T. Blake Ball

Effects of a mobile phone short message service on antiretroviral treatment adherence in Kenya (WelTel Kenya1): a randomised trial

BACKGROUND Mobile (cell) phone communication has been suggested as a method to improve delivery of health services. However, data on the effects of mobile health technology on patient outcomes in resource-limited settings are limited. We aimed to assess whether mobile phone communication between health-care workers and patients starting antiretroviral therapy in Kenya improved drug adherence and suppression of plasma HIV-1 RNA load. METHODS WelTel Kenya1 was a multisite randomised clinical trial of HIV-infected adults initiating antiretroviral therapy (ART) in three clinics in Kenya. Patients were randomised (1:1) by simple randomisation with a random number generating program to a mobile phone short message service (SMS) intervention or standard care. Patients in the intervention group received weekly SMS messages from a clinic nurse and were required to respond within 48 h. Randomisation, laboratory assays, and analyses were done by investigators masked to treatment allocation; however, study participants and clinic staff were not masked to treatment. Primary outcomes were self-reported ART adherence (>95% of prescribed doses in the past 30 days at both 6 and 12 month follow-up visits) and plasma HIV-1 viral RNA load suppression (<400 copies per mL) at 12 months. The primary analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, NCT00830622. FINDINGS Between May, 2007, and October, 2008, we randomly assigned 538 participants to the SMS intervention (n=273) or to standard care (n=265). Adherence to ART was reported in 168 of 273 patients receiving the SMS intervention compared with 132 of 265 in the control group (relative risk [RR] for non-adherence 0·81, 95% CI 0·69-0·94; p=0·006). Suppressed viral loads were reported in 156 of 273 patients in the SMS group and 128 of 265 in the control group, (RR for virologic failure 0·84, 95% CI 0·71-0·99; p=0·04). The number needed to treat (NNT) to achieve greater than 95% adherence was nine (95% CI 5·0-29·5) and the NNT to achieve viral load suppression was 11 (5·8-227·3). INTERPRETATION Patients who received SMS support had significantly improved ART adherence and rates of viral suppression compared with the control individuals. Mobile phones might be effective tools to improve patient outcome in resource-limited settings. FUNDING US Presidents Emergency Plan for AIDS Relief.

Influence of HLA supertypes on susceptibility and resistance to human immunodeficiency virus type 1 infection

Certain human leukocyte antigens, by presenting conserved immunogenic epitopes for T cell recognition, may, in part, account for the observed differences in human immunodeficiency virus type 1 (HIV-1) susceptibility. To determine whether HLA polymorphism influences HIV-1 susceptibility, a longitudinal cohort of highly HIV-1-exposed female sex workers based in Nairobi, Kenya, was prospectively analyzed. Decreased HIV-1 infection risk was strongly associated with possession of a cluster of closely related HLA alleles (A2/6802 supertype; incidence rate ratio [IRR], 0.45; 95% confidence interval [CI], 0.27-0.72; P=.0003). The alleles in this supertype are known in some cases to present the same peptide epitopes for T cell recognition. In addition, resistance to HIV-1 infection was independently associated with HLA DRB1*01 (IRR, 0.22; 95% CI, 0.06-0.60; P=.0003), which suggests that anti-HIV-1 class II restricted CD4 effector mechanisms may play an important role in protecting against viral challenge. These data provide further evidence that resistance to HIV-1 infection in this cohort of sex workers is immunologically mediated.

