Maternal vaccinations administered prior to conception or during pregnancy enhance the immune protection of newborn infants against many pathogens. A feasibility experiment was conducted to determine if monkeys can be used to model the placental transfer of maternal antibody against SARS-CoV-2. Six adult rhesus monkeys were immunized with adjuvanted recombinant-protein antigens comprised of receptor-binding domain human IgG1-Fc fusion proteins (RBD-Fc) containing protein sequences from the ancestral-Wuhan or Gamma variants. The female monkeys mounted robust and sustained anti-SARS-CoV-2 antibody responses. Blood samples collected from their infants after delivery verified prenatal transfer of high levels of spike-specific IgG, which were positively correlated with maternal IgG titers at term. In addition, an in vitro test of ACE2 neutralization indicated that the infants\' IgG demonstrated antigen specificity, reflecting prior maternal immunization with either Wuhan or Gamma-variant antigens. All sera showed stronger ACE2-RBD binding inhibition when variants in the assay more closely resembled the vaccine RBD sequence than with more distantly related variants (i.e., Delta and Omicron). Monkeys are a valuable animal model for evaluating new vaccines that can promote maternal and infant health. Further, the findings highlight the enduring nature and safety of the immune protection elicited by an adjuvanted recombinant RBD-Fc vaccine.

Three highly pathogenic coronaviruses (CoVs), SARS-CoV-2, SARS-CoV, and MERS-CoV, belonging to the genus beta-CoV, have caused outbreaks or pandemics. SARS-CoV-2 has evolved into many variants with increased resistance to the current vaccines. Spike (S) protein and its receptor-binding domain (RBD) fragment of these CoVs are important vaccine targets; however, the RBD of the SARS-CoV-2 Omicron variant is highly mutated, rending neutralizing antibodies elicited by ancestral-based vaccines targeting this region ineffective, emphasizing the need for effective vaccines with broad-spectrum efficacy against SARS-CoV-2 variants and other CoVs with pandemic potential. This study describes a pan-beta-CoV subunit vaccine, Om-S-MERS-RBD, by fusing the conserved and highly potent RBD of MERS-CoV into an RBD-truncated SARS-CoV-2 Omicron S protein, and evaluates its neutralizing immunogenicity and protective efficacy in mouse models. Om-S-MERS-RBD formed a conformational structure, maintained effective functionality and antigenicity, and bind efficiently to MERS-CoV receptor, human dipeptidyl peptidase 4, and MERS-CoV RBD or SARS-CoV-2 S-specific antibodies. Immunization of mice with Om-S-MERS-RBD and adjuvants (Alum plus monophosphoryl lipid A) induced broadly neutralizing antibodies against pseudotyped MERS-CoV, SARS-CoV, and SARS-CoV-2 original strain, as well as T-cell responses specific to RBD-truncated Omicron S protein. Moreover, the neutralizing activity against SARS-CoV-2 Omicron subvariants was effectively improved after priming with an Omicron-S-RBD protein. Adjuvanted Om-S-MERS-RBD protein protected mice against challenge with SARS-CoV-2 Omicron variant, MERS-CoV, and SARS-CoV, significantly reducing viral titers in the lungs. Overall, these findings indicated that Om-S-MERS-RBD protein could develop as an effective universal subunit vaccine to prevent infections with MERS-CoV, SARS-CoV, SARS-CoV-2, and its variants.
OBJECTIVE: Coronaviruses (CoVs), SARS-CoV-2, SARS-CoV, and MERS-CoV, the respective causative agents of coronavirus disease 2019, SARS, and MERS, continually threaten human health. The spike (S) protein and its receptor-binding domain (RBD) fragment of these CoVs are critical vaccine targets. Nevertheless, the highly mutated RBD of SARS-CoV-2 variants, especially Omicron, significantly reduces the efficacy of current vaccines against SARS-CoV-2 variants. Here a protein-based pan-beta-CoV subunit vaccine is designed by fusing the potent and conserved RBD of MERS-CoV into an RBD-truncated Omicron S protein. The resulting vaccine maintained effective functionality and antigenicity, induced broadly neutralizing antibodies against all of these highly pathogenic human CoVs, and elicited Omicron S-specific cellular immune responses, protecting immunized mice from SARS-CoV-2 Omicron, SARS-CoV, and MERS-CoV infections. Taken together, this study rationally designed a pan-beta-CoV subunit vaccine with broad-spectrum efficacy, which has the potential for development as an effective universal vaccine against SARS-CoV-2 variants and other CoVs with pandemic potential.

