No evidence for selection of HIV-1 with enhanced Gag-Protease or Nef function among breakthrough infections in the CAPRISA 004 tenofovir microbicide trial

BACKGROUND: Use of antiretroviral-based microbicides for HIV-1 prophylaxis could introduce a transmission barrier that inadvertently facilitates the selection of fitter viral variants among incident infections. To investigate this, we assessed the in vitro function of gag-protease and nef sequences from participants who acquired HIV-1 during the CAPRISA 004 1% tenofovir microbicide gel trial. Methods and RESULTS: We isolated the earliest available gag-protease and nef gene sequences from 83 individuals and examined their in vitro function using recombinant viral replication capacity assays and surface protein downregulation assays, respectively. No major phylogenetic clustering and no significant differences in gag-protease or nef function were observed in participants who received tenofovir gel versus placebo gel prophylaxis. CONCLUSION: Results indicate that the partial protective effects of 1% tenofovir gel use in the CAPRISA 004 trial were not offset by selection of transmitted/early HIV-1 variants with enhanced Gag-Protease or Nef fitness

False-negative HIV-1 polymerase chain reaction in a 15-month-old boy with HIV-1 subtype C infection

Polymerase chain reaction (PCR) testing is the gold standard for determining the HIV status in children <18 months of age. However, when clinicalmanifestations are not consistent with laboratory results, additional investigation is required. We report a 15-month-old HIV-exposed boy referredto our hospital after he had been admitted several times for infectious diseases. A rapid antibody test on the child was positive, while routinediagnostic HIV PCRs using the Roche COBAS Ampliprep/COBAS TaqMan HIV Qual Test were negative at 6 weeks, 6 months, 7 months and15 months. In addition, the same PCR test performed on the HIV-infected mother was also negative. Alternative PCR and viral load assays usingdifferent primer sets detected HIV RNA or proviral DNA in both child and mother. Gag sequences from the child and his mother classified bothinfections as HIV-1 subtype C, with very rare mutations that may have resulted in PCR assay primer/probe mismatch. Consequently, the child wascommenced on antiretroviral therapy and made a remarkable recovery. These findings indicate that more reliable PCR assays capable of detectinga wide range of HIV subtypes are desirable to circumvent the clinical problems created by false-negative PCR results

Early evolution of HLA-associated escape mutations in variable Gag proteins predicts CD4+ decline in HIV-1 subtype C infected women.

CAPRISA, 2017.Abstract available in pdf

Replication capacity of viruses from acute infection drives HIV-1 disease progression.

CAPRISA, 2017.Abstract available in pdf

No evidence for selection of HIV-1 with enhanced gag-protease or nef function among breakthrough infections in the CAPRISA 004 tenofovir microbicide trial.

CAPRISA, 2013.Background: Use of antiretroviral-based microbicides for HIV-1 prophylaxis could introduce a transmission barrier that inadvertently facilitates the selection of fitter viral variants among incident infections. To investigate this, we assessed the in vitro function of gag-protease and nef sequences from participants who acquired HIV-1 during the CAPRISA 004 1% tenofovir microbicide gel trial. Methods and Results: We isolated the earliest available gag-protease and nef gene sequences from 83 individuals and examined their in vitro function using recombinant viral replication capacity assays and surface protein down regulation assays, respectively. No major phylogenetic clustering and no significant differences in gag-protease or nef function were observed in participants who received tenofovir gel versus placebo gel prophylaxis. Conclusion: Results indicate that the partial protective effects of 1% tenofovir gel use in the CAPRISA 004 trial were not offset by selection of transmitted/early HIV-1 variants with enhanced Gag-Protease or Nef fitness

Structure and recognition of a novel HIV-1 gp120-gp41 interface antibody that caused MPER exposure through viral escape.

