Vaccination against COVID-19 among healthcare workers as a cocoon strategy for people living with HIV.

Introduction: Healthcare professionals working in infectious disease units are often engaged in the care of patients with HIV infection. A cocoon vaccination strategy may protect those who are immunocompromised from a severe course of COVID-19. Methods: The research was conducted between January 2021 and June 2022. The study participants were 450 healthcare workers (HCWs) from the Hospital for Infectious Diseases in Warsaw who were vaccinated against COVID-19 with the BNT162b2 mRNA vaccine (Pfizer-BioNTech) -, thefirst available type of vaccine in Poland. Sera were collected according to the schedule of the study. Statistical analyses were performed with non-parametric tests: Wilcoxon's test was used to compare dependent numerical variables, and Fisher's exact test and the Chi-squared test to compare categorical variables. A p value of <0.05 was considered statistically significant. Results: Among the 450 HCWs working in the Hospital for Infectious Diseases in Warsaw 412 (91,5 %) were vaccinated against COVID-19. In total 170 (41,3 %) vaccinated HCWs were included in the final analysis. Their median age was 51 years [interquartile range (IQR): 41-60 years] and median body mass index (BMI) was 25.10 [IQR: 22.68-29.03]. Most of the cohort consisted of women (n = 137, 80.59 %), with the majority working directly with patients (n = 137, 73.21 %). It was found that as early as 14 days after the second dose of the vaccine, 100 % of the study participants achieved a positive result for SARS CoV-2 S-RBD antibodies. There were 168 subjects who had had a COVID-19 diagnosis before entering study and after vaccination 65 HCWs was diagnosed with COVID-19. Conclusions: Due to the fact that people living with HIV with severe immunodeficiency may have an incomplete immune response to COVID vaccination and be at risk of a severe course of the disease, the cocoon strategy of vaccinating medical personnel may be beneficial for these patients.

A Dynamic and Effective Peptide-Based Strategy for Promptly Addressing Emerging SARS-CoV-2 Variants of Concern.

Genomic surveillance based on sequencing the entire genetic code of SARS-CoV-2 involves monitoring and studying genetic changes and variations in disease-causing organisms such as viruses and bacteria. By tracing the virus, it is possible to prevent epidemic spread in the community, ensuring a 'precision public health' strategy. A peptide-based design was applied to provide an efficacious strategy that is able to counteract any emerging viral variant of concern dynamically and promptly to affect the outcomes of a pandemic at an early stage while waiting for the production of the anti-variant-specific vaccine, which require longer times. The inhibition of the interaction between the receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and one of the cellular receptors (DPP4) that its receptors routinely bind to infect human cells is an intriguing therapeutic approach to prevent the virus from entering human cells. Among the other modalities developed for this purpose, peptides surely offer unique advantages, including ease of synthesis, serum stability, low immunogenicity and toxicity, and small production and distribution chain costs. Here, we obtained a potent new inhibitor based on the rearrangement of a previously identified peptide that has been rationally designed on a cell dipeptidyl peptidase 4 (DPP4) sequence, a ubiquitous membrane protein known to bind the RBD-SPIKE domain of the virus. This novel peptide (named DPP4-derived), conceived as an endogenous "drug", is capable of targeting the latest tested variants with a high affinity, reducing the VSV* DG-Fluc pseudovirus Omicron's infection capacity by up to 14%, as revealed by in vitro testing in human Calu-3 cells. Surface plasmon resonance (SPR) confirmed the binding affinity of the new DPP4-derived peptide with Omicron variant RBD.

Factors Influencing Breast Milk Antibody Titers during the Coronavirus Disease 2019 Pandemic: An Observational Study.