The receptor-binding domain (RBD) is crucial for understanding how severe acute respiratory syndrome coronavirus (SARS-CoV-2) recognizes and infects host cells. Chitooligosaccharide (CS) exhibits diverse antiviral activities, with its derivatives showing remarkable efficacy in blocking SARS-CoV-2 infection. Thus, this study employed spectroscopy, virus-infected cell experiments, and molecular simulation to investigate the molecular interactions between CS and SARS-CoV-2 RBD, as well as their mechanisms. In spectroscopic experiments, all four CS variants with different molecular weights formed interactions with the RBD. These variants increased the resistance of HEK293ACE2 cells to SARS-CoV-2 invasion. Molecular docking revealed that the four CS variants could bind to the RBD through hydrogen bonding or salt-bridge interactions, forming stable complexes. Chitotetraose provided stronger protection to HEK293ACE2 cells compared to other CS variants and displayed higher molecular docking scores. Further investigation into the optimal docking conformation of chitotetraose was conducted through molecular dynamics simulation methods. This study lays a solid theoretical foundation and provides a scientific basis for the development of targeted RBD inhibitors, as well as drug screening and application against novel coronaviruses.

Rapid evolution of SARS-CoV-2 has resulted in the emergence of numerous variants, posing significant challenges to public health surveillance. Clinical genome sequencing, while valuable, has limitations in capturing the full epidemiological dynamics of circulating variants in the general population. This study utilized receptor-binding domain (RBD) amplicon sequencing of wastewater samples to monitor the SARS-CoV-2 community dynamics and evolution in El Paso, TX. Over 17 months, we identified 91 variants and observed waves of dominant variants transitioning from BA.2 to BA.2.12.1, BA.4&5, BQ.1, and XBB.1.5. Our findings demonstrated early detection of variants and identification of unreported outbreaks, while showing strong consistency with clinical genome sequencing data at the local, state, and national levels. Alpha diversity analyses revealed significant periodical variations, with the highest diversity observed in winter and the outbreak lag phases, likely due to lower competition among variants before the outbreak growth phase. The data underscores the importance of low transmission periods for rapid mutation and variant evolution. This study highlights the effectiveness of integrating RBD amplicon sequencing with wastewater surveillance in tracking viral evolution, understanding variant emergence, and enhancing public health preparedness.

The highly pathogenic coronaviruses SARS-CoV-2 and SARS-CoV have led to the COVID-19 pandemic and SARS outbreak, respectively. The receptor-binding domain (RBD) of the spike (S) protein of SARS-CoV-2, particularly the Omicron variant, has frequent mutations, resulting in the reduced efficiency of current COVID-19 vaccines against new variants. Here, we designed two lipid nanoparticle-encapsulated mRNA vaccines by deleting the mutant RBD of the SARS-CoV-2 Omicron variant (SARS2-S (RBD-del)) or by replacing this mutant RBD with the conserved and potent RBD of SARS-CoV (SARS2-S (SARS-RBD)). Both mRNA vaccines were stable at various temperatures for different time periods. Unlike SARS2-S (RBD-del) mRNA, SARS2-S (SARS-RBD) mRNA elicited effective T-cell responses and potent antibodies specific to both SARS-CoV-2 S and SARS-CoV RBD proteins. It induced strong neutralizing antibodies against pseudotyped SARS-CoV-2 and SARS-CoV infections and protected immunized mice from the challenge of the SARS-CoV-2 Omicron variant and SARS-CoV by significantly reducing the viral titers in the lungs after Omicron challenge and by completely preventing SARS-CoV-induced weight loss and death. SARS2-S (SARS-RBD)-immunized serum antibodies protected naïve mice from the SARS-CoV challenge, with its protective efficacy positively correlating with the neutralizing antibody titers. These findings indicate that this mRNA vaccine has the potential for development as an effective vaccine against current and future SARS-CoV-2 variants and SARS-CoV.

