Oligomerization-driven avidity correlates with SARS-CoV-2 cellular binding and inhibition.

Cellular processes are controlled by the thermodynamics of the underlying biomolecular interactions. Frequently, structural investigations use one monomeric binding partner, while ensemble measurements of binding affinities generally yield one affinity representative of a 1:1 interaction, despite the majority of the proteome consisting of oligomeric proteins. For example, viral entry and inhibition in SARS-CoV-2 involve a trimeric spike surface protein, a dimeric angiotensin-converting enzyme 2 (ACE2) cell-surface receptor and dimeric antibodies. Here, we reveal that cooperativity correlates with infectivity and inhibition as opposed to 1:1 binding strength. We show that ACE2 oligomerizes spike more strongly for more infectious variants, while exhibiting weaker 1:1 affinity. Furthermore, we find that antibodies use induced oligomerization both as a primary inhibition mechanism and to enhance the effects of receptor-site blocking. Our results suggest that naive affinity measurements are poor predictors of potency, and introduce an antibody-based inhibition mechanism for oligomeric targets. More generally, they point toward a much broader role of induced oligomerization in controlling biomolecular interactions.

Evidence that the SARS-CoV-2 S protein undergoes a conformational change at the Golgi complex that leads to the formation of virus neutralising antibody binding epitopes in the S1 protein subunit.

Recombinant SARS-CoV-2 S protein expression was examined in Vero cells by imaging using the human monoclonal antibody panel (PD4, PD5, sc23, and sc29). The PD4 and sc29 antibodies recognised conformational specific epitopes in the S2 protein subunit at the Endoplasmic reticulum and Golgi complex. While PD5 and sc23 detected conformationally specific epitopes in the S1 protein subunit at the Golgi complex, only PD5 recognised the receptor binding domain (RBD). A comparison of the staining patterns of PD5 with non-conformationally specific antibodies that recognises the S1 subunit and RBD suggested the PD5 recognised a conformational structure within the S1 protein subunit. Our data suggests the antibody binding epitopes recognised by the human monoclonal antibodies formed at different locations in the secretory pathway during S protein transport, but a conformational change in the S1 protein subunit at the Golgi complex formed antibody binding epitopes that are recognised by virus neutralising antibodies.

Spike structures, receptor binding, and immune escape of recently circulating SARS-CoV-2 Omicron BA.2.86, JN.1, EG.5, EG.5.1, and HV.1 sub-variants.

The recently emerged BA.2.86, JN.1, EG.5, EG.5.1, and HV.1 variants have a growth advantage. In this study, we explore the structural bases of receptor binding and immune evasion for the Omicron BA.2.86, JN.1, EG.5, EG.5.1, and HV.1 sub-variants. Our findings reveal that BA.2.86 exhibits strong receptor binding, whereas its JN.1 sub-lineage displays a decreased binding affinity to human ACE2 (hACE2). Through complex structure analyses, we observed that the reversion of R493Q in BA.2.86 receptor binding domain (RBD) plays a facilitating role in receptor binding, while the L455S substitution in JN.1 RBD restores optimal affinity. Furthermore, the structure of monoclonal antibody (mAb) S309 complexed with BA.2.86 RBD highlights the importance of the K356T mutation, which brings a new N-glycosylation motif, altering the binding pattern of mAbs belonging to RBD-5 represented by S309. These findings emphasize the importance of closely monitoring BA.2.86 and its sub-lineages to prevent another wave of SARS-CoV-2 infections.

TRIM21 and Fc-engineered antibodies: decoding its complex antibody binding mode with implications for viral neutralization.

TRIM21 is a pivotal effector in the immune system, orchestrating antibody-mediated responses and modulating immune signaling. In this comprehensive study, we focus on the interaction of TRIM21 with Fc engineered antibodies and subsequent implications for viral neutralization. Through a series of analytical techniques, including biosensor assays, mass photometry, and electron microscopy, along with structure predictions, we unravel the intricate mechanisms governing the interplay between TRIM21 and antibodies. Our investigations reveal that the TRIM21 capacity to recognize, bind, and facilitate the proteasomal degradation of antibody-coated viruses is critically dependent on the affinity and avidity interplay of its interactions with antibody Fc regions. We suggest a novel binding mechanism, where TRIM21 binding to one Fc site results in the detachment of PRYSPRY from the coiled-coil domain, enhancing mobility due to its flexible linker, thereby facilitating the engagement of the second site, resulting in avidity due to bivalent engagement. These findings shed light on the dual role of TRIM21 in antiviral immunity, both in recognizing and directing viruses for intracellular degradation, and demonstrate its potential for therapeutic exploitation. The study advances our understanding of intracellular immune responses and opens new avenues for the development of antiviral strategies and innovation in tailored effector functions designed to leverage TRIM21s unique binding mode.