The COVID-19 pandemic has highlighted the role of breastfeeding in providing passive immunity to infants via specific anti-SARS-CoV-2 antibodies in breast milk. We aimed to quantify these antibodies across different lactation stages and identify influencing factors. This prospective study involved mother-child dyads from Innsbruck University Hospital, Austria, with a positive maternal SARS-CoV-2 test during pregnancy or peripartum between 2020 and 2023. We collected breast milk samples at various lactation stages and analyzed anti-Spike S1 receptor-binding domain (S1RBD) immunoglobulins (Ig). Maternal and neonatal data were obtained from interviews and medical records. This study included 140 mothers and 144 neonates. Anti-S1RBD-IgA (72.0%), -IgG (86.0%), and -IgM (41.7%) were highly present in colostrum and decreased as milk matured. Mothers with natural infection and vaccination exhibited higher anti-S1RBD-IgA and -IgG titers in all milk stages. Mothers with moderate to severe infections had higher concentrations of anti-S1RBD-IgA and -IgG in transitional milk and higher anti-S1RBD-IgA and -IgM in mature milk compared to those with mild or asymptomatic infections. Variations in antibody responses were also observed with preterm birth and across different virus waves. This study demonstrates the dynamic nature of breast milk Ig and underscores the importance of breastfeeding during a pandemic.

SARS-CoV-2 inhibitory potential of fish oil-derived 2-pyrone compounds by acquiring linoleic acid binding site on the spike protein.

Traditional medicines have reportedly treated SARS-CoV-2 infection. Substantial evidence shows that fish oil supplements promote human immune function, suggesting they may lessen susceptibility to SARS-CoV-2 infection and suppress viral replication by inducing interferon. Fish oil was subjected to partition chromatography and separated into two compounds (EP01 and DH01). Isolated compounds were purified and characterized using UV, FTIR, NMR, and mass spectrometry to confirm their identity. Molecular docking was studied on the SARS CoV-2 variants of concern; SARS CoV-2 WT (PDB: 6VXX), SARS CoV-2 Alpha variant (PDB: 7LWS), SARS CoV-2 Delta variant (PDB: 7TOU), SARS CoV-2 Gamma variant (PDB: 7V78), SARS CoV-2 Kappa variant (PDB: 7VX9), and SARS CoV-2 Omicron variant (PDB: 7QO7) and TMPRSS2 (PDB: 7Y0E). Further selected protein-ligand complexes were subjected to 100 ns MD simulations to predict their biological potential in the SARS-CoV-2 treatment. In-vitro biological studies were carried out to support in-silico findings. Isolated compounds EP01 and DH01 were identified as 5-Tridecyltetrahydro-2H-pyran-2-one and 5-Heptadecyltetrahydro-2H-pyran-2-one, respectively. The compound EP01 significantly reduced (93.24 %) the viral RNA copy number with an IC50 of ~8.661 µM. EP01 proved to be a potent antiviral by in-vitro method against the SARS-CoV-2 clinical isolate, making it a promising antiviral candidate, with a single dose capable of preventing viral replication.

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.

Humoral immune response against SARS-CoV-2 after adapted COVID-19 vaccine schedules in healthy adults: The IMCOVAS randomized clinical trial.

BACKGROUND: To overcome supply issues of COVID-19 vaccines, this partially single blind, multi-centric, vaccine trial aimed to evaluate humoral immunogenicity using lower vaccine doses, intradermal vaccination, and heterologous vaccine schedules. Also, the immunity after a booster vaccination was assessed. METHODOLOGY: 566 COVID-19-naïve healthy adults were randomized to 1 of 8 treatment arms consisting of combinations of BNT162b2, mRNA-1273, and ChAdOx1-S. Anti-Receptor-Binding Domain immunoglobulin G (RBD IgG) titers, neutralizing antibody titres, and avidity of the anti-RBD IgGs was assessed up to 1 year after study start. RESULTS: Prolonging the interval between vaccinations from 28 to 84 days and the use of a heterologous BNT162b2 + mRNA-1273 vaccination schedule led to a non-inferior immune response, compared to the reference schedule. A low dose of mRNA-1273 was sufficient to induce non-inferior immunity. Non-inferiority could not be demonstrated for intradermal vaccination. For all adapted vaccination schedules, anti-RBD IgG titres measured after a first booster vaccination were non-inferior to their reference schedule. CONCLUSION: This study suggests that reference vaccine schedules can be adapted without jeopardizing the development of an adequate immune response. Immunity after a booster vaccination did not depend on the dose or brand of the booster vaccine, which is relevant for future booster campaigns. The trial is registered in the European Union Clinical Trials Register (number 2021-001993-52) and on clinicaltrials.gov (NCT06189040).