Nanobodies, single-domain antibodies derived from variable domain of camelid or shark heavy-chain antibodies, have unique properties with small size, strong binding affinity, easy construction in versatile formats, high neutralizing activity, protective efficacy, and manufactural capacity on a large-scale. Nanobodies have been arisen as an effective research tool for development of nanobiotechnologies with a variety of applications. Three highly pathogenic coronaviruses (CoVs), SARS-CoV-2, SARS-CoV, and MERS-CoV, have caused serious outbreaks or a global pandemic, and continue to post a threat to public health worldwide. The viral spike (S) protein and its cognate receptor-binding domain (RBD), which initiate viral entry and play a critical role in virus pathogenesis, are important therapeutic targets. This review describes pathogenic human CoVs, including viral structures and proteins, and S protein-mediated viral entry process. It also summarizes recent advances in development of nanobodies targeting these CoVs, focusing on those targeting the S protein and RBD. Finally, we discuss potential strategies to improve the efficacy of nanobodies against emerging SARS-CoV-2 variants and other CoVs with pandemic potential. It will provide important information for rational design and evaluation of therapeutic agents against emerging and reemerging pathogens.

BACKGROUND: Immunization against SARS-CoV-2 is essential for vulnerable solid organ transplant (SOT) recipients who are at risk of infection. However, there are concerns about suboptimal immunogenicity, especially in humoral immunity (HMI), and limited exploration of cell-mediated immune (CMI) responses. The primary objective of this study was to assess the immunogenicity of ChAdOx1 nCoV-19 vaccination in SOT recipients. The secondary endpoint was to evaluate factors that affect immunogenicity and adverse events (AEs) following immunization in SOT recipients.
METHODS: All adult SOT recipients who received the two-dose ChAdOx1 nCoV-19 vaccine at a 12-week interval underwent measurements of HMI by evaluating anti-receptor-binding domain (RBD) IgG levels and CMI by investigating SARS-CoV-2-specific T cell and B cell responses before and after complete vaccination, around 2-4 weeks post-vaccination, and compared to controls. AEs were monitored in all participants.
RESULTS: The study included 63 SOT recipients: 44 kidney recipients, 16 liver recipients, and 3 heart transplant recipients, along with 11 immunocompetent controls. Among SOT recipients, 36% were female, and the median (IQR) age was 52 (42-61). The median (IQR) time since transplant was 55 (28-123) months. After the second dose, the median (IQR) anti-RBD antibody levels were significantly lower in SOT recipients compared to those in the control group (8.3 [0.4-46.0] vs. 272.2 [178.1-551.6] BAU/mL, p < 0.01). This resulted in a seroconversion rate (anti-RBD antibody > 7.1 BAU/mL) of 51% among SOT recipients and 100% among controls (p = 0.008). Receiving the vaccine beyond one year post-transplant significantly affected seroconversion (OR 9.04, 95% CI 1.04-78.56, p = 0.046), and low-dose mycophenolic acid marginally affected seroconversion (OR 2.67, 95% CI 0.89-7.96, p = 0.079). RBD-specific B cell responses were also significantly lower compared to those in the control group (0 [0-4] vs. 10 [6-22] SFUs/106 PBMCs, p = 0.001). Similarly, S1- and SNMO-specific T cell responses were significantly lower compared to those in the control group (48 [16-128] vs. 216 [132-356] SFUs/106 PBMCs, p = 0.004 and 20 [4-48] vs. 92 [72-320] SFUs/106 PBMCs, p = 0.004). AEs were generally mild and spontaneously resolved.
CONCLUSIONS: SOT recipients who received the full two-dose ChAdOx1 nCoV-19 vaccine demonstrated significantly diminished HMI and CMI responses compared to immunocompetent individuals. Consideration should be given to administering additional vaccine doses or optimizing immunosuppressant regimens during vaccination (Thai Clinical Trial Registry: TCTR20210523002).

