Glycan masking in vaccine design: Targets, immunogens and applications.

Glycan masking is a novel technique in reverse vaccinology in which sugar chains (glycans) are added on the surface of immunogen candidates to hide regions of low interest and thus focus the immune system on highly therapeutic epitopes. This shielding strategy is inspired by viruses such as influenza and HIV, which are able to escape the immune system by incorporating additional glycosylation and preventing the binding of therapeutic antibodies. Interestingly, the glycan masking technique is mainly used in vaccine design to fight the same viruses that naturally use glycans to evade the immune system. In this review we report the major successes obtained with the glycan masking technique in epitope-focused vaccine design. We focus on the choice of the target antigen, the strategy for immunogen design and the relevance of the carrier vector to induce a strong immune response. Moreover, we will elucidate the different applications that can be accomplished with glycan masking, such as shifting the immune response from hyper-variable epitopes to more conserved ones, focusing the response on known therapeutic epitopes, broadening the response to different viral strains/sub-types and altering the antigen immunogenicity to elicit higher or lower immune response, as desired.

Human Antibodies for Viral Infections.

Antibodies have been used to prevent or treat viral infections since the nineteenth century, but the full potential to use passive immunization for infectious diseases has yet to be realized. The advent of efficient methods for isolating broad and potently neutralizing human monoclonal antibodies is enabling us to develop antibodies with unprecedented activities. The discovery of IgG Fc region modifications that extend antibody half-life in humans to three months or more suggests that antibodies could become the principal tool with which we manage future viral epidemics. Antibodies for members of most virus families that cause severe disease in humans have been isolated, and many of them are in clinical development, an area that has accelerated during the effort to prevent or treat COVID-19 (coronavirus disease 2019). Broad and potently neutralizing antibodies are also important research reagents for identification of protective epitopes that can be engineered into active vaccines through structure-based reverse vaccinology.

Functional HIV-1/HCV cross-reactive antibodies isolated from a chronically co-infected donor.

Despite prolific efforts to characterize the antibody response to human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) mono-infections, the response to chronic co-infection with these two ever-evolving viruses is poorly understood. Here, we investigate the antibody repertoire of a chronically HIV-1/HCV co-infected individual using linking B cell receptor to antigen specificity through sequencing (LIBRA-seq). We identify five HIV-1/HCV cross-reactive antibodies demonstrating binding and functional cross-reactivity between HIV-1 and HCV envelope glycoproteins. All five antibodies show exceptional HCV neutralization breadth and effector functions against both HIV-1 and HCV. One antibody, mAb688, also cross-reacts with influenza and coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We examine the development of these antibodies using next-generation sequencing analysis and lineage tracing and find that somatic hypermutation established and enhanced this reactivity. These antibodies provide a potential future direction for therapeutic and vaccine development against current and emerging infectious diseases. More broadly, chronic co-infection represents a complex immunological challenge that can provide insights into the fundamental rules that underly antibody-antigen specificity.

Nebulized mRNA-Encoded Antibodies Protect Hamsters from SARS-CoV-2 Infection.

Despite the success of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccines, there remains a clear need for new classes of preventatives for respiratory viral infections due to vaccine hesitancy, lack of sterilizing immunity, and for at-risk patient populations, including the immunocompromised. While many neutralizing antibodies have been identified, and several approved, to treat COVID-19, systemic delivery, large doses, and high costs have the potential to limit their widespread use, especially in low- and middle-income countries. To use these antibodies more efficiently, an inhalable formulation is developed that allows for the expression of mRNA-encoded, membrane-anchored neutralizing antibodies in the lung to mitigate SARS-CoV-2 infections. First, the ability of mRNA-encoded, membrane-anchored, anti-SARS-CoV-2 antibodies to prevent infections in vitro is demonstrated. Next, it is demonstrated that nebulizer-based delivery of these mRNA-expressed neutralizing antibodies potently abrogates disease in the hamster model. Overall, these results support the use of nebulizer-based mRNA expression of neutralizing antibodies as a new paradigm for mitigating respiratory virus infections.