Ensemble-Based Mutational Profiling and Network Analysis of the SARS-CoV-2 Spike Omicron XBB Lineages for Interactions with the ACE2 Receptor and Antibodies: Cooperation of Binding Hotspots in Mediating Epistatic Couplings Underlies Binding Mechanism and Immune Escape.

In this study, we performed a computational study of binding mechanisms for the SARS-CoV-2 spike Omicron XBB lineages with the host cell receptor ACE2 and a panel of diverse class one antibodies. The central objective of this investigation was to examine the molecular factors underlying epistatic couplings among convergent evolution hotspots that enable optimal balancing of ACE2 binding and antibody evasion for Omicron variants BA.1, BA2, BA.3, BA.4/BA.5, BQ.1.1, XBB.1, XBB.1.5, and XBB.1.5 + L455F/F456L. By combining evolutionary analysis, molecular dynamics simulations, and ensemble-based mutational scanning of spike protein residues in complexes with ACE2, we identified structural stability and binding affinity hotspots that are consistent with the results of biochemical studies. In agreement with the results of deep mutational scanning experiments, our quantitative analysis correctly reproduced strong and variant-specific epistatic effects in the XBB.1.5 and BA.2 variants. It was shown that Y453W and F456L mutations can enhance ACE2 binding when coupled with Q493 in XBB.1.5, while these mutations become destabilized when coupled with the R493 position in the BA.2 variant. The results provided a molecular rationale of the epistatic mechanism in Omicron variants, showing a central role of the Q493/R493 hotspot in modulating epistatic couplings between convergent mutational sites L455F and F456L in XBB lineages. The results of mutational scanning and binding analysis of the Omicron XBB spike variants with ACE2 receptors and a panel of class one antibodies provide a quantitative rationale for the experimental evidence that epistatic interactions of the physically proximal binding hotspots Y501, R498, Q493, L455F, and F456L can determine strong ACE2 binding, while convergent mutational sites F456L and F486P are instrumental in mediating broad antibody resistance. The study supports a mechanism in which the impact on ACE2 binding affinity is mediated through a small group of universal binding hotspots, while the effect of immune evasion could be more variant-dependent and modulated by convergent mutational sites in the conformationally adaptable spike regions.

Molecular insights into the binding interactions and energetics of the omicron spike variant with hACE2 and a neutralizing antibody.

The global spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) since 2019 has led to a continuous evolution of viral variants, with the latest concern being the Omicron (B.1.1.529) variant. In this study, classical molecular dynamics simulations were conducted to elucidate the biophysical aspects of the Omicron spike protein's receptor-binding domain (RBD) in its interaction with human angiotensin-converting enzyme 2 (hACE2) and a neutralizing antibody, comparing it to the wildtype (WT). To model the Omicron variant, 15 in silico mutations were introduced in the RBD region of WT (retrieved from PDB). The simulations of WT spike-hACE2 and Omicron spike-hACE2 complexes revealed comparable binding stability and dynamics. Notably, the Q493R mutation in the Omicron spike increased interactions with hACE2, particularly with ASP38 and ASP355. Additionally, mutations such as N417K, T478K, and Y505H contributed to enhanced structural stability in the Omicron variant. Conversely, when comparing WT with Omicron in complex with a neutralizing antibody, simulation results demonstrated poorer binding dynamics and stability for the Omicron variant. The E484K mutation significantly decreased binding interactions, resulting in an overall decrease in binding energy (∼-57 kcal/mol) compared to WT (∼-84 kcal/mol). This study provides valuable molecular insights into the heightened infectivity of the Omicron variant, shedding light on the specific mutations influencing its interactions with hACE2 and neutralizing antibodies.

