Neutralizing antibody responses in HIV-1 dual infection : lessons for vaccine design

Includes abstract. Includes bibliographical references

Neutralizing antibody responses in HIV dual infection: lessons for vaccine design

The development of a safe, effective prophylactic HIV vaccine remains a major global health priority. Stabilized, soluble trimers that mimic the native functional HIV trimer have been developed that elicit strain-specific neutralizing HIV antibodies in animal models, and are currently being evaluated in several human clinical trials. Identifying whether multiple immunogens could be administered to facilitate the broadening of responses represents a pivotal challenge. In this thesis, we characterized the antibody response in individuals infected with multiple HIV strains to inform the development of polyvalent and sequential HIV vaccine regimens. We found that conventional approaches to detect HIV co- and superinfection are confounded by recombination. Therefore, we developed an automated, Bayesian approach to detect superinfection explicitly accounting for recombination. Using simulated and real sequence data, we demonstrated that this approach is sensitive, highly specific, and robust to recombination. Furthermore, analyzing previously published sequence datasets, we identified cases of superinfection that previously went undetected, indicating that superinfection occurs more frequently than previously estimated. We characterized the development of antibodies in five superinfected individuals identified in the CAPRISA 002 acute infection cohort. Specifically, we evaluated whether superinfection re-engaged cross-reactive memory B cells, promoting the development of cross-neutralizing antibodies. By comparing the breadth of the neutralizing antibody response in superinfected individuals to those that typically develop in singly infected individuals, we showed that HIV superinfection was not sufficient to broaden responses. By characterizing the kinetics and specificity of autologous neutralizing antibody responses, we show that responses to the superinfecting viruses failed to efficiently recruit neutralizing memory B cells. Instead, the secondary infection elicited strain-specific, de novo responses. This occurred even though the superinfecting viruses were relatively closely related (from the same subtype). To determine whether the co-exposure to diverse Env antigens favours the development of cross-neutralizing antibodies better than sequential exposure, we characterized the development of neutralizing antibodies in HIV co-infected individuals where several divergent viruses were transmitted prior to seroconversion. We identified three cases of co-infection that encompassed immunological exposure to: (i) two diverse, unlinked Envs, (ii) two related Envs with diversity uniformly distributed over the trimer, and (iii) two diverse but recombined Envs such that clusters of high homology were preserved in the presence of high diversity elsewhere. We found that, like superinfection, co-infection was not sufficient to broaden neutralizing antibody responses. Co-exposure to two HIV Env antigens did not necessarily produce additive or cross-neutralizing antibody responses, and in some cases was subject to immunological interference. This was most evident in the case of co-infection with two related Envs where diversity was uniformly distributed across the Env trimer; in this case neutralizing antibody responses to one variant arose to the near exclusion of responses to the other. However, in the case of co-exposure to diverse Envs but where the trimer apex was conserved in both variants through recombination, potent neutralization of both variants was evident. This was the co-infected participant who developed the broadest neutralizing antibody response, and we show that cross-neutralization was mediated, in part, by trimer apextargeting neutralizing antibodies. In conclusion, we find that HIV superinfection fails to efficiently recruit neutralizing memory B cells and, at best, results in additive nAb responses rather than a synergistic effect leading to cross-neutralization; a distinction that is highly relevant for vaccine design. While sequential immunizations with heterologous Env immunogens may be able to improve the potency of elicited responses, alone, they are unlikely to promote the development of bnAbs. Our observations from cases of co-infection suggests that cocktails of divergent stabilized Env trimers are unlikely to drive the development of cross-neutralizing antibodies, and may be subject to interference. However, the rational design of more similar immunogen cocktails where conserved epitopes are preserved across immunogens may be able to facilitate neutralizing antibodies to these targets, as seen in one individual. Thus, the use of related, stabilized Env trimers with diversity introduced in key regions together with strategies to reduce the immunogenicity of immunodominant, strain-specific epitopes may represent one path to a cross-neutralizing antibody response to multiple Envs within a cocktail

Extending Lunar Impact Flash Observations into the Daytime with Short-Wave Infrared

Lunar impact flash (LIF) observations typically occur in R, I, or unfiltered light, and are only possible during night, targeting the night side of a 10-60% illumination Moon, while >10{\deg} above the observers horizon. This severely limits the potential to observe, and therefore the number of lower occurrence, high energy impacts observed is reduced. By shifting from the typically used wavelengths to the J-Band Short-Wave Infrared, the greater spectral radiance for the most common temperature (2750 K) of LIFs and darker skies at these wavelengths enables LIF monitoring to occur during the daytime, and at greater lunar illumination phases than currently possible. Using a 40.0 cm f/4.5 Newtonian reflector with Ninox 640SU camera and J-band filter, we observed several stars and lunar nightside at various times to assess the theoretical limits of the system. We then performed LIF observations during both day and night to maximise the chances of observing a confirmed LIF to verify the methods. We detected 61 >5{\sigma} events, from which 33 candidate LIF events could not be discounted as false positives. One event was confirmed by multi-frame detection, and by independent observers observing in visible light. While this LIF was observed during the night, the observed signal can be used to calculate the equivalent Signal-to-Noise ratio for a similar daytime event. The threshold for daylight LIF detection was found to be between Jmag=+3.4+-0.18 and Jmag=+5.6+-0.18 (Vmag=+4.5 and Vmag=+6.7 respectively at 2750 K). This represents an increase in opportunity to observe LIFs by almost 500%

HIV-1 superinfection resembles primary infection.

