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

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

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

Probabilistic classification of anti-SARS-CoV-2 antibody responses improves seroprevalence estimates.

OBJECTIVES: Population-level measures of seropositivity are critical for understanding the epidemiology of an emerging pathogen, yet most antibody tests apply a strict cutoff for seropositivity that is not learnt in a data-driven manner, leading to uncertainty when classifying low-titer responses. To improve upon this, we evaluated cutoff-independent methods for their ability to assign likelihood of SARS-CoV-2 seropositivity to individual samples. METHODS: Using robust ELISAs based on SARS-CoV-2 spike (S) and the receptor-binding domain (RBD), we profiled antibody responses in a group of SARS-CoV-2 PCR+ individuals (n = 138). Using these data, we trained probabilistic learners to assign likelihood of seropositivity to test samples of unknown serostatus (n = 5100), identifying a support vector machines-linear discriminant analysis learner (SVM-LDA) suited for this purpose. RESULTS: In the training data from confirmed ancestral SARS-CoV-2 infections, 99% of participants had detectable anti-S and -RBD IgG in the circulation, with titers differing > 1000-fold between persons. In data of otherwise healthy individuals, 7.2% (n = 367) of samples were of uncertain serostatus, with values in the range of 3-6SD from the mean of pre-pandemic negative controls (n = 595). In contrast, SVM-LDA classified 6.4% (n = 328) of test samples as having a high likelihood (> 99% chance) of past infection, 4.5% (n = 230) to have a 50-99% likelihood, and 4.0% (n = 203) to have a 10-49% likelihood. As different probabilistic approaches were more consistent with each other than conventional SD-based methods, such tools allow for more statistically-sound seropositivity estimates in large cohorts. CONCLUSION: Probabilistic antibody testing frameworks can improve seropositivity estimates in populations with large titer variability

SARS-CoV-2 spike HexaPro formulated in aluminium hydroxide and administered in an accelerated vaccination schedule partially protects Syrian Hamsters against viral challenge despite low neutralizing antibody responses

SARS-CoV-2 continues to pose a threat to human health as new variants emerge and thus a diverse vaccine pipeline is needed. We evaluated SARS-CoV-2 HexaPro spike protein formulated in Alhydrogel® (aluminium oxyhydroxide) in Syrian hamsters, using an accelerated two dose regimen (given 10 days apart) and a standard regimen (two doses given 21 days apart). Both regimens elicited spike- and RBD-specific IgG antibody responses of similar magnitude, but in vitro virus neutralization was low or undetectable. Despite this, the accelerated two dose regimen offered reduction in viral load and protected against lung pathology upon challenge with homologous SARS-CoV-2 virus (Wuhan-Hu-1). This highlights that vaccine-induced protection against SARS-CoV-2 disease can be obtained despite low neutralizing antibody levels and suggests that accelerated vaccine schedules may be used to confer rapid protection against SARS-CoV-2 disease

Features of recently transmitted HIV-1 clade C viruses that impact antibody recognition : implications for active and passive immunization.

CAPRISA, 2016.Abstract available in PDF file

Developmental pathway for potent V1V2-directed HIV-neutralizing antibodies.

CAPRISA, 2014.Antibodies capable of neutralizing HIV-1 often target variable regions 1 and 2 (V1V2) of the HIV-1 envelope, but the mechanism of their elicitation has been unclear. Here we define the developmental pathway by which such antibodies are generated and acquire the requisite molecular characteristics for neutralization. Twelve somatically related neutralizing antibodies (CAP256-VRC26.01-12) were isolated from donor CAP256 (from the Centre for the AIDS Programme of Research in South Africa (CAPRISA)); each antibody contained the protruding tyrosine-sulphated, anionic antigen-binding loop (complementarity-determining region (CDR) H3) characteristic of this category of antibodies. Their unmutated ancestor emerged between weeks 30-38 post-infection with a 35-residue CDR H3, and neutralized the virus that superinfected this individual 15 weeks after initial infection. Improved neutralization breadth and potency occurred by week 59 with modest affinity maturation, and was preceded by extensive diversification of the virus population. HIV-1 V1V2-directed neutralizing antibodies can thus develop relatively rapidly through initial selection of B cells with a long CDR H3, and limited subsequent somatic hypermutation. These data provide important insights relevant to HIV-1 vaccine development