Interferon-γ/IL-2 ELISpot and mRNA Responses to the SARS-CoV2, Feline Coronavirus Serotypes 1 (FCoV1), and FCoV2 Receptor Binding Domains by the T Cells from COVID-19-Vaccinated Humans and FCoV1-Infected Cats.

The receptor binding domain (RBD) of SARS-CoV-2 (SCoV2) has been used recently to identify the RBD sequences of feline coronavirus serotypes 1 (FCoV1) and 2 (FCoV2). Cats naturally infected with FCoV1 have been shown to possess serum reactivities with FCoV1 and SCoV2 RBDs but not with FCoV2 RBD. In the current study, COVID-19-vaccinated humans and FCoV1-infected laboratory cats were evaluated for interferon-gamma (IFNγ) and interleukin-2 (IL-2 ELISpot responses by their peripheral blood mononuclear cells (PBMC) to SCoV2, FCoV1, and FCoV2 RBDs. Remarkably, the PBMC from COVID-19-vaccinated subjects developed IFNγ responses to SCoV2, FCoV1, and FCoV2 RBDs. The most vaccinated subject (five vaccinations over 2 years) appeared to produce hyperreactive IFNγ responses to all three RBDs, including the PBS media control. This subject lost IFNγ responses to all RBDs at 9 months (9 mo) post-last vaccination. However, her IL-2 responses to FCoV1 and FCoV2 RBDs were low but detectable at 10 mo post-last vaccination. This observation suggests that initially robust IFNγ responses to SCoV2 RBD may be an outcome of robust inflammatory IFNγ responses to SCoV2 RBD. Hence, the T-cell responses of vaccine immunity should be monitored by vaccine immunogen-specific IL-2 production. The PBMC from chronically FCoV1-infected cats developed robust IFNγ responses to SCoV2 and FCoV2 RBDs but had the lowest IFNγ responses to FCoV1 RBD. The constant exposure to FCoV1 reinfection may cause the IFNγ responses to be downregulated to the infecting virus FCoV1 but not to the cross-reacting epitopes on the SCoV2 and FCoV2 RBDs.

Polyvalent Nanobody Structure Designed for Boosting SARS-CoV-2 Inhibition.

Coronavirus transmission and mutations have brought intensive challenges on pandemic control and disease treatment. Developing robust and versatile antiviral drugs for viral neutralization is highly desired. Here, we created a new polyvalent nanobody (Nb) structure that shows the effective inhibition of SARS-CoV-2 infections. Our polyvalent Nb structure, called "PNS", is achieved by first conjugating single-stranded DNA (ssDNA) and the receptor-binding domain (RBD)-targeting Nb with retained binding ability to SARS-CoV-2 spike protein and then coalescing the ssDNA-Nb conjugates around a gold nanoparticle (AuNP) via DNA hybridization with a desired Nb density that offers spatial pattern-matching with that of the Nb binding sites on the trimeric spike. The surface plasmon resonance (SPR) assays show that the PNS binds the SARS-CoV-2 trimeric spike proteins with a ∼1000-fold improvement in affinity than that of monomeric Nbs. Furthermore, our viral entry inhibition assays using the PNS against SARS-CoV-2 WA/2020 and two recent variants of interest (BQ1.1 and XBB) show an over 400-fold enhancement in viral inhibition compared to free Nbs. Our PNS strategy built on a new DNA-protein conjugation chemistry provides a facile approach to developing robust virus inhibitors by using a corresponding virus-targeting Nb with a desired Nb density.

NS1: a promising novel target antigen with strong immunogenicity and protective efficacy for avian flavivirus vaccine development.