Aluminum hydroxide and immunostimulatory glycolipid adjuvant combination for enhanced COVID-19 subunit vaccine immunogenicity.

Protein-based subunit vaccines like RBD-Fc are promising tools to fight COVID-19. RBD-Fc fuses the receptor-binding domain (RBD) of the SARS-CoV-2 virus spike protein with the Fc region of human IgG1, making it more immunogenic than RBD alone. Earlier work showed that combining RBD-Fc with iNKT cell agonists as adjuvants improved neutralizing antibodies but did not sufficiently enhance T cell responses, a limitation RBD-Fc vaccines share with common adjuvants. Here we demonstrate that aluminum hydroxide combined with α-C-GC, a C-glycoside iNKT cell agonist, significantly improved the RBD-Fc vaccine's induction of RBD-specific T-cell responses. Additionally, aluminum hydroxide with α-GC-CPOEt, a phosphonate diester derivative, synergistically elicited more robust neutralizing antibodies. Remarkably, modifying αGC with phosphate (OPO3H2) or phosphonate (CPO3H2) to potentially enhance aluminum hydroxide interaction did not improve efficacy over unmodified αGC with aluminum hydroxide. These findings underscore the straightforward yet potent potential of this approach in advancing COVID-19 vaccine development and provide insights for iNKT cell-based immunotherapy.

The impact of N-glycans on the immune response of plant-produced SARS-CoV-2 RBD-Fc proteins.

Plant-based manufacturing has the advantage of post-translational modifications. While plant-specific N-glycans have been associated with allergic reactions, their effect on the specific immune response upon vaccination is not yet understood. In this study, we produced an RBD-Fc subunit vaccine in both wildtype (WT) and glycoengineered (∆XF) Nicotiana benthamiana plants. The N-glycan analysis: RBD-Fc carrying the ER retention peptide mainly displayed high mannose. When produced in WT RBD-Fc displayed complex-type (GnGnXF) N-glycans. In contrast, ∆XF plants produced RBD-Fc with humanized complex N-glycans that lack potentially immunogenic xylose and core fucose residues (GnGn). The three recombinant RBD-Fc glycovariants were tested. Immunization with any of the RBD-Fc proteins resulted in a similar titer of anti-RBD antibodies in mice. Likewise, antisera from subunit RBD-Fc vaccines also demonstrated comparable neutralization against SARS-CoV-2. Thus, we conclude that N-glycan modifications of the RBD-Fc protein have no impact on their capacity to activate immune responses and induce neutralizing antibody production.

Protection of K18-hACE2 Mice against SARS-CoV-2 Challenge by a Capsid Virus-like Particle-Based Vaccine.

The SARS-CoV-2 pandemic and the emergence of novel virus variants have had a dramatic impact on public health and the world economy, underscoring the need for detailed studies that explore the high efficacy of additional vaccines in animal models. In this study, we confirm the pathogenicity of the SARS-CoV-2/Leiden_008 isolate (GenBank accession number MT705206.1) in K18-hACE2 transgenic mice. Using this isolate, we show that a vaccine consisting of capsid virus-like particles (cVLPs) displaying the receptor-binding domain (RBD) of SARS-CoV-2 (Wuhan strain) induces strong neutralizing antibody responses and sterilizing immunity in K18-hACE2 mice. Furthermore, we demonstrate that vaccination with the RBD-cVLP vaccine protects mice from both a lethal infection and symptomatic disease. Our data also indicate that immunization significantly reduces inflammation and lung pathology associated with severe disease in mice. Additionally, we show that the survival of naïve animals significantly increases when sera from animals vaccinated with RBD-cVLP are passively transferred, prior to a lethal virus dose. Finally, the RBD-cVLP vaccine has a similar antigen composition to the clinical ABNCOV2 vaccine, which has shown non-inferiority to the Comirnaty mRNA vaccine in phase I-III trials. Therefore, our study provides evidence that this vaccine design is highly immunogenic and confers full protection against severe disease in mice.

