Insights on the mutational landscape of the SARS-CoV-2 Omicron variant

The SARS-COV2 Omicron variant has sparked global concern due to the possibility of enhanced transmissibility and escape from vaccines and therapeutics. In this study, we describe the mutational landscape of the Omicron variant using amino acid interaction (AAI) networks. AAI network analysis is particularly well suited for interrogating the impact of constellations of mutations as occur on Omicron that may function in an epistatic manner. Our analyses suggest that as compared to previous variants of concern, the Omicron variant has increased antibody escape breadth due to mutations in class 3 and 4 antibody epitopes as well as increased escape depth due to accumulated mutations in class 1 antibody epitopes. We note certain RBD mutations that might further enhance Omicron escape, and in particular advise careful surveillance of two subclades bearing R346S/K mutations. Further, AAI network analysis suggests that the function of certain therapeutic monoclonal antibodies may be disrupted by Omicron mutations as a result of the cumulative indirect perturbations to the epitope surface properties, despite point-mutation analyses suggesting these antibodies are tolerant of the set of Omicron mutations in isolation. Finally, for several Omicron mutations that do not appear to contribute meaningfully to antibody escape, we find evidence for a plausible role in enhanced transmissibility via disruption of RBD-down conformational stability at the RBD-RBD interface.

High-resolution epitope mapping and characterization of SARS-CoV-2 antibodies in large cohorts of subjects with COVID-19

As Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to spread, characterization of its antibody epitopes, emerging strains, related coronaviruses, and even the human proteome in naturally infected patients can guide the development of effective vaccines and therapies. Since traditional epitope identification tools are dependent upon pre-defined peptide sequences, they are not readily adaptable to diverse viral proteomes. The Serum Epitope Repertoire Analysis (SERA) platform leverages a high diversity random bacterial display library to identify proteome-independent epitope binding specificities which are then analyzed in the context of organisms of interest. When evaluating immune response in the context of SARS-CoV-2, we identify dominant epitope regions and motifs which demonstrate potential to classify mild from severe disease and relate to neutralization activity. We highlight SARS-CoV-2 epitopes that are cross-reactive with other coronaviruses and demonstrate decreased epitope signal for mutant SARS-CoV-2 strains. Collectively, the evolution of SARS-CoV-2 mutants towards reduced antibody response highlight the importance of data-driven development of the vaccines and therapies to treat COVID-19.

The SARS-CoV-2 spike residues 616/644 and 1138/1169 delineate two antibody epitopes in COVID-19 mRNA COMINARTY vaccine (Pfizer/BioNTech)

The newly identified coronavirus SARS-CoV-2 is responsible for the worldwide pandemic COVID-19. Considerable efforts have been made for the development of effective vaccine strategies against COVID-19. The SARS-CoV-2 spike protein has been assigned as major antigen candidate for the development of COVID-19 vaccines. The COVID-19 mRNA BNT162b2 vaccine (comirnaty, Pfizer/BioNTech) is a lipid nanoparticle-encapsulated mRNA encoding a full-length and prefusion-stabilized SARS-CoV-2 spike protein. In the present study, synthetic peptide-based ELISA assays were performed to identify linear B cell epitopes that contribute to elicitation of antibody response in vaccinated individuals with comirnaty. The synthetic S2P6 peptide containing the spike residues 1138/1169 and to a lesser extent, the synthetic S1P4 peptide containing the spike residues 616/644 were recognized by the immune sera from comirnaty recipients but not COVID-19 recovered patients. The S2P6 peptide has been identified as immunogenic peptide in adult BALB/c mice that received protein-peptide conjugates in a prime-boost schedule. Based on our data, we propose that the synthetic S2P6 peptide and to a lesser extent the synthetic S1P4 peptide, would be of interest to measure the dynamic of antibody response to comirnaty vaccine. The synthetic S2P6 peptide is a SARS-CoV-2 spike peptide candidate for the development of peptide-based vaccines against COVID-19.

Mapping SARS-CoV-2 Antibody Epitopes in COVID-19 Patients with a Multi-Coronavirus Protein Microarray

With novel mutant SARS-CoV-2 variants of concern on the rise, knowledge of immune specificities against SARS-CoV-2 proteins is increasingly important for understanding the impact of structural changes in antibody-reactive protein epitopes on naturally acquired and vaccine-induced immunity, as well as broader topics of cross-reactivity and viral evolution. A multi-coronavirus protein microarray used to map the binding of COVID-19 patient antibodies to SARS-CoV-2 proteins and protein fragments as well as to the proteins of four other coronaviruses that infect humans has shown specific regions of SARS-CoV-2 proteins that are highly reactive with patient antibodies and revealed cross-reactivity of these antibodies with other human coronaviruses.

Identification of Human Norovirus GII.3 Blockade Antibody Epitopes

Human noroviruses are a common pathogen causing acute gastroenteritis worldwide. Among all norovirus genotypes, GII.3 is particularly prevalent in the pediatric population. Here we report the identification of two distinct blockade antibody epitopes on the GII.3 capsid. We generated a panel of monoclonal antibodies (mAbs) from mice immunized with virus-like particle (VLP) of a GII.3 cluster 3 strain. Two of these mAbs, namely 8C7 and 8D1, specifically bound the parental GII.3 VLP but not VLPs of GII.4, GII.17, or GI.1. In addition, 8C7 and 8D1 efficiently blocked GII.3 VLP binding with its ligand, histo-blood group antigens (HBGA). These data demonstrate that 8C7 and 8D1 are GII.3-specific blockade antibodies. By using a series of chimeric VLPs, we mapped the epitopes of 8C7 and 8D1 to residues 385–400 and 401–420 of the VP1 capsid protein, respectively. These two blockade antibody epitopes are highly conserved among GII.3 cluster 3 strains. Structural modeling shows that the 8C7 epitope partially overlaps with the HBGA binding site (HBS) while the 8D1 epitope is spatially adjacent to HBS. These findings may enhance our understanding of the immunology and evolution of GII.3 noroviruses.

