Nonhlanhla N. Mkhize

Defining genital tract cytokine signatures of sexually transmitted infections and bacterial vaginosis in women at high risk of HIV infection: a cross-sectional study

Objectives Sexually transmitted infections (STI) and bacterial vaginosis (BV) cause female genital tract inflammation. This inflammation, which is often present in the absence of symptoms, is associated with increased susceptibility to HIV infection. We aimed to evaluate genital cytokine profiles and the degree of inflammation associated with common STIs and BV. Methods HIV-uninfected women (n=227) were screened for BV, Chlamydia trachomatis, Neisseria gonorrhoeae, Herpes simplex virus type 2 (HSV-2), and Trichomonas vaginalis. Concentrations of 42 cytokines in cervicovaginal lavages and 13 cytokines in plasma were measured using Luminex. Changes in cytokine profiles were evaluated using Mann–Whitney U test, logistic regression and factor analysis. p Values were adjusted for multiple comparisons using a false discovery rate step-down procedure. Results Women with chlamydia or gonorrhoea had the highest genital cytokine concentrations, with 17/42 and 14/42 cytokines upregulated compared with women with no infection, respectively. BV was associated with elevated proinflammatory cytokine concentrations, but lower chemokine and haematopoietic cytokine concentrations. HSV-2 reactivation was associated with lower levels of inflammation, while trichomoniasis did not cause significant differences in genital cytokine concentrations. Genital infections did not influence plasma cytokine concentrations. Although certain STIs, in particular chlamydia and gonorrhoea, were associated with high genital cytokine concentrations, only 19% of women with an STI/BV had clinical signs. Conclusions Chlamydia was associated with the highest genital cytokine levels, followed by gonorrhoea, HSV-2, trichomoniasis, and BV. In regions where HIV is prevalent and STIs are managed syndromically, better STI/BV screening is urgently needed, as certain infections were found to be highly inflammatory.

Reactivity of routine HIV antibody tests in children who initiated antiretroviral therapy in early infancy as part of the Children with HIV Early Antiretroviral Therapy (CHER) trial: a retrospective analysis

BACKGROUND Early antiretroviral therapy (ART) and virological suppression can affect evolving antibody responses to HIV infection. We aimed to assess frequency and predictors of seronegativity in infants starting early ART. METHODS We compared HIV antibody results between two of three treatment groups of the Children with HIV Early Antiretroviral Therapy (CHER) trial, done from July, 2005, until July, 2011, in which infants with HIV infection aged 5·7-12·0 weeks with a percentage of CD4-positive T lymphocytes of at least 25% were randomly assigned to immediate ART for 96 weeks (ART-96W) or deferred ART until clinical or immunological progression (ART-Def). We measured antibody from all available stored samples for ART-96W and ART-Def at trial week 84 using three assays: fourth-generation enzyme immunoassay HIV antigen-antibody combination, HIV-1 and HIV-2 rapid antibody test, and quantitative anti-gp120 IgG ELISA. We also assessed odds of seropositivity with respect to age of ART initiation and cumulative viral load. The CHER trial was registered with ClinicalTrials.gov, number NCT00102960. FINDINGS The median age of the infants from when samples were taken (184 samples from 268 infants) was 92 weeks (IQR 90·6-93·4). More specimens from the ART-96W group were seronegative than from the ART-Def group by enzyme immunoassay (ART-96W 49 [46%] of 107 vs ART-Def eight [11%] of 75; p<0·0001) and rapid antibody test (54 [53%] of 101 vs eight [11%] of 74; p<0·0001). Median anti-gp120 IgG concentration was lower in the ART-96W group (230 μg/μL [IQR 133-13 129]) than in the ART-Def group (6870 μg/μL [1706-53 645]; p<0·0001). If ART was started between 12 and 24 weeks of age, odds of seropositivity were increased 13·7 times (95% CI 3·1-60·2; p=0·001) compared with starting it between 0 and 12 weeks. All children starting ART aged older than 24 weeks were seropositive. Cumulative viral load to week 84 correlated with anti-gp120 IgG concentrations (coefficient 0·54; p<0·0001) and increased odds of seropositivity (odds ratio 1·59 [95% CI 1·1-2·3]) adjusted for ART initiation age. INTERPRETATION About half of children starting ART before 12 weeks of age were HIV seronegative by almost 2 years of age. HIV antibody tests cannot be used to reconfirm HIV diagnosis in children starting early ART. Long-term effects of seronegativity need further study. Clear guidelines are needed for retesting alongside improved diagnostic tests. FUNDING Wellcome Trust, Medical Research Council, and National Institutes of Health.