A bivalent SARS-CoV-2 monoclonal antibody combination does not affect the immunogenicity of a vector-based COVID-19 vaccine in macaques.

Human monoclonal antibodies (mAbs) that target the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) offer a promising approach for the prevention and treatment of coronavirus disease 2019 (COVID-19). Given suboptimal global vaccination rates, waning immunity in vaccinated individuals, and the emergence of SARS-CoV-2 variants of concern, the use of mAbs for COVID-19 prevention may increase and may need to be administered together with vaccines in certain settings. However, it is unknown whether administration of mAbs will affect the immunogenicity of SARS-CoV-2 vaccines. Using an adenovirus vector-based SARS-CoV-2 vaccine, we show that simultaneous administration of the vaccine with SARS-CoV-2 mAbs does not diminish vaccine-induced humoral or cellular immunity in cynomolgus macaques. These results suggest that SARS-CoV-2 mAbs and viral vector-based SARS-CoV-2 vaccines can be administered together without loss of potency of either product. Additional studies will be required to evaluate coadministration of mAbs with other vaccine platforms.

Resilience of S309 and AZD7442 monoclonal antibody treatments against infection by SARS-CoV-2 Omicron lineage strains.

Omicron variant strains encode large numbers of changes in the spike protein compared to historical SARS-CoV-2 isolates. Although in vitro studies have suggested that several monoclonal antibody therapies lose neutralizing activity against Omicron variants, the effects in vivo remain largely unknown. Here, we report on the protective efficacy against three SARS-CoV-2 Omicron lineage strains (BA.1, BA.1.1, and BA.2) of two monoclonal antibody therapeutics (S309 [Vir Biotechnology] monotherapy and AZD7442 [AstraZeneca] combination), which correspond to ones used to treat or prevent SARS-CoV-2 infections in humans. Despite losses in neutralization potency in cell culture, S309 or AZD7442 treatments reduced BA.1, BA.1.1, and BA.2 lung infection in susceptible mice that express human ACE2 (K18-hACE2) in prophylactic and therapeutic settings. Correlation analyses between in vitro neutralizing activity and reductions in viral burden in K18-hACE2 or human FcγR transgenic mice suggest that S309 and AZD7442 have different mechanisms of protection against Omicron variants, with S309 utilizing Fc effector function interactions and AZD7442 acting principally by direct neutralization. Our data in mice demonstrate the resilience of S309 and AZD7442 mAbs against emerging SARS-CoV-2 variant strains and provide insight into the relationship between loss of antibody neutralization potency and retained protection in vivo.

Single-cell profiling of the antigen-specific response to BNT162b2 SARS-CoV-2 RNA vaccine.

RNA-based vaccines against SARS-CoV-2 have proven critical to limiting COVID-19 disease severity and spread. Cellular mechanisms driving antigen-specific responses to these vaccines, however, remain uncertain. Here we identify and characterize antigen-specific cells and antibody responses to the RNA vaccine BNT162b2 using multiple single-cell technologies for in depth analysis of longitudinal samples from a cohort of healthy participants. Mass cytometry and unbiased machine learning pinpoint an expanding, population of antigen-specific memory CD4+ and CD8+ T cells with characteristics of follicular or peripheral helper cells. B cell receptor sequencing suggest progression from IgM, with apparent cross-reactivity to endemic coronaviruses, to SARS-CoV-2-specific IgA and IgG memory B cells and plasmablasts. Responding lymphocyte populations correlate with eventual SARS-CoV-2 IgG, and a participant lacking these cell populations failed to sustain SARS-CoV-2-specific antibodies and experienced breakthrough infection. These integrated proteomic and genomic platforms identify an antigen-specific cellular basis of RNA vaccine-based immunity.

Neutralizing COVID-19 Convalescent Plasma in Adults Hospitalized With COVID-19: A Blinded, Randomized, Placebo-Controlled Trial.