The emergence of the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) has posed a significant global health concern due to its severe respiratory illness and high fatality rate. Currently, despite the potential for resurgence, there are no specific treatments for MERS-CoV, and only supportive care is available. Our study aimed to address this therapeutic gap by developing a potent neutralizing bispecific antibody (bsAb) against MERS-CoV. Initially, we isolated four human monoclonal antibodies (mAbs) that specifically target the MERS-CoV receptor-binding domain (RBD) using phage display technology and an established human antibody library. Among these four selected mAbs, our intensive in vitro functional analyses showed that the MERS-CoV RBD-specific mAb K111.3 exhibited the most potent neutralizing activity against MERS-CoV pseudoviral infection and the molecular interaction between MERS-CoV RBD and human dipeptidyl peptidase 4. Consequently, we engineered a novel bsAb, K207.C, by utilizing K111.3 as the IgG base and fusing it with the single-chain variable fragment of its non-competing pair, K111.1. This engineered bsAb showed significantly enhanced neutralization potential against MERS-CoV compared to its parental mAb. These findings suggest that K207.C may serve as a potential candidate for effective MERS-CoV neutralization, further highlighting the promise of the bsAb dual-targeting approach in MERS-CoV neutralization.

The worldwide spread of SARS-CoV-2 has led to a significant economic and social burden on a global scale. Even though the pandemic has concluded, apprehension remains regarding the emergence of highly transmissible variants capable of evading immunity induced by either vaccination or prior infection. The success of viral penetration is due to the specific amino acid residues of the receptor-binding motif (RBM) involved in viral attachment. This region interacts with the cellular receptor ACE2, triggering a neutralizing antibody (nAb) response. In this study, we evaluated serum immunogenicity from individuals who received either a single dose or a combination of different vaccines against the original SARS-CoV-2 strain and a mutated linear RBM. Despite a modest antibody response to wild-type SARS-CoV-2 RBM, the Omicron variants exhibit four mutations in the RBM (S477N, T478K, E484A, and F486V) that result in even lower antibody titers. The primary immune responses observed were directed toward IgA and IgG. While nAbs typically target the RBD, our investigation has unveiled reduced seroreactivity within the RBD\'s crucial subregion, the RBM. This deficiency may have implications for the generation of protective nAbs. An evaluation of S1WT and S2WT RBM peptides binding to nAbs using microscale thermophoresis revealed a higher affinity (35 nM) for the S2WT sequence (GSTPCNGVEGFNCYF), which includes the FNCY patch. Our findings suggest that the linear RBM of SARS-CoV-2 is not an immunodominant region in vaccinated individuals. Comprehending the intricate dynamics of the humoral response, its interplay with viral evolution, and host genetics is crucial for formulating effective vaccination strategies, targeting not only SARS-CoV-2 but also anticipating potential future coronaviruses.

The spike protein receptor-binding domain (RBD) of SARS-CoV-2 is required for the infection of human cells. It is the main target that elicits neutralizing antibodies and also a major component of diagnostic kits. The large demand for this protein has led to the use of plants as a production platform. However, it is necessary to determine the N-glycan structures of an RBD to investigate its efficacy and functionality as a vaccine candidate or diagnostic reagent. Here, we analyzed the N-glycan profile of the RBD produced in rice callus. Of the two potential N-glycan acceptor sites, we found that one was not utilized and the other contained a mixture of complex-type N-glycans. This differs from the heterogeneous mixture of N-glycans found when an RBD is expressed in other hosts, including Nicotiana benthamiana. By comparing the glycosylation profiles of different hosts, we can select platforms that produce RBDs with the most beneficial N-glycan structures for different applications.