Quantitative Description of Surface Complementarity of Antibody-Antigen Interfaces

Antibodies have the remarkable ability to recognise their cognate antigens with extraordinary affinity and specificity. Discerning the rules that define antibody-antigen recognition is a fundamental step in the rational design and engineering of functional antibodies with desired properties. In this study we apply the 3D Zernike formalism to the analysis of the surface properties of the antibody complementary determining regions (CDRs). Our results show that shape and electrostatic 3DZD descriptors of the surface of the CDRs are predictive of antigen specificity, with classification accuracy of 81% and area under the receiver operating characteristic curve (AUC) of 0.85. Additionally, while in terms of surface size, solvent accessibility and amino acid composition, antibody epitopes are typically not distinguishable from non-epitope, solvent-exposed regions of the antigen, the 3DZD descriptors detect significantly higher surface complementarity to the paratope, and are able to predict correct paratope-epitope interaction with an AUC = 0.75.

Dengue virus serotype 1 conformational dynamics confers virus strain-dependent patterns of neutralization by polyclonal sera

Dengue virus co-circulates globally as four serotypes (DENV1-4) that vary up to 40% at the amino acid level. Viral strains within a serotype further cluster into multiple genotypes. Eliciting a protective tetravalent neutralizing antibody response is a major goal of vaccine design, and efforts to characterize epitopes targeted by polyclonal mixtures of antibodies are ongoing. Previously, we identified two E protein residues (126/157) that defined the serotype-specific antibody response to DENV1 genotype 4 strain West Pac-74. DENV1 and DENV2 human vaccine sera neutralized DENV1 viruses incorporating these substitutions equivalently. In this study, we explored the contribution of these residues in the neutralization of DENV1 strains representing distinct genotypes. While neutralization of the genotype 1 strain TVP2130 was similarly impacted by mutation at E residues 126/157, mutation of these residues in the genotype 2 strain 16007 did not markedly change neutralization sensitivity, indicating the existence of additional DENV1 type-specific antibody targets. The accessibility of antibody epitopes can be strongly influenced by the conformational dynamics of virions and modified allosterically by amino acid variation. We found that changes at E domain II residue 204, shown previously to impact access to a poorly accessible E domain III epitope, impacted sensitivity of DENV1 16007 to neutralization by vaccine immune sera. Our data identify a role for minor sequence variation in changes to the antigenic structure that impacts antibody recognition by polyclonal immune sera. Understanding how the many structures sampled by flaviviruses influences antibody recognition will inform the design and evaluation of DENV immunogens. IMPORTANCE Dengue virus (DENV) is an important human pathogen that co-circulates globally as four serotypes. Because sequential infection by different DENV serotypes is associated with more severe disease, eliciting a protective neutralizing antibody response against all four serotypes is a major goal of vaccine efforts. Here, we report that neutralization of DENV serotype 1 by polyclonal antibody is impacted by minor sequence variation among virus strains. Our data suggests mechanisms that control neutralization sensitivity extend beyond variation within antibody epitopes, but also include the influence of single amino acids on the ensemble of structural states sampled by structurally dynamic virions. A more detailed understanding of the antibody targets of DENV-specific polyclonal sera and factors that govern their access to antibody has important implications for flavivirus antigen design and evaluation.

Incorporating the Cluster A and V1V2 Targets into a Minimal Structural Unit of the HIV-1 Envelope to Elicit a Cross-Clade Response with Potent Fc-Effector Functions

The generation of a potent vaccine for the prevention and/or control of HIV-1 has been unsuccessful to date, despite decades of research. Existing evidence from both infected individuals and clinical trials support a role for non-neutralizing or weakly neutralizing antibodies with potent Fc-effector functions in the prevention and control of HIV-1 infection. Vaccination strategies that induce such antibodies have proven partially successful in preventing HIV-1 infection. This is largely thought to be due to the polyclonal response that is induced in a vaccine setting, as opposed to the infusion of a single therapeutic antibody, which is capable of diverse Fc-effector functions and targets multiple but highly conserved epitopes. Here, we build on the success of our inner domain antigen, ID2, which incorporates conformational CD4-inducible (CD4i) epitopes of constant region 1 and 2 (C1C2 or Cluster A), in the absence of neutralizing antibody epitopes, into a minimal structural unit of gp120. ID2 has been shown to induce Cluster A-specific antibodies in a BALB/c mouse model with Fc-effector functions against CD4i targets. In order to generate an immunogen that incorporates both epitope targets implicated in the protective Fc-effector functions of antibodies from the only partially successful human vaccine trial, RV144, we incorporated the V1V2 domain into our ID2 antigen generating ID2-V1V2, which we used to immunize in combination with ID2. Immunized BALB/c mice generated both Cluster A- and V1V2-specific antibodies, which synergized to significantly improve the Fc-mediated effector functions compared to mice immunized with ID2 alone. The sera were able to mediate both antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). We therefore conclude that ID2-V1V2 + ID2 represents a promising vaccine immunogen candidate for the induction of antibodies with optimal Fc-mediated effector functions against HIV-1.