Defining virus-antibody interplay during the development of HIV-1 neutralization breadth to inform vaccine design

A thesis submitted to the School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. Johannesburg, 2016Human Immunodeficiency Virus Type 1 (HIV-1) infects approximately two million people annually, highlighting the need for a preventative vaccine. An effective HIV-1 vaccine will likely need to elicit broadly neutralizing antibodies (bNAbs), which arise naturally in some infected individuals and recognize the envelopes (Env) of multiple HIV-1 strains. Understanding the molecular events that contribute to bNAb development during infection may provide a blueprint for vaccine strategies. Here we investigated the development of a V1V2-directed bNAb lineage in the context of viral co-evolution in an HIV-1 superinfected participant (CAP256). For this, clonally-related monoclonal antibodies (mAbs), with a range of neutralization breadth, were isolated. We determined their developmental pathway from strain-specificity towards neutralization breadth and identified viral variants responsible for initiating and maturing this bNAb lineage. MAbs were isolated by memory B cell culture or trimer-specific single B cell sorting and extensively characterized by Env-pseudotyped neutralization, cell surface-expressed Env binding, electron microscopy and epitope-predictive algorithms. Antibody next-generation sequencing (NGS) at multiple time-points enabled the inference of the unmutated common ancestor (UCA) of this lineage. Viral co-evolution was investigated using Env single genome amplification and V1V2 NGS. A family of 33 clonally-related mAbs, CAP256-VRC26.01-33, was isolated from samples spanning four years of infection. The UCA of this lineage possessed an unusually long heavy chain complementarity determining region 3 (CDRH3), which resulted from a unique recombination event. Surprisingly, this UCA potently neutralized later viral variants that had evolved from the superinfecting virus, which we termed bNAb-initiating Envs. Viral diversification, which peaked prior to the development of neutralization breadth, created multiple immunotypes at key residues in the V1V2 epitope. Exposure to these immunotypes allowed adaptation of some mAbs to tolerate this variation and thus mature towards neutralization breadth. Based on these data, we proposed a four-step immunization strategy which includes priming with bNAb-initiating Envs to engage rare B cells with a long CDRH3; followed by three sequential boosts (including select V1V2 immunotypes) to drive antibody maturation. In conclusion, this study has generated a testable HIV-1 vaccine immunization strategy through the delineation of mAb-virus co-evolution during the development of neutralization breadth.MT201

Broadly Neutralizing Antibodies as Treatment: Effects on Virus and Immune System

Purpose of Review The purpose of this study is to summarize recent advances in the use of broadly neutralizing antibodies (bNAbs) as therapeutics in human clinical trials and in non-human primate (NHP) models. We seek to highlight lessons from these studies with an emphasis on consequences to the virus and immune system. Recent Findings In the past 10 years, advances in HIV-1 trimer structure and B cell isolation methods have precipitated the identification of “new-generation” anti-HIV antibodies with broad and potent neutralization. In the past 2 years, the concept of using these bNAbs as therapeutic tools has moved from NHP models into human clinical trials. These trials have investigated the effects of bNAb infusions into patients chronically infected with HIV-1, while the NHP model has investigated treatment during acute infection. Summary Through this work, the relationship between in vitro breadth and potency and in vivo clinical effect, although unresolved, is gradually being elucidated. These results emphasize the need for combination antibody therapy

Results from the second WHO external quality assessment for the molecular detection of respiratory syncytial virus, 2019-2020