Toll-like receptor expression and responsiveness are increased in viraemic HIV-1 infection

Objectives:Toll-like receptors (TLR) are important in pathogen recognition and may play a role in HIV disease. We evaluated the effect of chronic untreated and treated HIV-1 infection on systemic TLR expression and TLR signalling. Methods:Two hundred HIV-infected and uninfected women from a Kenya cohort participated in the studies. TLR1 to TLR10 messenger RNA expression was determined by quantitative reverse transcriptase polymerase chain reaction in peripheral blood mononuclear cells (PBMC). TLR ligand responsiveness was determined in or using ex-vivo PBMC by cytokine production in culture supernatants. Results:Chronic, untreated HIV-1 infection was significantly associated with increased mRNA expression of TLR6, TLR7, and TLR8 and when analysis was limited to those with advanced disease (CD4 cell count < 200 cells/ml) TLR2, TLR3, and TLR4 were additionally elevated. TLR expression correlated with the plasma HIV-RNA load, which was significant for TLR6 and TLR7. In vitro HIV single-stranded RNA alone could enhance TLR mRNA expression. PBMC of HIV-infected subjects also demonstrated profoundly increased proinflammatory responsiveness to TLR ligands, suggesting sensitization of TLR signalling in HIV. Finally, viral suppression by HAART was associated with a normalization of TLR levels. Conclusion:Together, these data indicate that chronic viraemic HIV-1 is associated with increased TLR expression and responsiveness, which may perpetuate innate immune dysfunction and activation that underlies HIV pathogenesis, and thus reveal potential new targets for therapy.

HIV-1-specific cellular immune responses among HIV-1-resistant sex workers.

The goal of the present study was to determine whether there were HIV‐1 specific cellular immune responses among a subgroup of women within a cohort of Nairobi prostitutes (n = 1800) who, despite their intense sexual exposure to HIV‐1, are epidemiologically resistant to HIV‐1 infection. Of the 80 women defined to be resistant, 24 were recruited for immunological evaluation. The HIV‐1‐specific T‐helper responses were determined by IL‐2 production following stimulation with HIV‐1 envelope peptides and soluble gp120. Cytotoxic T lymphocyte responses were determined by lysis of autologous EBV‐transformed B cell lines infected with control vaccinia virus or recombinant vaccinia viruses containing the HIV‐1 structural genes env, gag and pol. Resistant women had significantly increased HIV‐1 specific T‐helper responses, as determined by in vitro IL‐2 production to HIV‐1 envelope peptides and soluble glycoprotein 120, compared with low‐risk seronegative and HIV‐1‐infected controls (P ≤ 0.01, Students t‐test). Seven of the 17 (41%) resistant women showed IL‐2 stimulation indices ≥ 2.0. HIV‐1‐specific CTL responses were detected among 15/22 (68.2%) resistant women compared with 0/12 low‐risk controls (Chi‐squared test, P < 0.001). In the two resistant individuals tested, the CTL activity was mediated by CD8+ effectors. Many HIV‐1‐resistant women show evidence of HIV‐1‐specific T‐helper and cytotoxic responses. These data support the suggestion that HIV‐1‐specific T‐cell responses contribute to protection against HIV‐1 infection.

Human Immunodeficiency Virus (HIV) Type 1 Proviral Hypermutation Correlates with CD4 Count in HIV-Infected Women from Kenya

ABSTRACT APOBEC3G is an important innate immune molecule that causes human immunodeficiency virus type 1 (HIV-1) hypermutation, which can result in detrimental viral genome mutations. The Vif protein of wild-type HIV-1 counteracts APOBEC3G activity by targeting it for degradation and inhibiting its incorporation into viral particles. Additional APOBEC cytidine deaminases have been identified, such as APOBEC3F, which has a similar mode of action but different sequence specificity. A relationship between APOBEC3F/G and HIV disease progression has been proposed. During HIV-1 sequence analysis of the vpu/env region of 240 HIV-infected subjects from Nairobi, Kenya, 13 drastically hypermutated proviral sequences were identified. Sequences derived from plasma virus, however, lacked hypermutation, as did proviral vif. When correlates of disease progression were examined, subjects with hypermutated provirus were found to have significantly higher CD4 counts than the other subjects. Furthermore, hypermutation as estimated by elevated adenine content positively correlated with CD4 count for all 240 study subjects. The sequence context of the observed hypermutation was statistically associated with APOBEC3F/G activity. In contrast to previous studies, this study demonstrates that higher CD4 counts correlate with increased hypermutation in the absence of obvious mutations in the APOBEC inhibiting Vif protein. This strongly suggests that host factors, such as APOBEC3F/G, are playing a protective role in these patients, modulating viral hypermutation and host disease progression. These findings support the potential of targeting APOBEC3F/G for therapeutic purposes.

