B cell genomics behind cross-neutralization of SARS-CoV-2 variants and SARS-CoV.

Monoclonal antibodies (mAbs) are a focus in vaccine and therapeutic design to counteract severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants. Here, we combined B cell sorting with single-cell VDJ and RNA sequencing (RNA-seq) and mAb structures to characterize B cell responses against SARS-CoV-2. We show that the SARS-CoV-2-specific B cell repertoire consists of transcriptionally distinct B cell populations with cells producing potently neutralizing antibodies (nAbs) localized in two clusters that resemble memory and activated B cells. Cryo-electron microscopy structures of selected nAbs from these two clusters complexed with SARS-CoV-2 spike trimers show recognition of various receptor-binding domain (RBD) epitopes. One of these mAbs, BG10-19, locks the spike trimer in a closed conformation to potently neutralize SARS-CoV-2, the recently arising mutants B.1.1.7 and B.1.351, and SARS-CoV and cross-reacts with heterologous RBDs. Together, our results characterize transcriptional differences among SARS-CoV-2-specific B cells and uncover cross-neutralizing Ab targets that will inform immunogen and therapeutic design against coronaviruses.

Structure of the membrane proximal external region of HIV-1 envelope glycoprotein.

The membrane-proximal external region (MPER) of the HIV-1 envelope glycoprotein (Env) bears epitopes of broadly neutralizing antibodies (bnAbs) from infected individuals; it is thus a potential vaccine target. We report an NMR structure of the MPER and its adjacent transmembrane domain in bicelles that mimic a lipid-bilayer membrane. The MPER lies largely outside the lipid bilayer. It folds into a threefold cluster, stabilized mainly by conserved hydrophobic residues and potentially by interaction with phospholipid headgroups. Antigenic analysis and comparison with published images from electron cryotomography of HIV-1 Env on the virion surface suggest that the structure may represent a prefusion conformation of the MPER, distinct from the fusion-intermediate state targeted by several well-studied bnAbs. Very slow bnAb binding indicates that infrequent fluctuations of the MPER structure give these antibodies occasional access to alternative conformations of MPER epitopes. Mutations in the MPER not only impede membrane fusion but also influence presentation of bnAb epitopes in other regions. These results suggest strategies for developing MPER-based vaccine candidates.

Neutralizing Antibody Responses following Long-Term Vaccination with HIV-1 Env gp140 in Guinea Pigs.

A vaccination regimen capable of eliciting potent and broadly neutralizing antibodies (bNAbs) remains an unachieved goal of the HIV-1 vaccine field. Here, we report the immunogenicity of longitudinal prime/boost vaccination regimens with a panel of HIV-1 envelope (Env) gp140 protein immunogens over a period of 200 weeks in guinea pigs. We assessed vaccine regimens that included a monovalent clade C gp140 (C97ZA012 [C97]), a tetravalent regimen consisting of four clade C gp140s (C97ZA012, 459C, 405C, and 939C [4C]), and a tetravalent regimen consisting of clade A, B, C, and mosaic gp140s (92UG037, PVO.4, C97ZA012, and Mosaic 3.1, respectively [ABCM]). We found that the 4C and ABCM prime/boost regimens were capable of eliciting greater magnitude and breadth of binding antibody responses targeting variable loop 2 (V2) over time than the monovalent C97-only regimen. The longitudinal boosting regimen conducted over more than 2 years increased the magnitude of certain tier 1 NAb responses but did not increase the magnitude or breadth of heterologous tier 2 NAb responses. These data suggest that additional immunogen design strategies are needed to induce broad, high-titer tier 2 NAb responses.IMPORTANCE The elicitation of potent, broadly neutralizing antibodies (bNAbs) remains an elusive goal for the HIV-1 vaccine field. In this study, we explored the use of a long-term vaccination regimen with different immunogens to determine if we could elicit bNAbs in guinea pigs. We found that longitudinal boosting over more than 2 years increased tier 1 NAb responses but did not increase the magnitude and breadth of tier 2 NAb responses. These data suggest that additional immunogen designs and vaccination strategies will be necessary to induce broad tier 2 NAb responses.

Circularized Nanodiscs for Multivalent Mosaic Display of SARS-CoV-2 Spike Protein Antigens.