Background: External quality assessments (EQAs) for the molecular detection of human respiratory syncytial virus (RSV) are necessary to ensure the standardisation of reliable results. The Phase II, 2019–2020 World Health Organization (WHO) RSV EQA included 28 laboratories in 26 countries. The EQA panel evaluated performance in the molecular detection and subtyping of RSV-A and RSV-B. This manuscript describes the preparation, distribution, and analysis of the 2019–2020 WHO RSV EQA. Methods: Panel isolates underwent whole genome sequencing and in silico primer matching. The final panel included nine contemporary, one historical virus and two negative controls. The EQA panel was manufactured and distributed by the UK National External Quality Assessment Service (UK NEQAS). National laboratories used WHO reference assays developed by the United States Centers for Disease Control and Prevention, an RSV subtyping assay developed by the Victorian Infectious Diseases Reference Laboratory (Australia), or other in-house or commercial assays already in use at their laboratories. Results: An in silico analysis of isolates showed a good match to assay primer/probes. The panel was distributed to 28 laboratories. Isolates were correctly identified in 98% of samples for detection and 99.6% for subtyping. Conclusions: The WHO RSV EQA 2019–2020 showed that laboratories performed at high standards. Updating the composition of RSV molecular EQAs with contemporary strains to ensure representation of circulating strains, and ensuring primer matching with EQA panel viruses, is advantageous in assessing diagnostic competencies of laboratories. Ongoing EQAs are recommended because of continued evolution of mismatches between current circulating strains and existing primer sets.Fil: Williams, Thomas. University of Edinburgh; Reino UnidoFil: Jackson, Sandra. Organizacion Mundial de la Salud; ArgentinaFil: Barr, Ian. University of Melbourne; Australia. Victorian Infectious Diseases Reference Laboratory; AustraliaFil: Bi, Shabana. University of Melbourne; AustraliaFil: Bhiman, Jinal. National Health Laboratory Service; AustraliaFil: Ellis, Joanna. National Health Laboratory Service; AustraliaFil: von Gottberg, Anne. University of the Witwatersrand; SudáfricaFil: Lindstrom, Stephen. Centers for Disease Control and Prevention; Estados UnidosFil: Peret, Teresa. University of Texas Medical Branch; Estados UnidosFil: Rughooputh, Sanjiv. National Health Laboratory Service; AustraliaFil: Viegas, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Hirve, Siddhivinayak. Organizacion Mundial de la Salud; ArgentinaFil: Zambon, Maria. National Health Laboratory Service; AustraliaFil: Zhang, Wenqing. Organizacion Mundial de la Salud; Argentin

Structure and Recognition of a Novel HIV-1 gp120-gp41 Interface Antibody that Caused MPER Exposure through Viral Escape

A comprehensive understanding of the regions on HIV-1 envelope trimers targeted by broadly neutralizing antibodies may contribute to rational design of an HIV-1 vaccine. We previously identified a participant in the CAPRISA cohort, CAP248, who developed trimer-specific antibodies capable of neutralizing 60% of heterologous viruses at three years post-infection. Here, we report the isolation by B cell culture of monoclonal antibody CAP248-2B, which targets a novel membrane proximal epitope including elements of gp120 and gp41. Despite low maximum inhibition plateaus, often below 50% inhibitory concentrations, the breadth of CAP248-2B significantly correlated with donor plasma. Site-directed mutagenesis, X-ray crystallography, and negative-stain electron microscopy 3D reconstructions revealed how CAP248-2B recognizes a cleavage-dependent epitope that includes the gp120 C terminus. While this epitope is distinct, it overlapped in parts of gp41 with the epitopes of broadly neutralizing antibodies PGT151, VRC34, 35O22, 3BC315, and 10E8. CAP248-2B has a conformationally variable paratope with an unusually long 19 amino acid light chain third complementarity determining region. Two phenylalanines at the loop apex were predicted by docking and mutagenesis data to interact with the viral membrane. Neutralization by CAP248-2B is not dependent on any single glycan proximal to its epitope, and low neutralization plateaus could not be completely explained by N- or O-linked glycosylation pathway inhibitors, furin co-transfection, or pre-incubation with soluble CD4. Viral escape from CAP248-2B involved a cluster of rare mutations in the gp120-gp41 cleavage sites. Simultaneous introduction of these mutations into heterologous viruses abrogated neutralization by CAP248-2B, but enhanced neutralization sensitivity to 35O22, 4E10, and 10E8 by 10-100-fold. Altogether, this study expands the region of the HIV-1 gp120-gp41 quaternary interface that is a target for broadly neutralizing antibodies and identifies a set of mutations in the gp120 C terminus that exposes the membrane-proximal external region of gp41, with potential utility in HIV vaccine design

Efficacy of primary series AZD1222 (ChAdOx1 nCoV-19) vaccination against SARS-CoV-2 variants of concern: Final analysis of a randomized, placebo-controlled, phase 1b/2 study in South African adults (COV005)