DNA-launched RNA replicon vaccines induce potent anti-SARS-CoV-2 immune responses in mice

Abstract The outbreak of the SARS-CoV-2 virus and its rapid spread into a global pandemic made the urgent development of scalable vaccines to prevent coronavirus disease (COVID-19) a global health and economic imperative. Here, we characterized and compared the immunogenicity of two alphavirus-based DNA-launched self-replicating (DREP) vaccine candidates encoding either SARS-CoV-2 spike glycoprotein (DREP-S) or a spike ectodomain trimer stabilized in prefusion conformation (DREP-Secto). We observed that the two DREP constructs were immunogenic in mice inducing both binding and neutralizing antibodies as well as T cell responses. Interestingly, the DREP coding for the unmodified spike turned out to be more potent vaccine candidate, eliciting high titers of SARS-CoV-2 specific IgG antibodies that were able to efficiently neutralize pseudotyped virus after a single immunization. In addition, both DREP constructs were able to efficiently prime responses that could be boosted with a heterologous spike protein immunization. These data provide important novel insights into SARS-CoV-2 vaccine design using a rapid response DNA vaccine platform. Moreover, they encourage the use of mixed vaccine modalities as a strategy to combat SARS-CoV-2

Picomolar SARS-CoV-2 Neutralization Using Multi-Arm PEG Nanobody Constructs

Multivalent antibody constructs have a broad range of clinical and biotechnological applications. Nanobodies are especially useful as components for multivalent constructs as they allow increased valency while maintaining a small molecule size. We here describe a novel, rapid method for the generation of bi- and multivalent nanobody constructs with oriented assembly by Cu-free strain promoted azide-alkyne click chemistry (SPAAC). We used sortase A for ligation of click chemistry functional groups site-specifically to the C-terminus of nanobodies before creating C-to-C-terminal nanobody fusions and 4-arm polyethylene glycol (PEG) tetrameric nanobody constructs. We demonstrated the viability of this approach by generating constructs with the SARS-CoV-2 neutralizing nanobody Ty1. We compared the ability of the different constructs to neutralize SARS-CoV-2 pseudotyped virus and infectious virus in neutralization assays. The generated dimers neutralized the virus similarly to a nanobody-Fc fusion variant, while a 4-arm PEG based tetrameric Ty1 construct dramatically enhanced neutralization of SARS-CoV-2, with an IC50 in the low picomolar range

Multiple Pathways Of Escape From HIV Broadly Cross-neutralizing V2-dependent Antibodies

Broadly cross-neutralizing (BCN) antibodies are likely to be critical for an effective HIV vaccine. However, the ontogeny of such antibodies and their relationship with autologous viral evolution is unclear. Here, we characterized viral evolution in CAP256, a subtype C-infected individual who developed potent BCN antibodies targeting positions R166 and K169 in the V2 region. CAP256 was superinfected at 3 months postinfection with a virus that was highly sensitive to BCN V2-dependent monoclonal antibodies. The autologous neutralizing response in CAP256 was directed at V1V2, reaching extremely high titers (\u3e1:40,000) against the superinfecting virus at 42 weeks, just 11 weeks prior to the development of the BCN response targeting the same region. Recombination between the primary and superinfecting viruses, especially in V2 and gp41, resulted in two distinct lineages by 4 years postinfection. Although neutralization of some CAP256 clones by plasma from as much as 2 years earlier suggested incomplete viral escape, nonetheless titers against later clones were reduced at least 40-fold to less than 1:1,000. Escape mutations were identified in each lineage, either at R166 or at K169, suggesting that strain-specific and BCN antibodies targeted overlapping epitopes. Furthermore, the early dependence of CAP256 neutralizing antibodies on the N160 glycan decreased with the onset of neutralization breadth, indicating a change in specificity. These data suggest rapid maturation, within 11 weeks, of CAP256 strain-specific antibodies to acquire breadth, with implications for the vaccine elicitation of BCN V2-dependent antibodies. Overall these studies demonstrate that ongoing viral escape is possible, even from BCN antibodies