Receptor-binding Domain Proteins Research Articles

Anti-RBD IgG antibodies from endemic coronaviruses do not protect against the acquisition of SARS-CoV-2 infection among exposed uninfected individuals

BackgroundThe Coronaviridae family comprises seven viruses known to infect humans, classified into alphacoronaviruses (HCoV-229E and HCoV-NL63) and betacoronaviruses (HCoV-OC43 and HCoV-HKU1), which are considered endemic. Additionally, it includes SARS-CoV (severe acute respiratory syndrome), MERS-CoV (Middle East respiratory syndrome), and the novel coronavirus SARS-CoV-2, responsible for COVID-19. SARS-CoV-2 induces severe respiratory complications, particularly in the elderly, immunocompromised individuals and those with underlying diseases. An essential question since the onset of the COVID-19 pandemic has been to determine whether prior exposure to seasonal coronaviruses influences immunity or protection against SARS-CoV-2.MethodsIn this study, we investigated a cohort of 47 couples (N=94), where one partner tested positive for SARS-CoV-2 infection via real-time PCR while the other remained negative. Plasma samples, collected at least 30 days post-PCR reaction, were assessed using indirect ELISA and competition assays to measure specific antibodies against the receptor-binding domain (RBD) portion of the Spike (S) protein from SARS-CoV-2, HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1.ResultsIgG antibody levels against the four endemic coronavirus RBD proteins were similar between the PCR-positive and PCR-negative individuals, suggesting that IgG against endemic coronavirus RBD regions was not associated with protection from infection. Moreover, we found no significant IgG antibody cross-reactivity between endemic coronaviruses and SARS-CoV-2 RBDs.ConclusionsTaken together, results suggest that anti-RBD antibodies induced by a previous infection with endemic HCoVs do not protect against acquisition of COVID-19 among exposed uninfected individuals.

Self-Assembling Sulfated Lactobacillus Exopolysaccharide Nanoparticles as Adjuvants for SARS-CoV-2 Subunit Vaccine Elicit Potent Humoral and Cellular Immune Responses.

Coronavirus disease 2019 (COVID-19) has caused a global pandemic since its onset in 2019, and the development of effective vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to induce potent and long-lasting immunity remains a priority. Herein, we prepared two Lactobacillus exopolysaccharide (EPS) nanoparticle adjuvants (NPs 7-4 and NPs 8-2) that were constructed by using sulfation-modified EPS and quaternization-modified chitosan. These two NPs displayed a spherical morphology with sizes of 39 and 47 nm. Furthermore, the zeta potentials of NPs 7-4 and NPs 8-2 were 50.40 and 44.40 mV, respectively. In vitro assays demonstrated that NPs could effectively adsorb antigenic proteins and exhibited a sustained release effect. Mouse immunization tests showed that the NPs induced the expression of cytokines and chemokines at the injection site and promoted the uptake of antigenic proteins by macrophages. Mechanically, the NPs upregulated the expression of pattern recognition receptors (toll-like receptors and nod-like receptors) and activated the immune response of T cells and the production of neutralizing antibodies. In addition, the NP adjuvants had favorable immune-enhancing effects in cats, which are of great significance for controlling the trans-host transmission and re-endemicity of SARS-CoV-2. Overall, we demonstrated that NP-adjuvanted SARS-CoV-2 receptor binding domain proteins could induce robust specific humoral and cellular immunity.

In silico antiviral properties of Spirulina platensis phycobiliprotein and phycobilin as natural inhibitor for SARS-CoV-2