BACKGROUND: Convalescent plasma has been one of the most common treatments for COVID-19, but most clinical trial data to date have not supported its efficacy. RESEARCH QUESTION: Is rigorously selected COVID-19 convalescent plasma with neutralizing anti-SARS-CoV-2 antibodies an efficacious treatment for adults hospitalized with COVID-19? STUDY DESIGN AND METHODS: This was a multicenter, blinded, placebo-controlled randomized clinical trial among adults hospitalized with SARS-CoV-2 infection and acute respiratory symptoms for < 14 days. Enrolled patients were randomly assigned to receive one unit of COVID-19 convalescent plasma (n = 487) or placebo (n = 473). The primary outcome was clinical status (disease severity) 14 days following study infusion measured with a seven-category ordinal scale ranging from discharged from the hospital with resumption of normal activities (lowest score) to death (highest score). The primary outcome was analyzed with a multivariable ordinal regression model, with an adjusted odds ratio (aOR) < 1.0 indicating more favorable outcomes with convalescent plasma than with placebo. In secondary analyses, trial participants were stratified according to the presence of endogenous anti-SARS-CoV-2 antibodies ("serostatus") at randomization. The trial included 13 secondary efficacy outcomes, including 28-day mortality. RESULTS: Among 974 randomized patients, 960 were included in the primary analysis. Clinical status on the ordinal outcome scale at 14 days did not differ between the convalescent plasma and placebo groups in the overall population (aOR, 1.04; one-seventh support interval [1/7 SI], 0.82-1.33), in patients without endogenous antibodies (aOR, 1.15; 1/7 SI, 0.74-1.80), or in patients with endogenous antibodies (aOR, 0.96; 1/7 SI, 0.72-1.30). None of the 13 secondary efficacy outcomes were different between groups. At 28 days, 89 of 482 (18.5%) patients in the convalescent plasma group and 80 of 465 (17.2%) patients in the placebo group had died (aOR, 1.04; 1/7 SI, 0.69-1.58). INTERPRETATION: Among adults hospitalized with COVID-19, including those seronegative for anti-SARS-CoV-2 antibodies, treatment with convalescent plasma did not improve clinical outcomes. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov; No.: NCT04362176; URL: www. CLINICALTRIALS: gov.

Epitope-focused immunogen design based on the ebolavirus glycoprotein HR2-MPER region.

The three human pathogenic ebolaviruses: Zaire (EBOV), Bundibugyo (BDBV), and Sudan (SUDV) virus, cause severe disease with high fatality rates. Epitopes of ebolavirus glycoprotein (GP) recognized by antibodies with binding breadth for all three ebolaviruses are of major interest for rational vaccine design. In particular, the heptad repeat 2 -membrane-proximal external region (HR2-MPER) epitope is relatively conserved between EBOV, BDBV, and SUDV GP and targeted by human broadly-neutralizing antibodies. To study whether this epitope can serve as an immunogen for the elicitation of broadly-reactive antibody responses, protein design in Rosetta was employed to transplant the HR2-MPER epitope identified from a co-crystal structure with the known broadly-reactive monoclonal antibody (mAb) BDBV223 onto smaller scaffold proteins. From computational analysis, selected immunogen designs were produced as recombinant proteins and functionally validated, leading to the identification of a sterile alpha motif (SAM) domain displaying the BDBV-HR2-MPER epitope near its C terminus as a promising candidate. The immunogen was fused to one component of a self-assembling, two-component nanoparticle and tested for immunogenicity in rabbits. Robust titers of cross-reactive serum antibodies to BDBV and EBOV GPs and moderate titers to SUDV GP were induced following immunization. To confirm the structural composition of the immunogens, solution NMR studies were conducted and revealed structural flexibility in the C-terminal residues of the epitope. Overall, our study represents the first report on an epitope-focused immunogen design based on the structurally challenging BDBV-HR2-MPER epitope.

An antibody targeting the N-terminal domain of SARS-CoV-2 disrupts the spike trimer.

