroconversion from 40 women enrolled in the CAPRISA 002 acute infection cohort. Seven of 40 participants had serum antibodies that neutralized more than 40% of viruses tested and were considered to have neutralization breadth. Among the samples with breadth, CAP257 serum neutralized 82% (36/44 variants) of the panel, while CAP256 serum neutralized 77% (33/43 variants) of the panel. Analysis of longitudinal samples showed that breadth developed gradually starting from year 2, with the number of viruses neutralized as well as the antibody titer increasing over time. Interestingly, neutralization breadth peaked at 4 years postinfection, with no increase thereafter. The extent of cross-neutralizing activity correlated with CD4(+) T cell decline, viral load, and CD4(+) T cell count at 6 months postinfection but not at later time points, suggesting that early events set the stage for the development of breadth. However, in a multivariate analysis, CD4 decline was the major driver of this association, as viral load was not an independent predictor of breadth. Mapping of the epitopes targeted by cross-neutralizing antibodies revealed that in one individual these antibodies recognized the membrane-proximal external region (MPER), while in two other individuals, cross-neutralizing activity was adsorbed by monomeric gp120 and targeted epitopes that involved the N-linked glycan at position 332 in the C3 region. Serum antibodies from the other four participants targeted quaternary epitopes, at least 2 of which were PG9/16-like and depended on the N160 and/or L165 residue in the V2 region. These data indicate that fewer than 20% of HIV-1 subtype C-infected individuals develop antibodies with cross-neutralizing activity after 3 years of infection and that these antibodies target different regions of the HIV-1 envelope, including as yet uncharacterized epitopes.

Potent and broad neutralization of HIV-1 subtype C by plasma antibodies targeting a quaternary epitope including residues in the V2 loop.

The targets of broadly cross-neutralizing (BCN) antibodies are of great interest in the HIV vaccine field. We have identified a subtype C HIV-1-superinfected individual, CAP256, with high-level BCN activity, and characterized the antibody specificity mediating breadth. CAP256 developed potent BCN activity peaking at 3 years postinfection, neutralizing 32 (76%) of 42 heterologous viruses, with titers of antibodies against some viruses exceeding 1:10,000. CAP256 showed a subtype bias, preferentially neutralizing subtype C and A viruses over subtype B viruses. CAP256 BCN serum targeted a quaternary epitope which included the V1V2 region. Further mapping identified residues F159, N160, L165, R166, D167, K169, and K171 (forming the FN/LRD-K-K motif) in the V2 region as crucial to the CAP256 epitope. However, the fine specificity of the BCN response varied over time and, while consistently dependent on R166 and K169, became gradually less dependent on D167 and K171, possibly contributing to the incremental increase in breadth over 4 years. The presence of an intact FN/LRD-K-K motif in heterologous viruses was associated with sensitivity, although the length of the adjacent V1 loop modulated the degree of sensitivity, with a shorter V1 region significantly associated with higher titers. Repair of the FN/LRD-K-K motif in resistant heterologous viruses conferred sensitivity, with titers sometimes exceeding 1:10,000. Comparison of the CAP256 epitope with that of the PG9/PG16 monoclonal antibodies suggested that these epitopes overlapped, adding to the mounting evidence that this may represent a common neutralization target that should be further investigated as a potential vaccine candidate.