South African HIV-1 subtype C transmitted variants with a specific V2 motif show higher dependence on α4β7 for replication

BackgroundThe integrin α4β7 mediates the trafficking of immune cells to the gut associated lymphoid tissue (GALT) and is an attachment factor for the HIV gp120 envelope glycoprotein. We developed a viral replication inhibition assay to more clearly evaluate the role of α4β7 in HIV infection and the contribution of viral and host factors.ResultsReplication of 60 HIV-1 subtype C viruses collected over time from 11 individuals in the CAPRISA cohort were partially inhibited by antibodies targeting α4β7. However, dependence on α4β7 for replication varied substantially among viral isolates from different individuals as well as over time in some individuals. Among 8 transmitted/founder (T/F) viruses, α4β7 reactivity was highest for viruses having P/SDI/V tri-peptide binding motifs. Mutation of T/F viruses that had LDI/L motifs to P/SDI/V resulted in greater α4β7 reactivity, whereas mutating P/SDI/V to LDI/L motifs was associated with reduced α4β7 binding. P/SDI/V motifs were more common among South African HIV subtype C viruses (35%) compared to subtype C viruses from other regions of Africa (<8%) and to other subtypes, due in part to a founder effect. In addition, individuals with bacterial vaginosis (BV) and who had higher concentrations of IL-7, IL-8 and IL-1α in the genital tract had T/F viruses with higher α4β7 dependence for replication, suggesting that viruses with P/SDI/V motifs may be preferentially transmitted in the presence of BV in this population.ConclusionsCollectively, these data suggest a role for α4β7 in HIV infection that is influenced by both viral and host factors including the sequence of the α4β7 binding motif, the cytokine milieu and BV in the genital tract. The higher frequency of P/SDI/V sequences among South African HIV-1 subtype C viruses may have particular significance for the role of α4β7 in this geographical region.

Stability and transport of cervical cytobrushes for isolation of mononuclear cells from the female genital tract

Cervical cytobrushing, biopsy, or lavages have previously been used to collect mononuclear cells from the female genital tract. Compared with blood, obtaining cells from the female genital tract is more invasive and generally yields few cells for subsequent immune studies. Because of the value of including mucosal sampling in HIV vaccine trials, standardisation of methods for collection, processing, and analysis of immunity from cells derived from the female genital tract is important. The aim of this study was to assess the effect of transport conditions on the viability, recovery and antigenic responsiveness of cervical T cells. This was investigated in cervical cytobrush specimens collected from 215 chronically HIV-infected women. Cytobrushes were either processed immediately, after cryopreservation, or after 24 h at 37 °C, 4 °C or room temperature. CD3+ T cell numbers were quantified using Guava automated cell counting. Viability was assessed using Trypan and Annexin/PI staining. Intracellular cytokine staining was used to evaluate IFN-γ responses to PMA, PHA and CEF peptides in cytobrush-derived T cells ex vivo and after delayed processing. In vitro polyclonal expansion of thawed cervical lymphocytes was conducted for 14 days in the presence of anti-CD3 and IL-2. We found that CD3+ T cell recovery and viability was similar in cytobrushes processed immediately or after 24 h irrespective of the conditions at which they were maintained. Fifty percent of the CD3+ T cells could be recovered after cryopreservation of cytobrushes and these could be polyclonally expanded in half of the cryopreserved samples. IFN-γ production following mitogenic stimulation was similar in ex vivo and delayed processing cytobrushes. Maintaining cytobrushes at 37 °C prior to processing significantly improved the detection of CEF-specific T cell responses compared to ex vivo. We conclude that cervical cytobrush-derived T cells are robust and can preserve their viability, phenotype and function over 24 h of mock transport.

Inflammatory cytokine biomarkers to identify women with asymptomatic sexually transmitted infections and bacterial vaginosis who are at high risk of HIV infection