CAPRISA, 2015.Abstract available in pdf

Frequent use of IGHV3-30-3 in SARS-CoV-2 neutralizing antibody responses

The antibody response to SARS-CoV-2 shows biased immunoglobulin heavy chain variable (IGHV) gene usage, allowing definition of genetic signatures for some classes of neutralizing antibodies. We investigated IGHV gene usage frequencies by sorting spike-specific single memory B cells from individuals infected with SARS-CoV-2 early in the pandemic. From two study participants and 703 spike-specific B cells, the most used genes were IGHV1-69, IGHV3-30-3, and IGHV3-30. Here, we focused on the IGHV3-30 group of genes and an IGHV3-30-3-using ultrapotent neutralizing monoclonal antibody, CAB-F52, which displayed broad neutralizing activity also in its germline-reverted form. IGHV3-30-3 is encoded by a region of the IGH locus that is highly variable at both the allelic and structural levels. Using personalized IG genotyping, we found that 4 of 14 study participants lacked the IGHV3-30-3 gene on both chromosomes, raising the question if other, highly similar IGHV genes could substitute for IGHV3-30-3 in persons lacking this gene. In the context of CAB-F52, we found that none of the tested IGHV3-33 alleles, but several IGHV3-30 alleles could substitute for IGHV3-30-3, suggesting functional redundancy between the highly homologous IGHV3-30 and IGHV3-30-3 genes for this antibody

South African HIV-1 Subtype C Transmitted Variants With A Specific V2 Motif Show Higher Dependence On aα4β7 For Replication

Background: The integrin aα4β7 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 aα4β7 in HIV infection and the contribution of viral and host factors. Results: Replication of 60 HIV-1 subtype C viruses collected over time from 11 individuals in the CAPRISA cohort were partially inhibited by antibodies targeting aα4β7. However, dependence on aα4β7 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, aα4β7 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 aα4β7 reactivity, whereas mutating P/SDI/V to LDI/L motifs was associated with reduced aα4β7 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

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

A bispecific monomeric nanobody induces spike trimer dimers and neutralizes SARS-CoV-2 in vivo

Experiments with replication-competent SARS-CoV-2 were performed in the Biomedicum BSL3 core facility, Karolinska Institutet. We thank Jonas Klingström for providing Calu-3 cells and sharing the Swedish SARS-CoV-2 isolate, and Alex Sigal from the Africa Health Research Institute for providing the beta variant (B.1.351/501Y.V2) isolate. We thank Penny Moore and the NICD (South Africa) for providing the B.1.351/beta variant spike plasmid, which was generated using funding from the South African Medical Research Council. We gratefully acknowledge the G2P-UK National Virology consortium funded by MRC/UKRI (grant ref: MR/W005611/1.) and the Barclay Lab at Imperial College for providing the B.1.617.2 spike plasmid. All cryo-EM data were collected in the Karolinska Institutet’s 3D-EM facility. We thank Agustin Ure for assistance with figure generation and Tomas Nyman (Protein Science Facility at KI) for providing access to SPR instruments. L.H. was supported by the David och Astrid Hageléns stiftelse, the Clas Groschinskys Minnesfond and a Jonas Söderquist’s scholarship. This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No. 101003653 (CoroNAb), to B.M. and G.M.M. B.M.H. is supported by the Knut and Alice Wallenberg Foundation (KAW 2017.0080 and KAW 2018.0080). The work was supported by project grants from the Swedish Research Council to E.S. (2020-02682), B.M.H. (2017-6702 and 2018-3808), B.M. (2018-02381) and to G.M.M. (2018-03914 and 2018-03843). E.S. is supported by Karolinska Institutet Foundation Grants, National Molecular Medicine Program Grants, and the grants from the SciLifeLab National COVID-19 Research Program, financed by the Knut and Alice Wallenberg Foundation. We thank National Microscopy Infrastructure, NMI (VR-RFI 2016-00968).N

Identification of broadly neutralizing antibody epitopes in the HIV-1 envelope glycoprotein using evolutionary models

Background: Identification of the epitopes targeted by antibodies that can neutralize diverse HIV-1 strains can provide important clues for the design of a preventative vaccine. Methods: We have developed a computational approach that can identify key amino acids within the HIV-1 envelope glycoprotein that influence sensitivity to broadly cross-neutralizing antibodies. Given a sequence alignment and neutralization titers for a panel of viruses, the method works by fitting a phylogenetic model that allows the amino acid frequencies at each site to depend on neutralization sensitivities. Sites at which viral evolution influences neutralization sensitivity were identified using Bayes factors (BFs) to compare the fit of this model to that of a null model in which sequences evolved independently of antibody sensitivity. Conformational epitopes were identified with a Metropolis algorithm that searched for a cluster of sites with large Bayes factors on the tertiary structure of the viral envelope. Results: We applied our method to ID50 neutralization data generated from seven HIV-1 subtype C serum samples with neutralization breadth that had been tested against a multi-clade panel of 225 pseudoviruses for which envelope sequences were also available. For each sample, between two and four sites were identified that were strongly associated with neutralization sensitivity (2ln(BF) > 6), a subset of which were experimentally confirmed using site-directed mutagenesis. Conclusions: Our results provide strong support for the use of evolutionary models applied to cross-sectional viral neutralization data to identify the epitopes of serum antibodies that confer neutralization breadth