The global pandemic COVID-19 in 2019 has led to a continued search for promising potential molecules to combat human viral diseases. An in silico strategy was attempted to narrow down the discovery of potentially active compounds for drug candidates. It has been reported that several bioactive molecules in cyanobacteria have displayed antiviral activities. This study aimed to investigate the potential of phycobiliprotein and phycobilin from Spirulina platensis, a nutrient-dense cyanobacterium, as anti-SARS-CoV-2 using in silico assessment. The docking of the drug candidate was performed by targeting three important SARS-CoV-2 protein receptors; main protease (Mpro), receptor binding domain (RBD), and RNA-dependent RNA polymerase (RdRp). The results revealed that phycoerythrobilin showed the high binding affinity against all target proteins, specially towards RBD and RdRp proteins that exceeded the chloroquine as well as remdesivir as control inhibitors. Inferiorly, phycocyanobilin showed also similar triple inhibitory effect against the three main proteins though lower affinity at SARS-CoV-2 Mpro when compared to inhibitor nelfinavir. Pharmacokinetics analysis was conducted for both compounds using SwissADME and pkCSM to provide the properties of bioactive candidates when designed as a drug. The molecular physicochemical evaluation showed that both phycocyanobilin and phycoerythrobilin have fulfilled the Lipinski's rule of five, suggesting that both molecules have drug-like properties and could act like a drug. Based on the simulation results, it can be concluded that phycobiliprotein and phycobilin from Spirulina platensis have the potential to be developed as source for the development of novel natural antiviral agent.

A superior heterologous prime-boost vaccination strategy against COVID-19: A bivalent vaccine based on yeast-derived RBD proteins followed by a heterologous vaccine.

Various vaccines have been challenged by SARS-CoV-2 variants. Here, we reported a yeast-derived recombinant bivalent vaccine (Bivalent wild-type [Wt]+De) based on the wtand Delta receptor-binding domain (RBD). Yeast derived RBD proteins based on the wtand Delta mutant were used as the prime vaccine. It was found that, in the presence of aluminium hydroxide (Alum) and unmethylated CpG-oligodeoxynucleotides (CpG) adjuvants, more cross-protective immunity against SARS-CoV-2 prototype and variants were elicited by bivalent vaccine than monovalent wtRBD or Delta RBD. Furthermore, a heterologous boosting strategy consisting of two doses of bivalent vaccines followed by one dose adenovirus vectored vaccine exhibited cross-neutralization capacity and specific T cell responses against Delta and Omicron (BA.1 and BA.4/5) variants in mice, superior to a homologous vaccination strategy. This study suggested that heterologous prime-boost vaccination with yeast-derived bivalent protein vaccine could be a potential approach to address the challenge of emerging variants.

Studying SARS-CoV-2 interactions using phage-displayed receptor binding domain as a model protein

SARS-CoV-2 receptor binding domain (RBD) mediates viral entry into human cells through its interaction with angiotensin converting enzyme 2 (ACE2). Most neutralizing antibodies elicited by infection or vaccination target this domain. Such a functional relevance, together with large RBD sequence variability arising during viral spreading, point to the need of exploring the complex landscape of interactions between RBD-derived variants, ACE2 and antibodies. The current work was aimed at developing a simple platform to do so. Biologically active and antigenic Wuhan-Hu-1 RBD, as well as mutated RBD variants found in nature, were successfully displayed on filamentous phages. Mutational scanning confirmed the global plasticity of the receptor binding motif within RBD, highlighted residues playing a critical role in receptor binding, and identified mutations strengthening the interaction. The ability of vaccine-induced antibodies to inhibit ACE2 binding of many mutated RBD variants, albeit at different extents, was shown. Amino acid replacements which could compromise such inhibitory potential were underscored. The expansion of our approach could be the starting point for a large-scale phage-based exploration of diversity within RBD of SARS-CoV-2 and related coronaviruses, useful to understand structure–function relationships, to engineer RBD proteins, and to anticipate changes to watch during viral evolution.

Persistence of SARS-CoV-2-antibodies against N, S and RBD after natural infection.

Coronavirus disease 2019 (COVID-19) pandemic has affected most countries in the world. Monitoring the humoral immune responses during the natural course of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection and the duration of them provide useful information for the development of vaccination strategies against this virus and its emerging variants. The importance of the antibody response especially neutralizing antibodies in long-term immunity to SARS-CoV-2 is significant. The present study is a cross-sectional study of sero-epidemiological type that has been proposed to compare the persistence of Immunoglobulin G (IgG) against N (nucleocapsid), S (spike) and RBD (receptor-binding domain) proteins in the community after the time of primary disease. A total of 652 serum samples were collected from hospital staff working in COVID wards, as well as a number of community members with different occupations, among those with positive antibody titers, 86 participated in the resampling test before vaccination. There was no association between antibody titer and disease severity (p>0.05). A significant decrease in Ab levels was observed in the paired second samples. The highest rate of decrease was related to anti-N, then anti-RBD and anti-S IgG levels, respectively. There is a significant relationship between the initial antibody titer and its reduction over time (p-value <0.05). Our data revealed that humoral immunity following natural infection of SARS-CoV-2 is detectable for at least 4 months, regardless of disease severity. The most decrease in antibody titer over time was related to anti-N IgG levels.

