Exceptional molecular and coreceptor-requirement properties of molecular clones isolated from an Human Immunodeficiency Virus Type-1 subtype C infection

<p>Abstract</p> <p>Background</p> <p>The pathogenic significance of coreceptor switch in the viral infection of HIV-1 is not completely understood. This situation is more complex in subtype C infection where coreceptor switch is either absent or extremely rare. To gain insights into the mechanisms that underlie coreceptor requirement of subtype C, we screened several primary viral isolates and identified a clinical sample that demonstrated a potential to grow on standard T-cell lines with no detectable CCR5 expression. The subject was diagnosed with HIV-1 associated dementia in the absence of opportunistic infections of the brain. To isolate molecular clones from this virus, we devised a novel strategy based on anchor primers that target a sequence in the reverse transcriptase, highly conserved among diverse subtypes of HIV-1.</p> <p>Results</p> <p>Using this strategy, we isolated 8 full-length molecular clones from the donor. Two of the eight molecular clones, 03In94_D17 and 03In94_D24, (D17 and D24) generated replication-competent viruses. Phylogenetic analysis of the full-length viral sequences revealed that both clones were non-recombinant subtype C viruses. They contain intact open reading frames in all the viral proteins. Both the viral clones are endowed with several unique molecular and biological properties. The viral promoter of the clones is characterized by the presence of four NF-kB binding elements, a feature rarely seen in the subtype C HIV-1 LTR. Interestingly, we identified the coexistence of two different forms of Rev, a truncated form common to subtype C and a full-length form less common for this subtype, in both proviral and plasma virus compartments. An exceptional property of the viruses, atypical of subtype C, is their ability to use a wide range of coreceptors including CCR5, CXCR4, and several others tested. Sequence analysis of Env of D17 and D24 clones identified differences within the variable loops providing important clues for the expanded coreceptor use. The V1, V2 and V4 loops in both of the molecular clones are longer due to the insertion of several amino acid residues that generated potential N-linked glycosylation sites.</p> <p>Conclusion</p> <p>The exceptional biological and molecular properties of these clones make them invaluable tools to understand the unique pathogenic characteristics of subtype C.</p

New member of the V1V2-directed CAP256-VRC26 lineage that shows increased breadth and exceptional potency.

CAPRISA, 2016.Abstract available in pdf

Enhancing durability of CIS43 monoclonal antibody by Fc mutation or AAV delivery for malaria prevention

CIS43 is a potent neutralizing human mAb that targets a highly conserved “junctional” epitope in the Plasmodium falciparum (Pf) circumsporozoite protein (PfCSP). Enhancing the durability of CIS43 in vivo will be important for clinical translation. Here, 2 approaches were used to improve the durability of CIS43 in vivo while maintaining potent neutralization. First, the Fc domain was modified with the LS mutations (CIS43LS) to increase CIS43 binding affinity for the neonatal Fc receptor (FcRn). CIS43LS and CIS43 showed comparable in vivo protective efficacy. CIS43LS had 9- to 13-fold increased binding affinity for human (6.2 nM versus 54.2 nM) and rhesus (25.1 nM versus 325.8 nM) FcRn at endosomal pH 6.0 compared with CIS43. Importantly, the half-life of CIS43LS in rhesus macaques increased from 22 days to 39 days compared with CIS43. The second approach for sustaining antibody levels of CIS43 in vivo is through adeno-associated virus (AAV) expression. Mice administered once with AAV-expressing CIS43 had sustained antibody levels of approximately 300 μg/mL and mediated protection against sequential malaria challenges up to 36 weeks. Based on these data, CIS43LS has advanced to phase I clinical trials, and AAV delivery provides a potential next-generation approach for malaria prevention

Polyfunctional CD4 T-cells correlating with neutralising antibody is a hallmark of COVISHIELDTM and COVAXIN® induced immunity in COVID-19 exposed Indians