The emergence of vaccine-evading SARS-CoV-2 variants urges the need for vaccines that elicit broadly neutralizing antibodies (bnAbs). Here, we assess covalently circularized nanodiscs decorated with recombinant SARS-CoV-2 spike glycoproteins from several variants for eliciting bnAbs with vaccination. Cobalt porphyrin-phospholipid (CoPoP) was incorporated into the nanodisc to allow for anchoring and functional orientation of spike trimers on the nanodisc surface through their His-tag. Monophosphoryl-lipid (MPLA) and QS-21 were incorporated as immunostimulatory adjuvants to enhance vaccine responses. Following optimization of nanodisc assembly, spike proteins were effectively displayed on the surface of the nanodiscs and maintained their conformational capacity for binding with human angiotensin-converting enzyme 2 (hACE2) as verified using electron microscopy and slot blot assay, respectively. Six different formulations were prepared where they contained mono antigens; four from the year 2020 (WT, Beta, Lambda, and Delta) and two from the year 2021 (Omicron BA.1 and BA.2). Additionally, we prepared a mosaic nanodisc displaying the four spike proteins from year 2020. Intramuscular vaccination of CD-1 female mice with the mosaic nanodisc induced antibody responses that not only neutralized matched pseudo-typed viruses, but also neutralized mismatched pseudo-typed viruses corresponding to later variants from year 2021 (Omicron BA.1 and BA.2). Interestingly, sera from mosaic-immunized mice did not effectively inhibit Omicron spike binding to human ACE-2, suggesting that some of the elicited antibodies were directed towards conserved neutralizing epitopes outside the receptor binding domain. Our results show that mosaic nanodisc vaccine displaying spike proteins from 2020 can elicit broadly neutralizing antibodies that can neutralize mismatched viruses from a following year, thus decreasing immune evasion of new emerging variants and enhancing healthcare preparedness.

Delayed and Attenuated Antibody Responses to Coronavirus Disease 2019 Vaccination With Poor Cross-Variant Neutralization in Solid-Organ Transplant Recipients-A Prospective Longitudinal Study.

Background: Therapeutically immunosuppressed transplant recipients exhibit attenuated responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines. To elucidate the kinetics and variant cross-protection of vaccine-induced antibodies in this population, we conducted a prospective longitudinal study in heart and lung transplant recipients receiving the SARS-CoV-2 messenger RNA (mRNA) 3-dose vaccination series. Methods: We measured longitudinal serum antibody and neutralization responses against the ancestral and major variants of SARS-CoV-2 in SARS-CoV-2-uninfected lung (n = 18) and heart (n = 17) transplant recipients, non-lung-transplanted patients with cystic fibrosis (n = 7), and healthy controls (n = 12) before, during, and after the primary mRNA vaccination series. Results: Among healthy controls, strong anti-spike responses arose immediately following vaccination and displayed cross-neutralization against all variants. In contrast, among transplant recipients, after the first 2 vaccine doses, increases in antibody concentrations occurred gradually, and cross-neutralization was completely absent against the Omicron B.1.1.529 variant. However, most (73%) of the transplant recipients had a significant response to the third vaccine dose, reaching levels comparable to those of healthy controls, with improved but attenuated neutralization of immune evasive variants, particularly Beta, Gamma, and Omicron. Responses in non-lung-transplanted patients with cystic fibrosis paralleled those in healthy controls. Conclusions: In this prospective, longitudinal analysis of variant-specific antibody responses, lung and heart transplant recipients display delayed and defective responses to the first 2 SARS-CoV-2 vaccine doses but significantly augmented responses to a third dose. Gaps in antibody-mediated immunity among transplant recipients are compounded by decreased neutralization against Omicron variants, leaving many patients with substantially weakened immunity against currently circulating variants.

Functional analyses and single cell immunoprofiling uncover sex-specific differences in SARS-CoV2 immune memory development.

SARS-CoV-2 infection leads to a broad range of outcomes and immune responses, with the development of neutralizing antibodies generally correlated with protection against reinfection. Here, we have characterized both neutralizing activity and T cell responses in a cluster of subjects with mild disease linked to a single spreading event. Surprisingly, we observed sex-specific associations between spike- and particularly nucleoprotein-specific T cell responses and neutralization, with pro-inflammatory cytokines being linked to higher titers only in males. Using single cell immunoprofiling, which provided matched transcriptome and T-cell receptor (TCR) profiles in restimulated CD4 + and CD8 + cells from these subjects, we identified differences in type I IFN signaling that may underlie this difference in antibody generation. Finally, we also identified several TCRs associated with cytokine producing T cells. Altogether, our work maps the breadth of immunological outcomes of SARS-CoV2 infections and highlight the potential role of sex-specific feedback loops during the generation of neutralizing antibodies.

Vaccine Breakthrough Infection with the SARS-CoV-2 Delta or Omicron (BA.1) Variant Leads to Distinct Profiles of Neutralizing Antibody Responses.

There is increasing evidence that the risk of SARS-CoV-2 infection among vaccinated individuals is variant-specific, suggesting that protective immunity against SARS-CoV-2 may differ by variant. We enrolled vaccinated (n = 39) and unvaccinated (n = 11) individuals with acute, symptomatic SARS-CoV-2 Delta or Omicron infection and performed SARS-CoV-2 viral load quantification, whole-genome sequencing, and variant-specific antibody characterization at the time of acute illness and convalescence. Viral load at the time of infection was inversely correlated with antibody binding and neutralizing antibody responses. Increases in antibody titers and neutralizing activity occurred at convalescence in a variant-specific manner. Across all variants tested, convalescent neutralization titers in unvaccinated individuals were markedly lower than in vaccinated individuals. For individuals infected with the Delta variant, neutralizing antibody responses were weakest against BA.2, whereas infection with Omicron BA.1 variant generated a broader response against all tested variants, including BA.2.

Vaccine breakthrough infection leads to distinct profiles of neutralizing antibody responses by SARS-CoV-2 variant.