Unraveling bioactive metabolites of mangroves as putative inhibitors of SARS-CoV-2 Mpro and RBD proteins: molecular dynamics and ADMET analysis

COVID-19 is a deadly pandemic caused by Corona virus leading to millions of deaths worldwide. Till today no medicine was available to cure this disease. This study selected 262 potential bioactive natural products derived from mangroves to inhibit the main protease (Mpro) and receptor-binding domain (RBD) protein of the COVID-19 virus. All the ligands were subjected to Adsorption Digestion Metabolism Excretion and Toxicity (ADMET) predictions and docking studies using AutodockVina. Among all the ligands, NP_143 (Shearinine A) and NP_242 (Amentoflavone), having the highest docking score of 10.2 and 10.1 Kj/mole, respectively, were picked for 100 ns of Molecular Dynamics using GROMACS. The trajectories generated were used to estimate Root mean square deviation (RMSD), Root mean square fluctuations (RMSF), Radius of Gyrations (RG), Solvent accessible surface area (SASA), and Hydrogen bonds. From the data generated, both the ligands have good binding ability at the active site of Mpro protein and do not deviate much. They have strong interactions with the amino acids during the 100 ns of simulations and can thus be considered potential drug candidates. Communicated by Ramaswamy H. Sarma

SARS-CoV-2 Protein Nanoparticle Vaccines Formed In Situ From Lyophilized Lipids.

The receptor binding domain (RBD) of the SARS-CoV-2 Spike (S) glycoprotein is an appealing immunogen, but associated vaccine approaches must overcome the hapten-like nature of the compact protein and adapt to emerging variants with evolving RBD sequences. Here, a vaccine manufacturing methodology is proposed comprising a sterile-filtered freeze-dried lipid cake formulation that can be reconstituted with liquid proteins to instantaneously form liposome-displayed protein nanoparticles. Mannitol is used as a bulking agent and a small amount of Tween-80 surfactant is required to achieve reconstituted submicron particles that do not precipitate prior to usage. The lipid particles include an E. coli-derived monophosphoryl lipid A (EcML) for immunogenicity, and cobalt porphyrin-phospholipid (CoPoP) for antigen display. Reconstitution of the lipid cake with aqueous protein results in rapid conversion of the RBD into intact liposome-bound format prior to injection. Protein particles can readily be formed with sequent-divergent RBD proteins derived from the ancestral or Omicron strains. Immunization of miceelicits antibodies that neutralize respective viral strains. When K18-hACE2 transgenic mice are immunized and challenged with ancestral SARS-CoV-2 or the Omicron BA.5 variant, both liquid liposomes displaying the RBD and rapid reconstituted particles protect mice from infection, as measured by the viral load in the lungs and nasal turbinates.

Trivalent mRNA Vaccine against SARS-CoV-2 and Variants with Effective Immunization.

mRNA vaccines encoding a single spike protein effectively prevent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, the emergence of SARS-CoV-2 variants leads to a wide range of immune evasion. Herein, a unique trivalent mRNA vaccine based on ancestral SARS-CoV-2, Delta, and Omicron variant spike receptor-binding domain (RBD) mRNAs was developed to tackle the immune evasion of the variants. First, three RBD mRNAs of SARS-CoV-2, Delta, and Omicron were coencapsulated into lipid nanoparticles (LNPs) by using microfluidic technology. After that, the physicochemical properties and time-dependent storage stability of the trivalent mRNA vaccine nanoformulation were tested by using dynamic light scattering (DLS). In vitro, the trivalent mRNA vaccine exhibited better lysosomal escape ability, transfection efficiency, and biocompatibility than did the commercial transfection reagent Lipo3000. In addition, Western blot analyses confirmed that the three RBD proteins can be detected in cells transfected with the trivalent mRNA vaccine. Furthermore, ex vivo imaging analysis indicated that the livers of BALB/c mice had the strongest protein expression levels after intramuscular (IM) injection. Using a prime-boost strategy, this trivalent vaccine elicited robust humoral and T-cell immune responses in both the high-dose and low-dose groups and showed no toxicity in BALB/c mice. Three specific IgG antibodies in the high-dose group against SARS-CoV-2, Delta, and Omicron variants approached ∼1/1,833,333, ∼1/1,866,667, and ∼1/925,000, respectively. Taken together, two doses of inoculation with the trivalent mRNA vaccine may provide broad and effective immunization responses against SARS-CoV-2 and variants.