Pyrosequencing of the chaperonin-60 universal target as a tool for determining microbial community composition.

ABSTRACT We compared dideoxy sequencing of cloned chaperonin-60 universal target (cpn60 UT) amplicons to pyrosequencing of amplicons derived from vaginal microbial communities. In samples pooled from a number of individuals, the pyrosequencing method produced a data set that included virtually all of the sequences that were found within the clone library and revealed an additional level of taxonomic richness. However, the relative abundances of the sequences were different in the two datasets. These observations were expanded and confirmed by the analysis of paired clone library and pyrosequencing datasets from vaginal swabs taken from four individuals. Both for individuals with a normal vaginal microbiota and for those with bacterial vaginosis, the pyrosequencing method revealed a large number of low-abundance taxa that were missed by the clone library approach. In addition, we showed that the pyrosequencing method generates a reproducible profile of microbial community structure in replicate amplifications from the same community. We also compared the taxonomic composition of a vaginal microbial community determined by pyrosequencing of 16S rRNA amplicons to that obtained using cpn60 universal primers. We found that the profiles generated by the two molecular targets were highly similar, with slight differences in the proportional representation of the taxa detected. However, the number of operational taxonomic units was significantly higher in the cpn60 data set, suggesting that the protein-encoding gene provides improved species resolution over the 16S rRNA target. These observations demonstrate that pyrosequencing of cpn60 UT amplicons provides a robust, reliable method for deep sequencing of microbial communities.

Development and characterisation of neutralising monoclonal antibody to the SARS-coronavirus

Abstract There is a global need to elucidate protective antigens expressed by the SARS-coronavirus (SARS-CoV). Monoclonal antibody reagents that recognise specific antigens on SARS-CoV are needed urgently. In this report, the development and immunochemical characterisation of a panel of murine monoclonal antibodies (mAbs) against the SARS-CoV is presented, based upon their specificity, binding requirements, and biological activity. Initial screening by ELISA, using highly purified virus as the coating antigen, resulted in the selection of 103mAbs to the SARS virus. Subsequent screening steps reduced this panel to seventeen IgG mAbs. A single mAb, F26G15, is specific for the nucleoprotein as seen in Western immunoblot while five other mAbs react with the Spike protein. Two of these Spike-specific mAbs demonstrate the ability to neutralise SARS-CoV in vitro while another four Western immunoblot-negative mAbs also neutralise the virus. The utility of these mAbs for diagnostic development is demonstrated. Antibody from convalescent SARS patients, but not normal human serum, is also shown to specifically compete off binding of mAbs to whole SARS-CoV. These studies highlight the importance of using standardised assays and reagents. These mAbs will be useful for the development of diagnostic tests, studies of SARS-CoV pathogenesis and vaccine development.

HIV-1 RNA Dysregulates the Natural TLR Response to Subclinical Endotoxemia in Kenyan Female Sex-Workers

Background Subclinical endotoxemia has been reported in HIV-1 infected persons and may drive systemic immune activation and pathogenesis. Proinflammatory responsiveness to endotoxin (LPS) is mediated by Toll-like receptor 4 (TLR4). We therefore examined the association between plasma LPS levels, HIV RNA, and TLR4 expression and cytokine responses in the blood of HIV infected and uninfected participants in a cohort of female sex-workers in Kenya. Methodology/Principal Findings Ex vivo plasma and peripheral blood mononuclear cells (PBMC) were assessed for LPS and TLR mRNA, respectively. The effects of HIV single stranded RNA, a TLR8 ligand, on TLR4 and LPS signaling were further assessed in short term PBMC culture. Both HIV uninfected and infected subjects frequently had low detectable LPS levels in their plasmas. Significantly increased LPS levels were associated with chronic HIV-1 infection, both treated and untreated, but not with other acute or semi-chronic conditions reported. In HIV-uninfected subjects, TLR4 mRNA expression levels correlated inversely with plasma LPS levels, suggesting chronic endotoxin ‘tolerance’ in vivo. A similar effect of reduced TLR4 mRNA was seen in short term PBMC culture after stimulation with LPS. Interestingly, the apparent in vivo tolerance effect was diminished in subjects with HIV infection. Additionally, pre-stimulation of PBMC with LPS lead to proinflammatory (TNF-α) tolerance to subsequent LPS stimulation; however, pre-treatment of PBMC with HIV single-stranded RNA40, could enhance TLR4-mediated LPS responsiveness in vitro. Conclusions/Significance Thus, dysregulation of endotoxin tolerance by HIV-1 RNA may exacerbate HIV chronic immune activation and pathogenesis.