Equal Maintenance of Anti-SARS-CoV-2 Antibody Levels Induced by Heterologous and Homologous Regimens of the BNT162b2, ChAdOx1, CoronaVac and Ad26.COV2.S Vaccines: A Longitudinal Study Up to the 4th Dose of Booster.

Several technological approaches have been used to develop vaccines against COVID-19, including those based on inactivated viruses, viral vectors, and mRNA. This study aimed to monitor the maintenance of anti-SARS-CoV-2 antibodies in individuals from Brazil according to the primary vaccination regimen, as follows: BNT162b2 (group 1; 22) and ChAdOx1 (group 2; 18). Everyone received BNT162b2 in the first booster while in the second booster CoronaVac, Ad26.COV2.S, or BNT162b2. Blood samples were collected from 2021 to 2023 to analyze specific RBD (ELISA) and neutralizing antibodies (PRNT50). We observed a progressive increase in anti-RBD and neutralizing antibodies in each subsequent dose, remaining at high titers until the end of follow-up. Group 1 had higher anti-RBD antibody titers than group 2 after beginning the primary regimen, with significant differences after the 2nd and 3rd doses. Group 2 showed a more expressive increase after the first booster with BNT162B2 (heterologous booster). Group 2 also presented high levels of neutralizing antibodies against the Gamma and Delta variants until five months after the second booster. In conclusion, the circulating levels of anti-RBD and neutralizing antibodies against the two variants of SARS-CoV-2 were durable even five months after the 4th dose, suggesting that periodic booster vaccinations (homologous or heterologous) induced long-lasting immunity.

Functional and structural insights into RAS effector proteins.

RAS proteins are conserved guanosine triphosphate (GTP) hydrolases (GTPases) that act as molecular binary switches and play vital roles in numerous cellular processes. Upon GTP binding, RAS GTPases adopt an active conformation and interact with specific proteins termed RAS effectors that contain a conserved ubiquitin-like domain, thereby facilitating downstream signaling. Over 50 effector proteins have been identified in the human proteome, and many have been studied as potential mediators of RAS-dependent signaling pathways. Biochemical and structural analyses have provided mechanistic insights into these effectors, and studies using model organisms have complemented our understanding of their role in physiology and disease. Yet, many critical aspects regarding the dynamics and biological function of RAS-effector complexes remain to be elucidated. In this review, we discuss the mechanisms and functions of known RAS effector proteins, provide structural perspectives on RAS-effector interactions, evaluate their significance in RAS-mediated signaling, and explore their potential as therapeutic targets.

Prevalence and correlates of probable rapid eye movement sleep behavior disorder among middle-aged and older adults in a psychiatric outpatient clinic: A cross-sectional survey.

OBJECTIVE: Rapid eye movement sleep behavior disorder (RBD) is often underdiagnosed among people living with mental disorders. The present study aimed to investigate the prevalence of probable RBD (pRBD) and its associated factors among middle-aged and older adults in a psychiatric outpatient clinic. METHODS: We conducted a cross-sectional survey among 2907 people aged 45-80 years who visited the outpatient clinic between March 1 and August 31, 2022 in a psychiatric hospital. A cutoff score ≥5 on the RBD Screening Questionnaire (RBDSQ) was used to indicate the presence of probable RBD (pRBD). Potential factors associated with pRBD were also assessed with a structured checklist. The association between these factors and the presence of pRBD was examined with logistic regression. RESULTS: The response rate was 64.3 %. Among 1868 respondents [age 58.5 ± 9.6 years, male n = 738 (39.5 %), female n = 1130 (60.5 %)], 15.9 % (95 % CI 14.2-17.6 %) screened positive for pRBD. Occupational exposure to chemicals; positive family history of psychotic disorders; a late start of mental health care; a medical history of autonomic dysfunction; mood problems; and use of antidepressants, hypnotics, and acetylcholinesterase inhibitors were associated with an increased likelihood of having pRBD (P < 0.05 for all). CONCLUSION: pRBD is common among outpatients with mental disorders, especially in mental disorders due to neurological diseases and physical conditions, mood disorders and anxiety or somatoform disorders. The findings highlight the importance of identifying sleep behavior disorders among people living with mental disorders in clinical practice.