Plant-produced RBD and cocktail-based vaccine candidates are highly effective against SARS-CoV-2, independently of its emerging variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel and highly pathogenic coronavirus that caused an outbreak in Wuhan City, China, in 2019 and then spread rapidly throughout the world. Although several coronavirus disease 2019 (COVID-19) vaccines are currently available for mass immunization, they are less effective against emerging SARS-CoV-2 variants, especially the Omicron (B.1.1.529). Recently, we successfully produced receptor-binding domain (RBD) variants of the spike (S) protein of SARS-CoV-2 and an antigen cocktail in Nicotiana benthamiana, which are highly produced in plants and elicited high-titer antibodies with potent neutralizing activity against SARS-CoV-2. In this study, based on neutralization ability, we demonstrate that plant-produced RBD and cocktail-based vaccine candidates are highly effective against SARS-CoV-2, independently of its emerging variants. These data demonstrate that plant-produced RBD and cocktail-based proteins are the most promising vaccine candidates and may protect against Delta and Omicron-mediated COVID-19. This is the first report describing vaccines against SARS-CoV-2, which demonstrate significant activities against Delta and Omicron variants.

Optimization of mammalian expression vector by cis-regulatory element combinations.

The regulation of gene expression in mammalian cells by combining various cis-regulatory features has rarely been discussed. In this study, we constructed expression vectors containing various combinations of regulatory elements to examine the regulation of gene expression by different combinations of cis-regulatory elements. The effects of four promoters (CMV promoter, PGK promoter, Polr2a promoter, and EF-1α core promoter), two enhancers (CMV enhancer and SV40 enhancer), two introns (EF-1α intron A and hybrid intron), two terminators (CYC1 terminator and TEF terminator), and their different combinations on downstream gene expression were compared in various mammalian cells using fluorescence microscopy to observe fluorescence, quantitative real-time PCR (qRT-PCR), and western blot. The receptor binding domain (RBD) sequence from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein was used to replace the eGFP sequence in the expression vector and the RBD expression was detected by qRT-PCR and western blot. The results showed that protein expression can be regulated by optimizing the combination of cis-acting elements. The vector with the CMV enhancer, EF-1α core promoter, and TEF terminator was found to express approximately threefold higher eGFP than the unmodified vector in different animal cells, as well as 2.63-fold higher recombinant RBD protein than the original vector in HEK-293T cells. Moreover, we suggest that combinations of multiple regulatory elements capable of regulating gene expression do not necessarily exhibit synergistic effects to enhance expression further. Overall, our findings provide insights into biological applications that require the regulation of gene expression and will help to optimize expression vectors for biosynthesis and other fields. Additionally, we provide valuable insights into the production of RBD proteins, which may aid in developing reagents for diagnosis and treatment during the COVID-19 pandemic.

Texture or Linker? Competitive Patterning of Receptor Assembly toward Ultra-Sensitive Impedimetric Detection of Viral Species at Gold-Nanotextured Titanium Surfaces.