The protective human antibody response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) focuses on the spike (S) protein, which decorates the virion surface and mediates cell binding and entry. Most SARS-CoV-2 protective antibodies target the receptor-binding domain or a single dominant epitope ("supersite") on the N-terminal domain (NTD). Using the single B cell technology called linking B cell receptor to antigen specificity through sequencing (LIBRA-Seq), we isolated a large panel of NTD-reactive and SARS-CoV-2-neutralizing antibodies from an individual who had recovered from COVID-19. We found that neutralizing antibodies against the NTD supersite were commonly encoded by the IGHV1-24 gene, forming a genetic cluster representing a public B cell clonotype. However, we also discovered a rare human antibody, COV2-3434, that recognizes a site of vulnerability on the SARS-CoV-2 S protein in the trimer interface (TI) and possesses a distinct class of functional activity. COV2-3434 disrupted the integrity of S protein trimers, inhibited the cell-to-cell spread of the virus in culture, and conferred protection in human angiotensin-converting enzyme 2-transgenic (ACE2-transgenic) mice against the SARS-CoV-2 challenge. This study provides insight into antibody targeting of the S protein TI region, suggesting this region may be a site of virus vulnerability.

Structural mapping of antibody landscapes to human betacoronavirus spike proteins.

Preexisting immunity against seasonal coronaviruses (CoVs) represents an important variable in predicting antibody responses and disease severity to severe acute respiratory syndrome CoV-2 (SARS-CoV-2) infections. We used electron microscopy-based polyclonal epitope mapping (EMPEM) to characterize the antibody specificities against ß-CoV spike proteins in prepandemic (PP) sera or SARS-CoV-2 convalescent (SC) sera. We observed that most PP sera had antibodies specific to seasonal human CoVs (HCoVs) OC43 and HKU1 spike proteins while the SC sera showed reactivity across all human ß-CoVs. Detailed molecular mapping of spike-antibody complexes revealed epitopes that were differentially targeted by preexisting antibodies and SC serum antibodies. Our studies provide an antigenic landscape to ß-HCoV spikes in the general population serving as a basis for cross-reactive epitope analyses in SARS-CoV-2-infected individuals.

Real-time cell analysis: A high-throughput approach for testing SARS-CoV-2 antibody neutralization and escape.

Real-time cell analysis (RTCA) enables high-throughput, quantitative kinetic measurements of cytopathic effect (CPE) in virus-infected cells. Here, we detail a RTCA approach for assessing antibody neutralization. We describe how to evaluate the neutralizing potency of monoclonal antibodies (mAbs) and identify viral escape mutants to antibody neutralization for severe respiratory syndrome coronavirus 2 (SARS-CoV-2). For complete details on the use and execution of this protocol, please refer to Zost et al. (2020) and Suryadevara et al. (2021).

A combination of two human neutralizing antibodies prevents SARS-CoV-2 infection in cynomolgus macaques.

BACKGROUND: Human monoclonal antibody (mAb) treatments are promising for COVID-19 prevention or therapy. The pre-exposure prophylactic efficacy of neutralizing antibodies that are engineered with mutations to extend their persistence in human serum and the neutralizing antibody titer in serum required for protection against SARS-CoV-2 infection remain poorly characterized. METHODS: The Fc region of two neutralizing mAbs (COV2-2130 and COV2-2381) targeting non-overlapping epitopes on the receptor binding domain of SARS-CoV-2 spike protein was engineered to extend their persistence in humans and reduce interactions with Fc gamma receptors. We assessed protection by individual antibodies or a combination of the two antibodies (designated ADM03820) given prophylactically by an intravenous or intramuscular route in a non-human primate (NHP) model of SARS-CoV-2 infection. FINDINGS: Passive transfer of individual mAbs or ADM03820 conferred virological protection in the NHP respiratory tract in a dose-dependent manner, and ADM03820 potently neutralized SARS-CoV-2 variants of concern in vitro. We defined a protective serum-neutralizing antibody titer and concentration in NHPs for passively transferred human antibodies that acted by direct viral neutralization. CONCLUSIONS: In summary, we demonstrate that neutralizing antibodies with extended half-life and lacking Fc-mediated effector functions are efficient for pre-exposure prophylaxis of SARS-CoV-2 infection in NHPs. These results support clinical development of ADM03820 for COVID-19 prevention. FUNDING: This research was supported by a contract from the JPEO-CBRND (W911QY-20-9-003, 20-05); the Joint Sciences and Technology Office and Joint Program Executive Office (MCDC-16-01-002 JSTO, JPEO); a DARPA grant (HR0011-18-2-0001); an NIH grant (R01 AI157155); and the 2019 Future Insight Prize from Merck KGaA.