Protective immunity against SARS-CoV-2 infection after COVID-19 vaccination may differ by variant. We enrolled vaccinated (n = 39) and unvaccinated (n = 11) individuals with acute, symptomatic SARS-CoV-2 Delta or Omicron infection and performed SARS-CoV-2 viral load quantification, whole-genome sequencing, and variant-specific antibody characterization at the time of acute illness and convalescence. Viral load at the time of infection was inversely correlated with antibody binding and neutralizing antibody responses. Across all variants tested, convalescent neutralization titers in unvaccinated individuals were markedly lower than in vaccinated individuals. Increases in antibody titers and neutralizing activity occurred at convalescence in a variant-specific manner. For example, among individuals infected with the Delta variant, neutralizing antibody responses were weakest against BA.2, whereas infection with Omicron BA.1 variant generated a broader response against all tested variants, including BA.2.

Structural basis of transmembrane coupling of the HIV-1 envelope glycoprotein.

The prefusion conformation of HIV-1 envelope protein (Env) is recognized by most broadly neutralizing antibodies (bnAbs). Studies showed that alterations of its membrane-related components, including the transmembrane domain (TMD) and cytoplasmic tail (CT), can reshape the antigenic structure of the Env ectodomain. Using nuclear magnetic resonance (NMR) spectroscopy, we determine the structure of an Env segment encompassing the TMD and a large portion of the CT in bicelles. The structure reveals that the CT folds into amphipathic helices that wrap around the C-terminal end of the TMD, thereby forming a support baseplate for the rest of Env. NMR dynamics measurements provide evidences of dynamic coupling across the TMD between the ectodomain and CT. Pseudovirus-based neutralization assays suggest that CT-TMD interaction preferentially affects antigenic structure near the apex of the Env trimer. These results explain why the CT can modulate the Env antigenic properties and may facilitate HIV-1 Env-based vaccine design.

Optimization and qualification of a functional anti-drug antibody assay for HIV-1 bnAbs.

The recent identification of human monoclonal antibodies with broad and potent neutralizing activity against HIV-1 (bnAbs) has resulted in substantial efforts to develop these molecules for clinical use in the prevention and treatment of HIV-1 infection. As with any protein therapeutic drug product, it is imperative to have qualified assays that can accurately detect and quantify anti-drug antibodies (ADA) that may develop in patients receiving passive administration of HIV-1 bnAbs. Here, we have optimized and qualified a functional assay to assess the potential of ADA to inhibit the neutralizing function of HIV-1 bnAbs. Using a modified version of the validated TZM-bl HIV-1 neutralization assay, murine anti-idiotype antibodies were utilized to optimize and evaluate parameters of linearity, range, limit of detection, specificity, and precision for measuring inhibitory ADA activity against multiple HIV-1 bnAbs that are in clinical development. We further demonstrate the utility of this assay for detecting naturally occurring ADA responses in non-human primates receiving passive administration of human bnAbs. This functional assay format complements binding-antibody ADA strategies being developed for HIV-1 bnAbs, and when utilized together, will support a multi-tiered approach for ADA testing that is compliant with Good Clinical Laboratory Practice (GCLP) procedures and FDA guidance.

HIV-1 Neutralizing Antibody Signatures and Application to Epitope-Targeted Vaccine Design.

Eliciting HIV-1-specific broadly neutralizing antibodies (bNAbs) remains a challenge for vaccine development, and the potential of passively delivered bNAbs for prophylaxis and therapeutics is being explored. We used neutralization data from four large virus panels to comprehensively map viral signatures associated with bNAb sensitivity, including amino acids, hypervariable region characteristics, and clade effects across four different classes of bNAbs. The bNAb signatures defined for the variable loop 2 (V2) epitope region of HIV-1 Env were then employed to inform immunogen design in a proof-of-concept exploration of signature-based epitope targeted (SET) vaccines. V2 bNAb signature-guided mutations were introduced into Env 459C to create a trivalent vaccine, and immunization of guinea pigs with V2-SET vaccines resulted in increased breadth of NAb responses compared with Env 459C alone. These data demonstrate that bNAb signatures can be utilized to engineer HIV-1 Env vaccine immunogens capable of eliciting antibody responses with greater neutralization breadth.

Structural basis for membrane anchoring of HIV-1 envelope spike.

HIV-1 envelope spike (Env) is a type I membrane protein that mediates viral entry. We used nuclear magnetic resonance to determine an atomic structure of the transmembrane (TM) domain of HIV-1 Env reconstituted in bicelles that mimic a lipid bilayer. The TM forms a well-ordered trimer that protects a conserved membrane-embedded arginine. An amino-terminal coiled-coil and a carboxyl-terminal hydrophilic core stabilize the trimer. Individual mutations of conserved residues did not disrupt the TM trimer and minimally affected membrane fusion and infectivity. Major changes in the hydrophilic core, however, altered the antibody sensitivity of Env. These results show how a TM domain anchors, stabilizes, and modulates a viral envelope spike and suggest that its influence on Env conformation is an important consideration for HIV-1 immunogen design.