In this work, we study the electrodes with a periodic matrix of gold particles pattered by titanium dimples and modified by 3-mercaptopropionic acid (MPA) followed by CD147 receptor grafting for specific impedimetric detection of SARS-CoV-2 viral spike proteins. The synergistic DFT and MM/MD modeling revealed that MPA adsorption geometries on the Au-Ti surface have preferential and stronger binding patterns through the carboxyl bond inducing an enhanced surface coverage with CD147. Control of bonding at the surface is essential for oriented receptor assembling and boosted sensitivity. The complex Au-Ti electrode texture along with optimized MPA concentration is a crucial parameter, enabling to reach the detection limit of ca. 3 ng mL-1. Scanning electrochemical microscopy imaging and quantum molecular modeling were performed to understand the electrochemical performance and specific assembly of MPA displaying a free stereo orientation and not disturbed by direct interactions with closely adjacent receptors. This significantly limits nonspecific interceptor reactions, strongly decreasing the detection of receptor-binding domain proteins by saturation of binding groups. This method has been demonstrated for detecting the SARS virus but can generally be applied to a variety of protein-antigen systems. Moreover, the raster of the pattern can be tuned using various anodizing processes at the titania surfaces.

Longitudinal analysis of SARS-CoV-2 infection and vaccination in the LA-SPARTA cohort reveals increased risk of infection in vaccinated Hispanic participants.

SARS-CoV-2 is the etiologic agent of coronavirus disease 2019 (COVID-19). Questions remain regarding correlates of risk and immune protection against COVID-19. We prospectively enrolled 200 participants with a high risk of SARS-CoV-2 occupational exposure at a U.S. medical center between December 2020 and April 2022. Participant exposure risks, vaccination/infection status, and symptoms were followed longitudinally at 3, 6, and 12 months, with blood and saliva collection. Serological response to the SARS-CoV-2 spike holoprotein (S), receptor binding domain (RBD) and nucleocapsid proteins (NP) were quantified by ELISA assay. Based on serology, 40 of 200 (20%) participants were infected. Healthcare and non-healthcare occupations had equivalent infection incidence. Only 79.5% of infected participants seroconverted for NP following infection, and 11.5% were unaware they had been infected. The antibody response to S was greater than to RBD. Hispanic ethnicity was associated with 2-fold greater incidence of infection despite vaccination in this cohort. Overall, our findings demonstrate: 1) variability in the antibody response to SARS-CoV-2 infection despite similar exposure risk; 2) the concentration of binding antibody to the SARS-CoV-2 S or RBD proteins is not directly correlated with protection against infection in vaccinated individuals; and 3) determinants of infection risk include Hispanic ethnicity despite vaccination and similar occupational exposure.

Incorporation of SARS-CoV-2 spike NTD to RBD protein vaccine improves immunity against viral variants.

Emerging SARS-CoV-2 variants pose a threat to human health worldwide. SARS-CoV-2 receptor binding domain (RBD)-based vaccines are suitable candidates for booster vaccines, eliciting a focused antibody response enriched for virus neutralizing activity. Although RBD proteins are manufactured easily, and have excellent stability and safety properties, they are poorly immunogenic compared to the full-length spike protein. We have overcome this limitation by engineering a subunit vaccine composed of an RBD tandem dimer fused to the N-terminal domain (NTD) of the spike protein. We found that inclusion of the NTD (1) improved the magnitude and breadth of the Tcell and anti-RBD response, and (2) enhanced T follicular helper cell and memory B cell generation, antibody potency, and cross-reactive neutralization activity against multiple SARS-CoV-2 variants, including B.1.1.529 (Omicron BA.1). In summary, our uniquely engineered RBD-NTD-subunit protein vaccine provides a promising booster vaccination strategy capable of protecting against known SARS-CoV-2 variants of concern.

SARS-CoV-2 spike protein induces IL-18-mediated cardiopulmonary inflammation via reduced mitophagy