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

Background: The 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. Methods: In 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. Results: IgG 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. Conclusions: Taken 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.

A Nasal Vaccine Candidate, Containing Three Antigenic Regions from SARS-CoV-2, to Induce a Broader Response.

A chimeric protein, formed by two fragments of the conserved nucleocapsid (N) and S2 proteins from SARS-CoV-2, was obtained as a recombinant construct in Escherichia coli. The N fragment belongs to the C-terminal domain whereas the S2 fragment spans the fibre structure in the post-fusion conformation of the spike protein. The resultant protein, named S2NDH, was able to form spherical particles of 10 nm, which forms aggregates upon mixture with the CpG ODN-39M. Both preparations were recognized by positive COVID-19 human sera. The S2NDH + ODN-39M formulation administered by the intranasal route resulted highly immunogenic in Balb/c mice. It induced cross-reactive anti-N humoral immunity in both sera and bronchoalveolar fluids, under a Th1 pattern. The cell-mediated immunity (CMI) was also broad, with positive response even against the N protein of SARS-CoV-1. However, neither neutralizing antibodies (NAb) nor CMI against the S2 region were obtained. As alternative, the RBD protein was included in the formulation as inducer of NAb. Upon evaluation in mice by the intranasal route, a clear adjuvant effect was detected for the S2NDH + ODN-39M preparation over RBD. High levels of NAb were induced against SARS-CoV-2 and SARS-CoV-1. The bivalent formulation S2NDH + ODN-39M + RBD, administered by the intranasal route, constitutes an attractive proposal as booster vaccine of sarbecovirus scope.

COVID-19 Vaccination in Pregnancy: Pilot Study of Plasma MicroRNAs Associated with Inflammatory Cytokines after COVID-19 mRNA Vaccination.

BACKGROUND: The impact of mRNA COVID-19 vaccines on the immunological profiles of pregnant women remains a crucial area of study. This research aims to explore the specific immunological changes triggered by these vaccines in this demographic. METHODS: In a focused investigation, we examined the effects of mRNA COVID-19 vaccination on microRNA expression in pregnant women. Key microRNAs, including miR-451a, miR-23a-3p, and miR-21-5p, were analyzed for expression changes post-vaccination. Additionally, we assessed variations in S1RBD IgG levels and specific cytokines to gauge the broader immunological response. RESULTS: Post-vaccination, significant expression shifts in the targeted microRNAs were observed. Alongside these changes, we noted alterations in S1RBD IgG and various cytokines, indicating an adapted inflammatory response. Notably, these immunological markers displayed no direct correlation with S1RBD IgG concentrations, suggesting a complex interaction between the vaccine and the immune system in pregnant women. CONCLUSIONS: Our pilot study provides valuable insights into the nuanced effects of the mRNA COVID-19 vaccine on immune dynamics in pregnant women, particularly emphasizing the role of microRNAs. The findings illuminate the intricate interplay between vaccines, microRNAs, and immune responses, enhancing our understanding of these relationships in the context of pregnancy. This research contributes significantly to the growing body of knowledge regarding mRNA COVID-19 vaccines and their specific impact on maternal immunology, offering a foundation for further studies in this vital area.

Development of Broad-Spectrum Nanobodies for the Therapy and Diagnosis of SARS-CoV-2 and Its Multiple Variants.