Abstract Detailed characterisation of immune responses induced by COVID-19 vaccines rolled out in India: COVISHIELDTM (CS) and COVAXIN® (CO) in a pre-exposed population is only recently being discovered. We addressed this issue in subjects who received their primary series of vaccination between November 2021 and January 2022. Both vaccines are capable of strongly boosting Wuhan Spike-specific neutralising antibody, polyfunctional Th1 cytokine producing CD4+ T-cells and single IFN-γ + CD8+ T-cells. Consistent with inherent differences in vaccine platform, the vector-based CS vaccine-induced immunity was of greater magnitude, breadth, targeting Delta and Omicron variants compared to the whole-virion inactivated vaccine CO, with CS vaccinees showing persistent CD8+ T-cells responses until 3 months post primary vaccination. This study provides detailed evidence on the magnitude and quality of CS and CO vaccine induced responses in subjects with pre-existing SARS-CoV-2 immunity in India, thereby mitigating vaccine hesitancy arguments in such a population, which remains a global health challenge

Improved pharmacokinetics of HIV-neutralizing VRC01-class antibodies achieved by reduction of net positive charge on variable domain

ABSTRACTThe amino-acid composition of the immunoglobulin variable region has been observed to impact antibody pharmacokinetics (PK). Here, we sought to improve the PK of the broad HIV−1-neutralizing VRC01-class antibodies, VRC07-523LS and N6LS, by reducing the net positive charge in their variable domains. We used a structure-guided approach to generate a panel of antibody variants incorporating select Arg or Lys substituted to Asp, Gln, Glu, or Ser. The engineered variants exhibited reduced affinity to heparin, reduced polyreactivity, and improved PK in human FcRn-transgenic mice. One variant, VRC07-523LS.v34, with three charge substitutions, had an observed in vivo half-life and an estimated human half-life of 10.8 and 60 days, respectively (versus 5.4 and 38 days for VRC07-523LS) and retained functionality, neutralizing 92% of a 208-strain panel at a geometric mean IC80 <1 µg/mL. Another variant, N6LS.C49, with two charge substitutions, had an observed in vivo half-life and an estimated human half-life of 14.5 and 80 days (versus 9.0 and 44 days for N6LS) and neutralized ~80% of 208 strains at a geometric mean IC80 <1 µg/mL. Since Arg and Lys residues are prevalent in human antibodies, we propose substitution of select Arg or Lys with Asp, Gln, Glu, or Ser in the framework region as a general means to improve PK of therapeutic antibodies

Surface-Matrix Screening Identifies Semi-specific Interactions that Improve Potency of a Near Pan-reactive HIV-1-Neutralizing Antibody

SUMMARY Highly effective HIV-1-neutralizing antibodies could have utility in the prevention or treatment of HIV-1 infection. To improve the potency of 10E8, an antibody capable of near pan-HIV-1 neutralization, we engineered 10E8-surface mutants and screened for improved neutralization. Variants with the largest functional enhancements involved the addition of hydrophobic or positively charged residues, which were positioned to interact with viral membrane lipids or viral glycan-sialic acids, respectively. In both cases, the site of improvement was spatially separated from the region of antibody mediating molecular contact with the protein component of the antigen, thereby improving peripheral semi-specific interactions while maintaining unmodified dominant contacts responsible for broad recognition. The optimized 10E8 antibody, with mutations to phenylalanine and arginine, retained the extraordinary breadth of 10E8 but with ~10-fold increased potency. We propose surface-matrix screening as a general method to improve antibodies, with improved semi-specific interactions between antibody and antigen enabling increased potency without compromising breadth

Vaccine-Induced Antibodies that Neutralize Group 1 and Group 2 Influenza A Viruses

Antibodies capable of neutralizing divergent influenza A viruses could form the basis of a universal vaccine. Here, from subjects enrolled in an H5N1 DNA/MIV-prime-boost influenza vaccine trial, we sorted hemagglutinin cross-reactive memory B cells and identified three antibody classes, each capable of neutralizing diverse subtypes of group 1 and group 2 influenza A viruses. Co-crystal structures with hemagglutinin revealed that each class utilized characteristic germline genes and convergent sequence motifs to recognize overlapping epitopes in the hemagglutinin stem. All six analyzed subjects had sequences from at least one multidonor class, and—in half the subjects—multidonor-class sequences were recovered from >40% of cross-reactive B cells. By contrast, these multidonor-class sequences were rare in published antibody datasets. Vaccination with a divergent hemagglutinin can thus increase the frequency of B cells encoding broad influenza A-neutralizing antibodies. We propose the sequence signature-quantified prevalence of these B cells as a metric to guide universal influenza A immunization strategies