Cardiopulmonary complications are major drivers of mortality caused by the SARS-CoV-2 virus. Interleukin-18, an inflammasome-induced cytokine, has emerged as a novel mediator of cardiopulmonary pathologies but its regulation via SARS-CoV-2 signaling remains unknown. Based on a screening panel, IL-18 was identified amongst 19 cytokines to stratify mortality and hospitalization burden in patients hospitalized with COVID-19. Supporting clinical data, administration of SARS-CoV-2 Spike 1 (S1) glycoprotein or receptor-binding domain (RBD) proteins into human angiotensin-converting enzyme 2 (hACE2) transgenic mice induced cardiac fibrosis and dysfunction associated with higher NF-κB phosphorylation (pNF-κB) and cardiopulmonary-derived IL-18 and NLRP3 expression. IL-18 inhibition via IL-18BP resulted in decreased cardiac pNF-κB and improved cardiac fibrosis and dysfunction in S1- or RBD-exposed hACE2 mice. Through in vivo and in vitro work, both S1 and RBD proteins induced NLRP3 inflammasome and IL-18 expression by inhibiting mitophagy and increasing mitochondrial reactive oxygenation species. Enhancing mitophagy prevented Spike protein-mediated IL-18 expression. Moreover, IL-18 inhibition reduced Spike protein-mediated pNF-κB and EC permeability. Overall, the link between reduced mitophagy and inflammasome activation represents a novel mechanism during COVID-19 pathogenesis and suggests IL-18 and mitophagy as potential therapeutic targets.

Baculovirus displaying SARS-CoV-2 spike RBD promotes neutralizing antibody production in a mouse model

BackgroundThere is always a need for a safe and efficient vaccine platform, especially when facing a pandemic such as COVID-19. Most of the SARS-CoV-2-based vaccines are based on the full spike protein, which is presented as a trimerized protein, and many viral vector vaccines express the spike protein into the host cells and do not display it on virus surfaces. However, the spike receptor-binding domain (RBD)-based vaccines are efficient and are currently under investigation and clinical trials. MethodologyIn this study, we are testing the efficacy of the RBD displayed on a baculovirus as a mean to formulate a safe and stable carrier to induce the immune system against SARS-CoV-2. Therefore, two pseudotyped baculoviruses were constructed to display the RBD, AcRBD-sfGFP-64, and AcRBD-sfGFP-V, using two different displaying strategies based on gp64 and VSV-G envelope glycoproteins, from Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and vesicular stomatitis virus (VSV), respectively. BALB/C mice were immunized with the pseudotyped baculoviruses in a dose-optimized manner. Dot blot and Western blot were used to screen and validate the polyclonal antibodies’ specificity to the SARS-CoV-2 RBD. A plaque reduction neutralization test (PRNT) was used to measure the sera neutralization capacity against a SARS-CoV-2 wild-type isolate from Egypt. ELISA was used to quantify certain cytokines for the assessment of the immune response. ResultThe outcome of our investigation showed that the monomeric RBD proteins were properly displayed on baculovirus and efficiently triggered the mouse immune system. The produced sera efficiently neutralized about 50% of SARS-CoV-2 in more than 100-fold serum dilution. The immunized mice showed a significant increase (p<0.01) in the levels of IL-2 and IFN-γ and a significant decrease (p<0.01) and (p<0.001) in the levels of IL-4 and IL-10, respectively, which suggest that AcRBD-sfGFP-64 and AcRBD-sfGFP-V induce Th1 cellular immune response. ConclusionThe produced recombinant viruses can induce the immune response without adjuvant, which needs dose optimization and further stability tests. Neutralizing antibodies were induced without affecting the health of immunized mice. Th1 response can be attainable through the system, which is of great benefit in SARS CoV-2 infection and the system can be tested for future applications including vaccine development and polyclonal antibody production.

1960. Mucosal rAd5 Immunization against SARS-CoV-2 Spike Elicits Cross-Reactive Nasal and Serum Neutralizing Antibodies and Protects Against Beta Variant Challenge in African Green Monkeys