Background Untreated sexually transmitted infections (STIs) and bacterial vaginosis (BV) cause genital inflammation and increase the risk of HIV infection. WHO-recommended syndromic STI and BV management is severely limited as many women with asymptomatic infections go untreated. The purpose of this cross-sectional study was to evaluate genital cytokine profiles as a biomarker of STIs and BV to identify women with asymptomatic, treatable infections. Methods Concentrations of 42 cytokines in cervicovaginal lavages from 227 HIV-uninfected women were measured using Luminex. All women were screened for BV by microscopy and STIs using molecular assays. Multivariate analyses were used to identify cytokine profiles associated with STIs/BV. Results A multivariate profile of seven cytokines (interleukin (IL)-1α, IL-1β, tumour necrosis factor-β, IL-4, fractalkine, macrophage-derived chemokine, and interferon-γ) most accurately predicted the presence of a treatable genital condition, with 77% classification accuracy and 75% cross-validation accuracy (sensitivity 72%; specificity 81%, positive predictive value (PPV) 86%, negative predictive value (NPV) 64%). Concomitant increased IL-1β and decreased IP-10 concentrations predicted the presence of a treatable genital condition without a substantial reduction in predictive value (sensitivity 77%, specificity 72%, PPV 82% and NPV 65%), correctly classifying 75% of the women. This approach performed substantially better than clinical signs (sensitivity 19%, specificity 92%, PPV 79% and NPV 40%). Conclusions Supplementing syndromic management with an assessment of IL-1β and IP-10 as biomarkers of genital inflammation may improve STI/BV management for women, enabling more effective treatment of asymptomatic infections and potentially reducing their risk of HIV infection.

Structure of an N276-Dependent HIV-1 Neutralizing Antibody Targeting a Rare V5 Glycan Hole Adjacent to the CD4 Binding Site

ABSTRACT All HIV-1-infected individuals develop strain-specific neutralizing antibodies to their infecting virus, which in some cases mature into broadly neutralizing antibodies. Defining the epitopes of strain-specific antibodies that overlap conserved sites of vulnerability might provide mechanistic insights into how broadly neutralizing antibodies arise. We previously described an HIV-1 clade C-infected donor, CAP257, who developed broadly neutralizing plasma antibodies targeting an N276 glycan-dependent epitope in the CD4 binding site. The initial CD4 binding site response potently neutralized the heterologous tier 2 clade B viral strain RHPA, which was used to design resurfaced gp120 antigens for single-B-cell sorting. Here we report the isolation and structural characterization of CAP257-RH1, an N276 glycan-dependent CD4 binding site antibody representative of the early CD4 binding site plasma response in donor CAP257. The cocrystal structure of CAP257-RH1 bound to RHPA gp120 revealed critical interactions with the N276 glycan, loop D, and V5, but not with aspartic acid 368, similarly to HJ16 and 179NC75. The CAP257-RH1 monoclonal antibody was derived from the immunoglobulin-variable IGHV3-33 and IGLV3-10 genes and neutralized RHPA but not the transmitted/founder virus from donor CAP257. Its narrow neutralization breadth was attributed to a binding angle that was incompatible with glycosylated V5 loops present in almost all HIV-1 strains, including the CAP257 transmitted/founder virus. Deep sequencing of autologous CAP257 viruses, however, revealed minority variants early in infection that lacked V5 glycans. These glycan-free V5 loops are unusual holes in the glycan shield that may have been necessary for initiating this N276 glycan-dependent CD4 binding site B-cell lineage. IMPORTANCE The conserved CD4 binding site on gp120 is a major target for HIV-1 vaccine design, but key events in the elicitation and maturation of different antibody lineages to this site remain elusive. Studies have shown that strain-specific antibodies can evolve into broadly neutralizing antibodies or in some cases act as helper lineages. Therefore, characterizing the epitopes of strain-specific antibodies may help to inform the design of HIV-1 immunogens to elicit broadly neutralizing antibodies. In this study, we isolate a narrowly neutralizing N276 glycan-dependent antibody and use X-ray crystallography and viral deep sequencing to describe how gp120 lacking glycans in V5 might have elicited these early glycan-dependent CD4 binding site antibodies. These data highlight how glycan holes can play a role in the elicitation of B-cell lineages targeting the CD4 binding site.

CD4 T cell depletion at the cervix during HIV infection is associated with accumulation of terminally differentiated T cells

ABSTRACT In blood, the accumulation of terminally differentiated (TD) T cells during HIV infection is associated with CD4 T cell loss and HIV disease progression. Here, we investigated the maintenance and functional characteristics of memory T cells at the cervix. We found that CD4 T cell depletion at the cervix mirrors CD4 depletion in blood. In all women, depletion of CD4 T cells at the cervix was associated with significant reductions in CD45RA− CCR7+ (central memory [CM]) T cells and the accumulation of CD45RA+ CCR7− (TD T cells). We determined whether inflammation in the genital tract was associated with the local differentiation of T cells at the cervix. In uninfected women, genital tract inflammation was associated with the accumulation of CD45RA− CCR7+ CM CD4 T cells and reduced frequencies of CD45RA+ CCR7− TD cells at the cervix. This finding may reflect the fact that, in the absence of HIV infection, TD T cells may be slowly lost in the presence of genital inflammation, while CD45RA− CCR7+ CM T cells are recruited to replenish the diminishing CD4 T cell pool. Following global stimulation with phorbol myristate acetate (PMA)-ionomycin, we noted a significant interleukin 2 (IL-2) deficit in both cervical and blood CD4 T cells from HIV-infected women compared to uninfected women, while gamma interferon (IFN-γ) production was similar, irrespective of HIV status. Few HIV-infected women had detectable IFN-γ and IL-2 HIV-specific T cell responses at the cervix, and these responses were significantly lower in magnitude than the corresponding responses in blood. These data suggest that CD4 depletion was associated with the accumulation of terminally differentiated T cell phenotypes at the cervical mucosa defective in their ability to produce IL-2. CD4 depletion and compromised immunity at the cervix may be accompanied by progressive decline of central memory-like T cells and development of T cells toward terminally differentiated phenotypes.