Tembusu virus (TMUV), an avian pathogenic flavivirus, has emerged as a significant threat to the duck industry in Southeast Asia, causing substantial economic losses. Due to the antibody-dependent enhancement (ADE) effect of TMUV subneutralizing antibodies, there is a pressing need to further develop new TMUV vaccine target antigens that ensure both safety and efficacy. Here, the TMUV non-structural protein 1 (NS1) as a target for development of effective anti-TMUV vaccines was unveiled. The amino acid sequences of TMUV NS1 exhibit a high degree of conservation across different strains (92.63-100%). To investigate the potential of TMUV NS1 as a vaccine target, the TMUV NS1-based plasmids were constructed and identified the C-terminal 30 amino acids residues of TMUV E (EC30) as an effective signal peptide for promoting NS1 expression and secretion. Subsequently, the plasmid pVAX1-EC30-NS1 was employed to immunize ducks, resulting in specific anti-NS1 IgG responses being stimulated, while without inducing anti-TMUV neutralizing antibodies. Furthermore, the cellular immune responses triggered by the TMUV NS1 were evaluated, observing a notable increase in lymphocyte proliferation at 4 wk and 6 wk postinjection with the pVAX1-EC30-NS1. Additionally, there was a significant up-regulation of NS1-specific Il-4 and Ifnγ levels at these time points. Following this, ducks from different groups were challenged with TMUV, and remarkably, those immunized with the NS1 vaccine displayed significantly lower viral copies both at 3 d postinfection (dpi) and 7 dpi (P < 0.05) compared to ducks immunized with the control vector. Notably, the NS1 demonstrated remarkable protection against TMUV challenge without causing severe gross lesions. Collectively, these findings highlighted the impressive immunogenicity and protectivity of the TMUV NS1. Consequently, NS1 holds great promise as a novel antigen target for the development of efficient and safe TMUV vaccines.

SARS-CoV-2 BA.2.86 enters lung cells and evades neutralizing antibodies with high efficiency.

BA.2.86, a recently identified descendant of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron BA.2 sublineage, contains ∼35 mutations in the spike (S) protein and spreads in multiple countries. Here, we investigated whether the virus exhibits altered biological traits, focusing on S protein-driven viral entry. Employing pseudotyped particles, we show that BA.2.86, unlike other Omicron sublineages, enters Calu-3 lung cells with high efficiency and in a serine- but not cysteine-protease-dependent manner. Robust lung cell infection was confirmed with authentic BA.2.86, but the virus exhibited low specific infectivity. Further, BA.2.86 was highly resistant against all therapeutic antibodies tested, efficiently evading neutralization by antibodies induced by non-adapted vaccines. In contrast, BA.2.86 and the currently circulating EG.5.1 sublineage were appreciably neutralized by antibodies induced by the XBB.1.5-adapted vaccine. Collectively, BA.2.86 has regained a trait characteristic of early SARS-CoV-2 lineages, robust lung cell entry, and evades neutralizing antibodies. However, BA.2.86 exhibits low specific infectivity, which might limit transmissibility.

Microneme-located VP2 in Eimeria acervulina elicits effective protective immunity against infectious bursal disease virus.

Using transgenic Eimeria spp. to deliver exogenous antigens is a viable option for developing multivalent live vaccines. Previous research revealed that the location of antigen expression in recombinant Eimeria dictates the magnitude and type of immune responses. In this study, we constructed genetically modified Eimeria acervulina that expressed VP2 protein, a protective antigen from infectious bursal disease virus (IBDV), on the surface or in the microneme of sporozoites. After vaccination, VP2-specific antibody was readily detected in specific pathogen-free chickens receiving transgenic E. acervulina parasites expressing VP2 in microneme, but animals vaccinated with which expressing VP2 on surface failed to produce detectable antibody after two times immunizations. Moreover, the bursal lesion of microneme-located VP2 transgenic E. acervulina immunized chickens was less severe compared with un-immunized animals after IBDV challenge infection. Therefore, genetically modified E. acervulina that express IBDV-derived VP2 in micronemes are effective in inducing specific antibody responses against VP2, while parasites that have VP2 expression on cell surface are not suitable. Thus, the use of Eimeria parasites as vaccine vectors needs to consider the proper targeting of exogenous immunogens. Our results have implications for the design of other vector vaccines.

Computational design of scFv anti-receptor binding domain of SARS-CoV-2 spike protein based on antibody S230 anti-SARS-CoV-1.