COVID-19 vaccine efficacy (VE) has been observed to vary against antigenically distinct SARS-CoV-2 variants of concern (VoC). Here we report the final analysis of VE and safety from COV005: a phase 1b/2, multicenter, double-blind, randomized, placebo-controlled study of primary series AZD1222 (ChAdOx1 nCoV-19) vaccination in South African adults aged 18-65 years. South Africa's first, second, and third waves of SARS-CoV-2 infections were respectively driven by the ancestral SARS-CoV-2 virus (wild type, WT), and SARS-CoV-2 Beta and Delta VoCs. VE against asymptomatic and symptomatic infection was 90.6% for WT, 6.7% for Beta and 77.1% for Delta. No cases of severe COVID-19 were documented ahead of unblinding. Safety was consistent with the interim analysis, with no new safety concerns identified. Notably, South Africa's Delta wave occurred ≥ 9 months after primary series vaccination, suggesting that primary series AZD1222 vaccination offers a good durability of protection, potentially due to an anamnestic response. Clinical trial identifier: CT.gov NCT04444674

Omicron extensively but incompletely escapes Pfizer BNT162b2 neutralization

The emergence of Omicron (Pango lineage B.1.1.529), first identified in Botswana and South Africa, may compromise vaccine effectiveness and lead to re-infections1. We investigated whether Omicron escapes antibody neutralization in South Africans vaccinated with Pfizer BNT162b2. We also investigated if Omicron requires the ACE2 receptor to infect cells. We isolated and sequence confirmed live Omicron virus from an infected person in South Africa and compared plasma neutralization of Omicron relative to an ancestral SARS-CoV-2 strain, observing that Omicron still required ACE2 to infect. For neutralization, blood samples were taken soon after vaccination from participants who were vaccinated and previously infected or vaccinated with no evidence of previous infection. Neutralization of ancestral virus was much higher in infected and vaccinated versus vaccinated only participants but both groups showed a 22-fold escape from vaccine elicited neutralization by the Omicron variant. However, in the previously infected and vaccinated group, the level of residual neutralization of Omicron was similar to the level of neutralization of ancestral virus observed in the vaccination only group. These data support the notion that, provided high neutralization capacity is elicited by vaccination/boosting approaches, reasonable effectiveness against Omicron may be maintained

Cross-reactive neutralizing antibody responses elicited by SARS-CoV-2 501Y.V2 (B.1.351)

No abstract available.The South African Medical Research Council, the Centers for Disease Control and Prevention, the ELMA South Africa Foundation, the Wellcome Trust, the Fogarty International Center of the National Institutes of Health, the FLAIR Fellowship program, the European and Developing Countries Clinical Trials Partnership 2 of the European Union Horizon 2020 program, the South African Research Chairs Initiative of the Department of Science and Innovation and the National Research Foundation.http://www.nejm.orgam2022Internal Medicin

Omicron infection enhances Delta antibody immunity in vaccinated persons

The extent to which Omicron infection(1–9), with or without previous vaccination, elicits protection against the previously dominant Delta (B.1.617.2) variant is unclear. Here we measured the neutralization capacity against variants of severe acute respiratory syndrome coronavirus 2 in 39 individuals in South Africa infected with the Omicron sublineage BA.1 starting at a median of 6 (interquartile range 3–9) days post symptom onset and continuing until last follow-up sample available, a median of 23 (interquartile range 19–27) days post symptoms to allow BA.1-elicited neutralizing immunity time to develop. Fifteen participants were vaccinated with Pfizer's BNT162b2 or Johnson & Johnson's Ad26.CoV2.S and had BA.1 breakthrough infections, and 24 were unvaccinated. BA.1 neutralization increased from a geometric mean 50% focus reduction neutralization test titre of 42 at enrolment to 575 at the last follow-up time point (13.6-fold) in vaccinated participants and from 46 to 272 (6.0-fold) in unvaccinated participants. Delta virus neutralization also increased, from 192 to 1,091 (5.7-fold) in vaccinated participants and from 28 to 91 (3.0-fold) in unvaccinated participants. At the last time point, unvaccinated individuals infected with BA.1 had low absolute levels of neutralization for the non-BA.1 viruses and 2.2-fold lower BA.1 neutralization, 12.0-fold lower Delta neutralization, 9.6-fold lower Beta variant neutralization, 17.9-fold lower ancestral virus neutralization and 4.8-fold lower Omicron sublineage BA.2 neutralization relative to vaccinated individuals infected with BA.1. These results indicate that hybrid immunity formed by vaccination and Omicron BA.1 infection should be protective against Delta and other variants. By contrast, infection with Omicron BA.1 alone offers limited cross-protection despite moderate enhancement

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

Structural constraints of vaccine-induced tier-2 autologous HIV neutralizing antibodies targeting the receptor-binding site.

CAPRISA, 2017.Abstract available in pdf