The continuous evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evaded the efficacy of previously developed antibodies and vaccines, thus remaining a significant global public health threat. Therefore, it is imperative to develop additional antibodies that are capable of neutralizing emerging variants. Nanobodies, as the smallest functional single-domain antibodies, exhibit enhanced stability and penetration ability, enabling them to recognize numerous concealed epitopes that are inaccessible to conventional antibodies. Herein, we constructed an immune library based on the immunization of alpaca with the S1 subunit of the SARS-CoV-2 spike protein, from which two nanobodies, Nb1 and Nb2, were selected using phage display technology for further characterization. Both nanobodies, with the binding residues residing within the receptor-binding domain (RBD) region of the spike, exhibited high affinity toward the S1 subunit. Moreover, they displayed cross-neutralizing activity against both wild-type SARS-CoV-2 and 10 ο variants, including BA.1, BA.2, BA.3, BA.5, BA.2.75, BF.7, BQ.1, EG.5.1, XBB.1.5, and JN.1. Molecular modeling and dynamics simulations predicted that both nanobodies interacted with the viral RBD through their complementarity determining region 1 (CDR1) and CDR2. These two nanobodies are novel tools for the development of therapeutic and diagnostic countermeasures targeting SARS-CoV-2 variants and potentially emerging coronaviruses.

Genetic characterization of the first Deltacoronavirus from wild birds around Qinghai Lake.

Deltacoronavirus, widely distributed among pigs and wild birds, pose a significant risk of cross-species transmission, including potential human epidemics. Metagenomic analysis of bird samples from Qinghai Lake, China in 2021 reported the presence of Deltacoronavirus. A specific gene fragment of Deltacoronavirus was detected in fecal samples from wild birds at a positive rate of 5.94% (6/101). Next-generation sequencing (NGS) identified a novel Deltacoronavirus strain, which was closely related to isolates from the United Arab Emirates (2018), China (2022), and Poland (2023). Subsequently the strain was named A/black-headed gull/Qinghai/2021(BHG-QH-2021) upon confirmation of the Cytochrome b gene of black-headed gull in the sample. All available genome sequences of avian Deltacoronavirus, including the newly identified BHG-QH-2021 and 5 representative strains of porcine Deltacoronavirus (PDCoV), were classified according to ICTV criteria. In contrast to Coronavirus HKU15, which infects both mammals and birds and shows the possibility of cross-species transmission from bird to mammal host, our analysis revealed that BHG-QH-2021 is classified as Putative species 4. Putative species 4 has been reported to infect 5 species of birds but not mammals, suggesting that cross-species transmission of Putative species 4 is more prevalent among birds. Recombination analysis traced BHG-QH-2021 origin to dut148cor1 and MW01_1o strains, with MW01_1o contributing the S gene. Surprisingly, SwissModle prediction showed that the optimal template for receptor-binding domain (RBD) of BHG-QH-2021 is derived from the human coronavirus 229E, a member of the Alphacoronavirus, rather than the anticipated RBD structure of PDCoV of Deltacoronavirus. Further molecular docking analysis revealed that substituting the loop 1-2 segments of HCoV-229E significantly enhanced the binding capability of BHG-QH-2021 with human Aminopeptidase N (hAPN), surpassing its native receptor-binding domain (RBD). Most importantly, this finding was further confirmed by co-immunoprecipitation experiment that loop 1-2 segments of HCoV-229E enable BHG-QH-2021 RBD binding to hAPN, indicating that the loop 1-2 segment of the RBD in Putative species 4 is a probable key determinant for the virus ability to spill over into humans. Our results summarize the phylogenetic relationships among known Deltacoronavirus, reveal an independent putative avian Deltacoronavirus species with inter-continental and inter-species transmission potential, and underscore the importance of continuous surveillance of wildlife Deltacoronavirus.

Impact of mutations in SARS-CoV-2 recombinant sub-variant XBB.1.16 on the binding affinity with human ACE2 receptor.