The SARS-CoV-2 monoclonal antibody combination, AZD7442, is protective in nonhuman primates and has an extended half-life in humans.

Despite the success of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines, there remains a need for more prevention and treatment options for individuals remaining at risk of coronavirus disease 2019 (COVID-19). Monoclonal antibodies (mAbs) against the viral spike protein have potential to both prevent and treat COVID-19 and reduce the risk of severe disease and death. Here, we describe AZD7442, a combination of two mAbs, AZD8895 (tixagevimab) and AZD1061 (cilgavimab), that simultaneously bind to distinct, nonoverlapping epitopes on the spike protein receptor binding domain to neutralize SARS-CoV-2. Initially isolated from individuals with prior SARS-CoV-2 infection, the two mAbs were designed to extend their half-lives and reduce effector functions. The AZD7442 mAbs individually prevent the spike protein from binding to angiotensin-converting enzyme 2 receptor, blocking virus cell entry, and neutralize all tested SARS-CoV-2 variants of concern. In a nonhuman primate model of SARS-CoV-2 infection, prophylactic AZD7442 administration prevented infection, whereas therapeutic administration accelerated virus clearance from the lung. In an ongoing phase 1 study in healthy participants (NCT04507256), a 300-mg intramuscular injection of AZD7442 provided SARS-CoV-2 serum geometric mean neutralizing titers greater than 10-fold above those of convalescent serum for at least 3 months, which remained threefold above those of convalescent serum at 9 months after AZD7442 administration. About 1 to 2% of serum AZD7442 was detected in nasal mucosa, a site of SARS-CoV-2 infection. Extrapolation of the time course of serum AZD7442 concentration suggests AZD7442 may provide up to 12 months of protection and benefit individuals at high-risk of COVID-19.

Efficient discovery of SARS-CoV-2-neutralizing antibodies via B cell receptor sequencing and ligand blocking.

Although several monoclonal antibodies (mAbs) targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been approved for coronavirus disease 2019 (COVID-19) therapy, development was generally inefficient, with lead generation often requiring the production and testing of numerous antibody candidates. Here, we report that the integration of target-ligand blocking with a previously described B cell receptor-sequencing approach (linking B cell receptor to antigen specificity through sequencing (LIBRA-seq)) enables the rapid and efficient identification of multiple neutralizing mAbs that prevent the binding of SARS-CoV-2 spike (S) protein to angiotensin-converting enzyme 2 (ACE2). The combination of target-ligand blocking and high-throughput antibody sequencing promises to increase the throughput of programs aimed at discovering new neutralizing antibodies.

An infectious SARS-CoV-2 B.1.1.529 Omicron virus escapes neutralization by therapeutic monoclonal antibodies.

The emergence of the highly transmissible B.1.1.529 Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is concerning for antibody countermeasure efficacy because of the number of mutations in the spike protein. In this study, we tested a panel of anti-receptor-binding domain monoclonal antibodies (mAbs) corresponding to those in clinical use by Vir Biotechnology (S309, the parent mAb of VIR-7831 (sotrovimab)), AstraZeneca (COV2-2196 and COV2-2130, the parent mAbs of AZD8895 and AZD1061), Regeneron (REGN10933 and REGN10987), Eli Lilly (LY-CoV555 and LY-CoV016) and Celltrion (CT-P59) for their ability to neutralize an infectious B.1.1.529 Omicron isolate. Several mAbs (LY-CoV555, LY-CoV016, REGN10933, REGN10987 and CT-P59) completely lost neutralizing activity against B.1.1.529 virus in both Vero-TMPRSS2 and Vero-hACE2-TMPRSS2 cells, whereas others were reduced (COV2-2196 and COV2-2130 combination, ~12-fold decrease) or minimally affected (S309). Our results suggest that several, but not all, of the antibodies in clinical use might lose efficacy against the B.1.1.529 Omicron variant.