Developing therapeutics such as neutralizing antibodies targeting the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is essential to halt the Covid-19 infection. However, antibody production is expensive and relatively inaccessible to many low-income countries. Therefore, a more efficient and smaller antibody fragment, such as a single-chain variable fragment (scFv), derived from a known neutralizing antibody structure, is of interest due to the lower cost of recombinant protein production and the ability to tailor scFvs against circulating viruses. In this study, we used computational design to create an scFv based on the structure of a known neutralizing antibody, S230, for SARS-CoV-1. By analyzing the interaction of S230 with the RBD of both SARS-CoV-1 and SARS-CoV-2, five mutations were introduced to improve the binding of the scFv to the RBD of SARS-CoV-2. These mutations were Ser32Thr, Trp99Val, Asn57Val, Lys65Glu, and Tyr106Ile. Molecular dynamics simulations were used to evaluate the stability and affinity of the designed scFv. Our results showed that the designed scFv improved binding to the RBD of SARS-CoV-2 compared to the original S230, as indicated by principal component analysis, distance analysis, and MM/GBSA interaction energy. Furthermore, a positive result in a spot test lateral flow assay of the expressed scFv against the RBD indicated that the mutations did not alter the protein's structure. The designed scFv showed a negative result when tested against human serum albumin as a negative control, indicating reasonable specificity. We hope that this study will be useful in designing a specific and low-cost therapeutic agent, particularly during early outbreaks when information on neutralizing antibodies is limited.Communicated by Ramaswamy H. Sarma.

Unveiling the multifaceted landscape of N-glycosylation in antibody variable domains: Insights and implications.

N-glycosylation at the antibody variable domain has emerged as an important modification influencing antibody function. Despite its significance, information regarding its role and regulation remains limited. To address this gap, we comprehensively explored antibody structures housing N-glycosylation within the Protein Data Bank, yielding fresh insights into this intricate landscape. Our findings revealed that among 208 structures, N-glycosylation was more prevalent in human and mouse antibodies containing IGHV1-8 and IGHV2-2 germline genes, respectively. Moreover, our research highlights the potential for somatic hypermutation to introduce N-glycosylation sites by substituting polar residues (Ser or Thr) in germline variable genes with asparagine. Notably, our study underscores the prevalence of N-glycosylation in antiviral antibodies, especially anti-HIV. Besides antigen-antibody interaction, our findings suggest that N-glycosylation may impact antibody specificity, affinity, and avidity by influencing Fab dimer formation and complementary-determining region orientation. We also identified different glycan structures in HIV and SARS-CoV-2 antibody proteomic datasets, highlighting disparities from the N-glycan structures between PDB antibodies and biological repertoires further highlighting the complexity of N-glycosylation patterns. Our findings significantly enrich our understanding of the N-glycosylation's multifaceted characteristics within the antibody variable domain. Additionally, they underscore the pressing imperative for a more comprehensive characterization of its impact on antibody function.

Impact of SARS-CoV-2 vaccination on FcγRIIIA/CD16 dynamics in Natural Killer cells: relevance for antibody-dependent functions.

Introduction: Natural Killer (NK) cells contribute to the protective effects of vaccine-induced antibodies thanks to the low affinity receptor for IgG, FcγRIIIA/CD16, whose aggregation leads to the killing of infected cells and IFNγ release, through which they potentiate adaptive immune responses. Methods: Forty-seven healthy young individuals undergoing either homologous (ChAdOx1-S/ChAdOx1-S) or heterologous (ChAdOx1-S/BNT162B2) SARS-CoV-2 vaccination settings were recruited. Peripheral blood samples were collected immediately prior to vaccination and 8 weeks after the booster dose. The phenotypic and functional profile of NK cells was evaluated by flow cytometry at both time points. Serum samples were tested to evaluate circulating anti-Spike IgG levels and cytomegalovirus serostatus. CD16 F158V polymorphism was assessed by sequencing analysis. Results: The downregulation of CD16 and the selective impairment of antibody-dependent cytotoxicity and IFNγ production in CD56dim NK population, persisting 8 weeks after boosting, were observed in heterologous, but not in homologous SARS-CoV-2 vaccination scheme. While the magnitude of CD16-dependent functions of the global CD56dim pool correlated with receptor levels before and after vaccination, the responsivity of NKG2C+ subset, that displays amplified size and functionality in HCMV+ individuals, resulted intrinsically insensitive to CD16 levels. Individual CD16 responsiveness was also affected by CD16F158V polymorphism; F/F low affinity individuals, characterized by reduced CD16 levels and functions independently of vaccination, did not show post-vaccinal functional impairment with respect to intermediate and high affinity ones, despite a comparable CD16 downregulation. Further, CD16 high affinity ligation conditions by means of afucosylated mAb overcame vaccine-induced and genotype-dependent functional defects. Finally, the preservation of CD16 expression directly correlated with anti-Spike IgG titer, hinting that the individual magnitude of receptor-dependent functions may contribute to the amplification of the vaccinal response. Conclusion: This study demonstrates a durable downmodulation of CD16 levels and Ab-dependent NK functions after SARS-CoV-2 heterologous vaccination, and highlights the impact of genetic and environmental host-related factors in modulating NK cell susceptibility to post-vaccinal Fc-dependent functional impairment.