Abstract Background Mucosal vaccination may offer increased protection against SARS-CoV-2 compared to parental immunization. Here, we describe immunogenicity and efficacy following viral challenge in non-human primates after intranasal delivery of three unique non-replicating adenoviral vector vaccine (rAd5) candidates. Methods African green monkeys (AMG) were prime boost immunized 29 days apart with vaccine candidates either expressing the parental spike protein alone (Wuhan-S), spike plus nucleocapsid (Wuhan-S-N), or the spike protein from the beta variant (beta-S). Serum and nasal swabs were collected every 14 days and humoral responses to full length spike (S) and receptor binding domain (RBD) were assessed. All AMGs were challenged with SARS-CoV-2 B.1.351 (beta variant) on day 56. Viral loads measured every two days by TCID50 in nasal washes and bronchial lavage fluid post challenge. Results Mucosal immunization with Wuhan-S induced significant increases in serum IgG and IgA responses against the homologous parental lineage, as well as beta, delta, and omicron variants. In nasal samples, Wuhan-S immunization elicited over 500-fold increases in in cross-reactive IgA against multiple variants of concern including delta and omicron. While the beta-S rAd5 vaccine candidate induced enhanced serum IgG responses to homologous S and RBD proteins, this approach resulted in less cross-reactive antibodies to other variants compared to Wuhan-S rAd5 vaccine. Despite the differences in the ability to elicit cross-reactive antibody responses, all vaccinated AMGs challenged with SARS-CoV-2 B.1.351 (beta variant), had a significant reduction in viral titers by TCID50 in the nasal passages and reduced viral load in bronchial lavage fluid compared to unvaccinated controls. Conclusion These results demonstrate mucosal administration of rAd5 clinical candidate vaccine, Wuhan-S, is immunogenic and offers cross-protective humoral responses in both serum and nasal compartments against a mismatched SARS-CoV-2 challenge virus. Disclosures Becca A. Flitter, PhD, MPH, Vaxart Inc: Stocks/Bonds Sarah N. Tedjakusuma, n/a, Vaxart Inc: Stocks/Bonds Colin A. Lester, n/a, Vaxart Inc: Stocks/Bonds Elena D. Neuhaus, n/a, Vaxart Inc: Stocks/Bonds Susan Johnson, PhD, Vaxart Inc: Stocks/Bonds Emery G. Dora, n/a, Vaxart Inc: Stocks/Bonds Nadine Peinovich, MPH, Vaxart Inc: Stocks/Bonds Clara B. Jegede, n/a, Vaxart Inc: Stocks/Bonds Sean N. Tucker, PhD, Vaxart Inc: Stocks/Bonds.

Development of in House ELISAs to Detect Antibodies to SARS-CoV-2 in Infected and Vaccinated Humans by Using Recombinant S, S1 and RBD Proteins.

(1) Background: The aim of this study was to produce in-house ELISAs which can be used to determine SARS-CoV-2-specific antibody levels directed against the spike protein (S), the S1 subunit of S and the receptor binding domain (RBD) of S in SARS-CoV-2 vaccinated and infected humans. (2) Methods: Three in-house ELISAs were developed by using recombinant proteins of SARS-CoV-2, namely the S, S1 and RBD proteins. Specificity and sensitivity evaluations of these tests were performed using sera from SARS-CoV-2-infected (n = 70) and SARS-CoV-2-vaccinated (n = 222; CoronaVac vaccine) humans in Istanbul, Turkey. The analyses for the presence of SARS-CoV-2-specific antibodies were performed using the in-house ELISAs, a commercial ELISA (Abbott) and a commercial surrogate virus neutralization test (sVNT). We also analyzed archival human sera (n = 50) collected before the emergence of COVID-19 cases in Turkey. (3) Results: The sensitivity of the in-house S, S1 and RBD ELISAs was found to be 88.44, 90.17 and 95.38%, while the specificity was 72.27, 89.08 and 89.92%, respectively, when compared to the commercial SARS-CoV-2 antibody test kit. The area under curve (AUC) values were 0.777 for the in-house S ELISA, 0.926 for the S1 ELISA, and 0.959 for the RBD ELISA. The kappa values were 0.62, 0.79 and 0.86 for the S, S1 and RBD ELISAs, respectively. (4) Conclusions: The in-house S1 and RBD ELISAs developed in this study have acceptable performance characteristics in terms of sensitivity, specificity, AUC and kappa values. In particular, the RBD ELISA seems viable to determine SARS-CoV-2-specific antibody levels, both in infected and vaccinated people, and help mitigate SARS-CoV-2 outbreaks and spread.

SARS-CoV-2 Antibody Effectiveness Is Influenced by Non-Epitope Mutation/Binding-Induced Denaturation of the Epitope 3D Architecture.