Rapid discovery of diverse neutralizing SARS-CoV-2 antibodies from large-scale synthetic phage libraries.

Coronavirus disease 2019 (COVID-19) is an evolving global public health crisis in need of therapeutic options. Passive immunization of monoclonal antibodies (mAbs) represents a promising therapeutic strategy capable of conferring immediate protection from SARS-CoV-2 infection. Herein, we describe the discovery and characterization of neutralizing SARS-CoV-2 IgG and VHH antibodies from four large-scale phage libraries. Each library was constructed synthetically with shuffled complementarity-determining region loops from natural llama and human antibody repertoires. While most candidates targeted the receptor-binding domain of the S1 subunit of SARS-CoV-2 spike protein, we also identified a neutralizing IgG candidate that binds a unique epitope on the N-terminal domain. A select number of antibodies retained binding to SARS-CoV-2 variants Alpha, Beta, Gamma, Kappa and Delta. Overall, our data show that synthetic phage libraries can rapidly yield SARS-CoV-2 S1 antibodies with therapeutically desirable features, including high affinity, unique binding sites, and potent neutralizing activity in vitro, and a capacity to limit disease in vivo.

Standardized two-step testing of antibody activity in COVID-19 convalescent plasma.

The COVID-19 pandemic revealed an urgent need for rapid profiling of neutralizing antibody responses and development of antibody therapeutics. The current Food and Drug Administration-approved serological tests do not measure antibody-mediated viral neutralization, and there is a need for standardized quantitative neutralization assays. We report a high-throughput two-step profiling approach for identifying neutralizing convalescent plasma. Screening and downselection for serum antibody binding to the receptor-binding domain are followed by quantitative neutralization testing using a chimeric vesicular stomatitis virus expressing spike protein of SARS-CoV-2 in a real-time cell analysis assay. This approach enables a predictive screening process for identifying plasma units that neutralize SARS-CoV-2. To calibrate antibody neutralizing activity in serum from convalescent plasma donors, we introduce a neutralizing antibody standard reagent composed of two human antibodies that neutralize SARS-CoV strains, including SARS-CoV-2 variants of concern. Our results provide a framework for establishing a standardized assessment of antibody-based interventions against COVID-19.

Potent neutralization of SARS-CoV-2 variants of concern by an antibody with an uncommon genetic signature and structural mode of spike recognition.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages that are more transmissible and resistant to currently approved antibody therapies poses a considerable challenge to the clinical treatment of coronavirus disease (COVID-19). Therefore, the need for ongoing discovery efforts to identify broadly reactive monoclonal antibodies to SARS-CoV-2 is of utmost importance. Here, we report a panel of SARS-CoV-2 antibodies isolated using the linking B cell receptor to antigen specificity through sequencing (LIBRA-seq) technology from an individual who recovered from COVID-19. Of these antibodies, 54042-4 shows potent neutralization against authentic SARS-CoV-2 viruses, including variants of concern (VOCs). A cryoelectron microscopy (cryo-EM) structure of 54042-4 in complex with the SARS-CoV-2 spike reveals an epitope composed of residues that are highly conserved in currently circulating SARS-CoV-2 lineages. Further, 54042-4 possesses uncommon genetic and structural characteristics that distinguish it from other potently neutralizing SARS-CoV-2 antibodies. Together, these findings provide motivation for the development of 54042-4 as a lead candidate to counteract current and future SARS-CoV-2 VOCs.