Molecular definition of vaginal microbiota in East African commercial sex workers.

ABSTRACT Resistance to HIV infection in a cohort of commercial sex workers living in Nairobi, Kenya, is linked to mucosal and antiinflammatory factors that may be influenced by the vaginal microbiota. Since bacterial vaginosis (BV), a polymicrobial dysbiosis characterized by low levels of protective Lactobacillus organisms, is an established risk factor for HIV infection, we investigated whether vaginal microbiology was associated with HIV-exposed seronegative (HESN) or HIV-seropositive (HIV+) status in this cohort. A subset of 44 individuals was selected for deep-sequencing analysis based on the chaperonin 60 (cpn60) universal target (UT), including HESN individuals (n = 16), other HIV-seronegative controls (HIV-N, n = 16), and HIV+ individuals (n = 12). Our findings indicate exceptionally high phylogenetic resolution of the cpn60 UT using reads as short as 200 bp, with 54 species in 29 genera detected in this group. Contrary to our initial hypothesis, few differences between HESN and HIV-N women were observed. Several HIV+ women had distinct profiles dominated by Escherichia coli. The deep-sequencing phylogenetic profile of the vaginal microbiota corresponds closely to BV+ and BV− diagnoses by microscopy, elucidating BV at the molecular level. A cluster of samples with intermediate abundance of Lactobacillus and dominant Gardnerella was identified, defining a distinct BV phenotype that may represent a transitional stage between BV+ and BV−. Several alpha- and betaproteobacteria, including the recently described species Variovorax paradoxus, were found to correlate positively with increased Lactobacillus levels that define the BV− (“normal”) phenotype. We conclude that cpn60 UT is ideally suited to next-generation sequencing technologies for further investigation of microbial community dynamics and mucosal immunity underlying HIV resistance in this cohort.

Multiplex Detection of Bacteria Associated with Normal Microbiota and with Bacterial Vaginosis in Vaginal Swabs by Use of Oligonucleotide-Coupled Fluorescent Microspheres

ABSTRACT Bacterial vaginosis (BV) is a recurrent condition that is associated with a range of negative outcomes, including the acquisition of human immunodeficiency virus and other sexually transmitted diseases, preterm births, and pelvic inflammatory disease. In contrast to the Lactobacillus-dominated normal vaginal microbiota, BV is characterized by a lack of lactobacilli and an abundance of anaerobic and gram-negative organisms, including Gardnerella vaginalis and Atopobium vaginae. To date, the laboratory diagnosis of BV has relied upon the fulfillment of criteria determined by microscopic observation of Gram-stained vaginal swabs. We describe a molecular-based method for the easy determination of the species profile within the vaginal microbiota based on the amplification of the chaperonin-60 genes of all bacteria present in the swab and hybridization of the amplicon to species-specific oligonucleotide-coupled fluorescent beads that are identified by flow cytometry with a Luminex instrument. We designed a nineplex Luminex array for characterization of the vaginal microbiota and applied it to the analysis of vaginal swabs from individuals from Africa and North America. Using the presence of A. vaginae or G. vaginalis, or both, as the defining criterion for BV, we found that the method was highly specific and sensitive for the diagnosis of BV using microscopy as a gold standard.