Persistence of Genital Tract T Cell Responses in HIV-Infected Women on Highly Active Antiretroviral Therapy

ABSTRACT Initiation of highly active antiretroviral therapy (HAART) for HIV-infected individuals is associated with control of viremia, improved CD4 counts, and declining systemic HIV-specific immune responses. While HAART effectively reduces plasma viremia, it remains unclear how effectively antiretroviral drugs reach mucosal surfaces, such as those of the genital tract. The aim of this study was to determine the effect of HAART on genital tract CD4 T cell reconstitution, HIV shedding, and HIV-specific T cell responses. Cervical cytobrush and blood specimens were obtained from 35 HIV-infected, HAART-naïve women and 27 women on HAART in order to investigate HIV Gag-specific T cell responses by intracellular gamma interferon (IFN-γ) staining. Interleukin 1β (IL-1β), IL-6, and IL-8 concentrations were measured by enzyme-linked immunosorbent assays (ELISA). We show that for HIV-infected women, HAART is associated with significantly improved CD4 T cell counts both in blood and at the cervix. While HAART effectively suppressed both blood and cervical viremia, HIV-specific CD8 T cell responses in blood were lost, while those at the cervix were preserved.

Evaluation of CD103 (αEβ7) integrin expression by CD8 T cells in blood as a surrogate marker to predict cervical T cell responses in the female genital tract during HIV infection.

Mucosal homing receptors expressed by blood T cells may be useful surrogates for measuring mucosal T cell immune responses at the site of HIV transmission. Here, we investigated whether HIV-specific responses by T cells expressing the mucosal integrin receptor CD103 in blood reliably predicted corresponding HIV-specific responses at the cervix. The frequency of CD8+ T cells expressing CD103 in blood correlated significantly with the number of CD103+CD8+ T cells from the cervix suggesting that CD103 was involved in trafficking of T cells from blood to the cervical mucosa. TGF-β concentrations in plasma were significantly associated with the frequency of CD103 expression by blood but not cervical CD8 T cells. The majority of Gag-responsive CD8 T cells were CD103+ in both blood and at the cervix. Despite this, the magnitude of Gag-specific IFN-γ responses by CD103+CD8+ T cells in blood did not predict similar Gag-specific responses at the cervix.

HIV-specific Fc effector function early in infection predicts the development of broadly neutralizing antibodies.

While the induction of broadly neutralizing antibodies (bNAbs) is a major goal of HIV vaccination strategies, there is mounting evidence to suggest that antibodies with Fc effector function also contribute to protection against HIV infection. Here we investigated Fc effector functionality of HIV-specific IgG plasma antibodies over 3 years of infection in 23 individuals, 13 of whom developed bNAbs. Antibody-dependent cellular phagocytosis (ADCP), complement deposition (ADCD), cellular cytotoxicity (ADCC) and cellular trogocytosis (ADCT) were detected in almost all individuals with levels of activity increasing over time. At 6 months post-infection, individuals with bNAbs had significantly higher levels of ADCD and ADCT that correlated with antibody binding to C1q and FcγRIIa respectively. In addition, antibodies from individuals with bNAbs showed more IgG subclass diversity to multiple HIV antigens which also correlated with Fc polyfunctionality. Germinal center activity represented by CXCL13 levels and expression of activation-induced cytidine deaminase (AID) was found to be associated with neutralization breadth, Fc polyfunctionality and IgG subclass diversity. Overall, multivariate analysis by random forest classification was able to group bNAb individuals with 85% sensitivity and 80% specificity based on the properties of their antibody Fc early in HIV infection. Thus, the Fc effector function profile predicted the development of neutralization breadth in this cohort, suggesting that intrinsic immune factors within the germinal center provide a mechanistic link between the Fc and Fab of HIV-specific antibodies.