Liver macrophages and sinusoidal endothelial cells execute vaccine-elicited capture of invasive bacteria.

Vaccination has substantially reduced the morbidity and mortality of bacterial diseases, but mechanisms of vaccine-elicited pathogen clearance remain largely undefined. We report that vaccine-elicited immunity against invasive bacteria mainly operates in the liver. In contrast to the current paradigm that migrating phagocytes execute vaccine-elicited immunity against blood-borne pathogens, we found that invasive bacteria are captured and killed in the liver of vaccinated host via various immune mechanisms that depend on the protective potency of the vaccine. Vaccines with relatively lower degrees of protection only activated liver-resident macrophage Kupffer cells (KCs) by inducing pathogen-binding immunoglobulin M (IgM) or low amounts of IgG. IgG-coated pathogens were directly captured by KCs via multiple IgG receptors FcγRs, whereas IgM-opsonized bacteria were indirectly bound to KCs via complement receptors of immunoglobulin superfamily (CRIg) and complement receptor 3 (CR3) after complement C3 activation at the bacterial surface. Conversely, the more potent vaccines engaged both KCs and liver sinusoidal endothelial cells by inducing higher titers of functional IgG antibodies. Endothelial cells (ECs) captured densely IgG-opsonized pathogens by the low-affinity IgG receptor FcγRIIB in a "zipper-like" manner and achieved bacterial killing predominantly in the extracellular milieu via an undefined mechanism. KC- and endothelial cell-based capture of antibody-opsonized bacteria also occurred in FcγR-humanized mice. These vaccine protection mechanisms in the liver not only provide a comprehensive explanation for vaccine-/antibody-boosted immunity against invasive bacteria but also may serve as in vivo functional readouts of vaccine efficacy.

The heat-labile enterotoxin B subunit bio-adjuvant linked to Newcastle disease virus recombinant hemagglutinin neuraminidase elicited a humoral immune response in animal model.

Newcastle disease is a highly contagious viral infection primarily affecting poultry, leading to significant economic losses worldwide due to its high morbidity and mortality rates. Given the severity of the disease and its impact on the poultry industry, there is an urgent need for a preventative approach to tackle this issue. Developing an efficient and effective vaccine is a valuable step toward reducing the burden of this virus. Consequently, investing in preventive measures, such as vaccination programs, is a top priority to mitigate the economic losses associated with Newcastle disease and protect the livelihoods of those relying on the poultry industry. Despite many vaccines against this viral disease, it still infects many wild and domestic birds worldwide. In this work, chimeric proteins, composed of the recombinant B subunit of Enterotoxigenic E. coli with one or two HN (Hemagglutinin-neuraminidase) subunits of NDV (LHN and LHN2, respectively), expressed using E.coli host. In-silico, in-vitro, and In-vivo procedures were performed to evaluate the immunogenicity of these proteins. The sera from immunized mice were analyzed using Western Blotting and ELISA. The LHN2 protein with an extra HN subunit elicited a higher antibody titer than the LHN protein (P<0.05). Both products could effectively elicit an immune response against NDV and can be considered a component of Newcastle disease vaccine candidates.

Enhanced immunogenicity elicited by a novel DNA vaccine encoding the SARS-CoV-2 S1 protein fused to the optimized flagellin of Salmonella typhimurium in mice.