The public health threat from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to intensify with emerging variants of concern (VOC) aiming to render COVID-19 vaccines/infection-induced antibodies redundant. The SARS-CoV-2 spike protein is responsible for receptor binding and infection of host cells making it a legitimate antibody target. Antibodies mostly target epitopes in the receptor binding domain (RBD). Mutations occurring within epitopes influence antibody specificity and function by altering their 3D architecture. However, the mechanisms by which non-epitope mutations in the RBD influence antibody specificity and function remain a mystery. We used Protein Data Bank (PDB) deposited 3D structures for the original, Beta, Delta, BA.1, and BA.2 RBD proteins in complex with either neutralizing antibodies or Angiotensin-Converting Enzyme 2 (ACE2) to elucidate the structural and mechanistic basis for neutralizing antibody evasion driven by non-epitope amino acid substitutions in the RBD. Since the mechanism behind the extensively reported functional discrepancies between the same antibody when used individually and when used in an antibody cocktail is lacking, we explored the structural basis for this inconsistency. Finally, since SARS-CoV-2 antibodies are viral mutagens, we deciphered determinants for antibody-pressured amino acid substitutions. On the one hand, we show that non-epitope mutations in the RBD domain of SARS-CoV-2 VOC influence the formation of hydrogen bonds in the paratope-epitope interface by repositioning RBD amino-acid sidechains (AASCs). This increases the distance between complementary donor/acceptor atoms on paratope and epitope AASCs leading to weaker or the complete prevention of the formation of hydrogen bonds in the paratope-epitope interface. On the other hand, we show that SARS-CoV-2 VOC employ the same strategy to simultaneously search for complementary donor/acceptor atoms on ACE2 AASCs to form new interactions, potentially favoring increased viral transmission. Additionally, we illustrate that converting the spike protein to an RBD, a deletion mutation, also repositions epitope AASCs and that AASC interactions in the paratope-epitope interface vary when an antibody is used individually versus when utilized as a cocktail with other antibodies. Finally, we show that the process of substituting immunogenic RBD amino acids begins with the repositioning of their AASCs induced by immune/antibody pressure. We show that donor/acceptor atoms from any amino acid can determine cross-reactivity instead, provided they possess and present spatially pairing donor/acceptor atoms. By studying structural alignments for PDB deposited antibody-RBD 3D structures and relating them to published binding and neutralization profiles of the same antibodies, we demonstrate that minor structural alterations such as epitope AASC repositioning have a major impact on antibody effectiveness and, hence, should receive adequate attention given that protein structure dictates protein function.

COVID-19 vaccine immunogenicity in people with HIV.

Many vaccines require higher/additional doses or adjuvants to provide adequate protection for people with HIV (PWH). Our objective was to compare COVID-19 vaccine immunogenicity in PWH to HIV-negative individuals. In a Canadian multi-center prospective, observational cohort of PWH receiving at least two COVID-19 vaccinations, we measured vaccine-induced immunity at 3 and 6 months post 2nd and 1-month post 3rd doses. The primary outcome was the percentage of PWH mounting vaccine-induced immunity [co-positivity for anti-IgG against SARS-CoV2 Spike(S) and receptor-binding domain proteins] 6 months post 2nd dose. Univariable and multivariable logistic regressions were used to compare COVID-19-specific immune responses between groups and within subgroups. Data from 294 PWH and 267 controls were analyzed. Immunogenicity was achieved in over 90% at each time point in both groups. The proportions of participants achieving comparable anti-receptor-binding domain levels were similar between the group at each time point. Anti-S IgG levels were similar by group at month 3 post 2nd dose and 1-month post 3rd dose. A lower proportion of PWH vs. controls maintained vaccine-induced anti-S IgG immunity 6 months post 2nd dose [92% vs. 99%; odds ratio: 0.14 (95% confidence interval: 0.03, 0.80; P = 0.027)]. In multivariable analyses, neither age, immune non-response, multimorbidity, sex, vaccine type, or timing between doses were associated with reduced IgG response. Vaccine-induced IgG was elicited in the vast majority of PWH and was overall similar between groups. A slightly lower proportion of PWH vs. controls maintained vaccine-induced anti-S IgG immunity 6 months post 2nd dose demonstrating the importance of timely boosting in this population.