CAPRISA, 2017.Abstract available in pdf

Neutralization profiles of HIV-1 viruses from the VRC01 Antibody Mediated Prevention (AMP) trials

The VRC01 Antibody Mediated Prevention (AMP) efficacy trials conducted between 2016 and 2020 showed for the first time that passively administered broadly neutralizing antibodies (bnAbs) could prevent HIV-1 acquisition against bnAb-sensitive viruses. HIV-1 viruses isolated from AMP participants who acquired infection during the study in the sub-Saharan African (HVTN 703/HPTN 081) and the Americas/European (HVTN 704/HPTN 085) trials represent a panel of currently circulating strains of HIV-1 and offer a unique opportunity to investigate the sensitivity of the virus to broadly neutralizing antibodies (bnAbs) being considered for clinical development. Pseudoviruses were constructed using envelope sequences from 218 individuals. The majority of viruses identified were clade B and C; with clades A, D, F and G and recombinants AC and BF detected at lower frequencies. We tested eight bnAbs in clinical development (VRC01, VRC07-523LS, 3BNC117, CAP256.25, PGDM1400, PGT121, 10–1074 and 10E8v4) for neutralization against all AMP placebo viruses (n = 76). Compared to older clade C viruses (1998–2010), the HVTN703/HPTN081 clade C viruses showed increased resistance to VRC07-523LS and CAP256.25. At a concentration of 1μg/ml (IC80), predictive modeling identified the triple combination of V3/V2-glycan/CD4bs-targeting bnAbs (10-1074/PGDM1400/VRC07-523LS) as the best against clade C viruses and a combination of MPER/V3/CD4bs-targeting bnAbs (10E8v4/10-1074/VRC07-523LS) as the best against clade B viruses, due to low coverage of V2-glycan directed bnAbs against clade B viruses. Overall, the AMP placebo viruses represent a valuable resource for defining the sensitivity of contemporaneous circulating viral strains to bnAbs and highlight the need to update reference panels regularly. Our data also suggests that combining bnAbs in passive immunization trials would improve coverage of global viruses

Recombinant expression of the pRb- and p53-interacting domains from the human RBBP6 protein for in vitro binding studies

Magister Scientiae - MScThis thesis describes the cloning and recombinant expression of domains from the human RBBP6 protein for future in vitro binding studies with pRb and p53. RBBP6 is a splicing-associated protein that is known to interact with both p53 and the Retinoblastoma gene product (pRb), and has recently been shown to be highly upregulated in oesophageal cancer. The pRb binding domain (RbBD) and the p53 binding domain (p53BD) were each expressed using the glutathione-S-transferase (GST) tag affinity system, and affinity purified using a glutathione-linked agarose column. Purified fusion proteins were cleaved to separate the target protein from GST using PreScission™ Protease, for which there is a recognition sequence located immediately upstream of the multiple cloning site on the pGEX-6P series of plasmids. The pRb binding and p53 binding domains were further purified using cation exchange chromatography. Mass spectrometry confirmed that the RbBD was expressed as a single species of the expected molecular weight. However preliminary NMR analysis suggested that the domain was not fully folded. A total yield of 8 mg of protein was achieved from 1l of culture, which make it feasible to express 15N and 12C labelled samples for NMR. The p53BD was found to be expressed at lower levels and subject to C-terminal degradation, which suggest that the C-terminus is unstructured most likely due to the presence of poly-lysine tail. Human pRb protein was also successfully expressed and purified using the GST affinity system. Human p53 protein was expressed but was found to be insoluble and attempts to purify it were not pursued. Attempts to confirm the interactions between human RBBP6 and p53 and pRb proteins are on-going but fall outside the scope of this thesis. Expression constructs for the RING and zinc finger domains from human RBBP6 were also cloned into the pGEX system for future structural studies using NMR. Both domains were found to be expressed as soluble fusion proteins in preliminary expression studies.South Afric

Brief report: selection of HIV-1 variants with higher transmission potential by 1% tenofovir gel microbicide.

CAPRISA, 2017.Abstract available in pdf

Functional analyses of Nef clones from treatment and placebo arms.

<p>Panel A: Selected Western blot results depict control SF2 Nef, empty (delta-Nef) plasmid and four samples each obtained from participants in the 1% tenofovir gel and placebo study arms. Panel B: Results for the CD4 downregulation activity of participant-derived <i>nef</i> isolates are shown, normalized to control SF2 Nef (which is equal to 1.0). Panel C: Results for the HLA-A*02 downregulation activity of participant-derived <i>nef</i> isolates are shown, normalized to control SF2 Nef (equal to 1.0). No significant differences in Nef-mediated CD4 or HLA downregulation function were observed between study arms (Mann-Whitney, p = 0.2 and p = 0.06, respectively).</p