IMPORTANCE: The development of safe and effective vaccines is needed to control the transmission of coronavirus disease 2019 (COVID-19). Synthetic DNA vaccines represent a promising platform in response to such outbreaks. Here, DNA vaccine candidates were developed using an optimized antibiotic-resistance gene-free asd-pVAX1 vector. An optimized flagellin (FliC) adjuvant was designed by fusion expression to increase the immunogenicity of the S1 antigen. S1 and S1-FliCΔD2D3 proteins were strongly expressed in mammalian cells. The FliCΔD2D3-adjuvanted DNA vaccine induced Th1/Th2-mixed immune responses and high titers of neutralizing antibodies. This study provides crucial information regarding the selection of a safer DNA vector and adjuvant for vaccine development. Our FliCΔD2D3-adjuvanted S1 DNA vaccine is more potent at inducing both humoral and cellular immune responses than S1 alone. This finding provides a new idea for the development of novel DNA vaccines against COVID-19 and could be further applied for the development of other vaccines.

Cryptic-site-specific antibodies to the SARS-CoV-2 receptor binding domain can retain functional binding affinity to spike variants.

IMPORTANCE: Multiple SARS-CoV-2 variants of concern have emerged and caused a significant number of infections and deaths worldwide. These variants of concern contain mutations that might significantly affect antigen-targeting by antibodies. It is therefore important to further understand how antibody binding and neutralization are affected by the mutations in SARS-CoV-2 variants. We highlighted how antibody epitope specificity can influence antibody binding to SARS-CoV-2 spike protein variants and neutralization of SARS-CoV-2 variants. We showed that weakened spike binding and neutralization of Beta (B.1.351) and Omicron (BA.1) variants compared to wildtype are not universal among the panel of antibodies and identified antibodies of a specific binding footprint exhibiting consistent enhancement of spike binding and retained neutralization to Beta variant. These data and analysis can inform how antigen-targeting by antibodies might evolve during a pandemic and prepare for potential future sarbecovirus outbreaks.

Computational design of novel nanobodies targeting the receptor binding domain of variants of concern of SARS-CoV-2.

The COVID-19 pandemic has created an urgent need for effective therapeutic and diagnostic strategies to manage the disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the emergence of numerous variants of concern (VOCs) has made it challenging to develop targeted therapies that are broadly specific in neutralizing the virus. In this study, we aimed to develop neutralizing nanobodies (Nbs) using computational techniques that can effectively neutralize the receptor-binding domain (RBD) of SARS-CoV-2 VOCs. We evaluated the performance of different protein-protein docking programs and identified HDOCK as the most suitable program for Nb/RBD docking with high accuracy. Using this approach, we designed 14 novel Nbs with high binding affinity to the VOC RBDs. The Nbs were engineered with mutated amino acids that interacted with key amino acids of the RBDs, resulting in higher binding affinity than human angiotensin-converting enzyme 2 (ACE2) and other viral RBDs or haemagglutinins (HAs). The successful development of these Nbs demonstrates the potential of molecular modeling as a low-cost and time-efficient method for engineering effective Nbs against SARS-CoV-2. The engineered Nbs have the potential to be employed in RBD-neutralizing assays, facilitating the identification of novel treatment, prevention, and diagnostic strategies against SARS-CoV-2.

Structural basis for cross-group recognition of an influenza virus hemagglutinin antibody that targets postfusion stabilized epitope.

Plasticity of influenza virus hemagglutinin (HA) conformation increases an opportunity to generate conserved non-native epitopes with unknown functionality. Here, we have performed an in-depth analysis of human monoclonal antibodies against a stem-helix region that is occluded in native prefusion yet exposed in postfusion HA. A stem-helix antibody, LAH31, provided IgG Fc-dependent cross-group protection by targeting a stem-helix kinked loop epitope, with a unique structure emerging in the postfusion state. The structural analysis and molecular modeling revealed key contact sites responsible for the epitope specificity and cross-group breadth that relies on somatically mutated light chain. LAH31 was inaccessible to the native prefusion HA expressed on cell surface; however, it bound to the HA structure present on infected cells with functional linkage to the Fc-mediated clearance. Our study uncovers a novel non-native epitope that emerges in the postfusion HA state, highlighting the utility of this epitope for a broadly protective antigen design.