Despite the waning threat of the COVID-19 pandemic, its detrimental impact on global health persists. Regardless of natural immunity or immunity obtained through vaccination, emerging variants of the virus continue to undergo mutations and propagate globally. The persistent mutations in SARS-CoV-2, along with the subsequent formation of recombinant sub-variants has become a challenge for researchers and health professionals, raising concerns about the efficacy of current vaccines. Gaining a better understanding of the biochemical interactions between the Spike Protein (RBD) of SARS-CoV-2 variants and the human ACE2 receptor can prove to be beneficial in designing and developing antiviral therapeutics that are equally effective against all strains and emerging variants. Our objective in this study was to investigate the interfacial binding pattern of the SARS-CoV-2 RBD-ACE2 complex of the Wild Type (WT), Omicron, and the Omicron recombinant sub-variant XBB.1.16. We aimed to examine the atomic level factors and observe how mutations influence the interaction between the virus and its host using Molecular Dynamics simulation, MM/GBSA energy calculations, and Principal Component Analysis. Our findings reveal a higher degree of structural deviation and flexibility in XBB.1.16 compared to WT and Omicron. PCA indicated a wider cluster and significant flexibility in the movements of XBB.1.16 which can also be observed in free energy landscapes, while the normal mode analysis revealed converging motions within the RBD-ACE2 complexes which can facilitate the interaction between them. A pattern of decreased binding affinity was observed in case of XBB.1.16 when compared to the WT and Omicron. These observed deviations in XBB.1.16 when compared to its parent lineage Omicron, and WT can be attributed to the mutations specific to it. Collectively, these results enhance our understanding of the impact of mutations on the interaction between this strain and the host, taking us one step closer to designing effective antiviral therapeutics against the continually mutating strains.

Molecular basis for receptor recognition and broad host tropism for merbecovirus MjHKU4r-CoV-1.

A novel pangolin-origin MERS-like coronavirus (CoV), MjHKU4r-CoV-1, was recently identified. It is closely related to bat HKU4-CoV, and is infectious in human organs and transgenic mice. MjHKU4r-CoV-1 uses the dipeptidyl peptidase 4 (DPP4 or CD26) receptor for virus entry and has a broad host tropism. However, the molecular mechanism of its receptor binding and determinants of host range are not yet clear. Herein, we determine the structure of the MjHKU4r-CoV-1 spike (S) protein receptor-binding domain (RBD) complexed with human CD26 (hCD26) to reveal the basis for its receptor binding. Measuring binding capacity toward multiple animal receptors for MjHKU4r-CoV-1, mutagenesis analyses, and homology modeling highlight that residue sites 291, 292, 294, 295, 336, and 344 of CD26 are the crucial host range determinants for MjHKU4r-CoV-1. These results broaden our understanding of this potentially high-risk virus and will help us prepare for possible outbreaks in the future.

Role of N343 glycosylation on the SARS-CoV-2 S RBD structure and co-receptor binding across variants of concern.

Glycosylation of the SARS-CoV-2 spike (S) protein represents a key target for viral evolution because it affects both viral evasion and fitness. Successful variations in the glycan shield are difficult to achieve though, as protein glycosylation is also critical to folding and structural stability. Within this framework, the identification of glycosylation sites that are structurally dispensable can provide insight into the evolutionary mechanisms of the shield and inform immune surveillance. In this work, we show through over 45 µs of cumulative sampling from conventional and enhanced molecular dynamics (MD) simulations, how the structure of the immunodominant S receptor binding domain (RBD) is regulated by N-glycosylation at N343 and how this glycan's structural role changes from WHu-1, alpha (B.1.1.7), and beta (B.1.351), to the delta (B.1.617.2), and omicron (BA.1 and BA.2.86) variants. More specifically, we find that the amphipathic nature of the N-glycan is instrumental to preserve the structural integrity of the RBD hydrophobic core and that loss of glycosylation at N343 triggers a specific and consistent conformational change. We show how this change allosterically regulates the conformation of the receptor binding motif (RBM) in the WHu-1, alpha, and beta RBDs, but not in the delta and omicron variants, due to mutations that reinforce the RBD architecture. In support of these findings, we show that the binding of the RBD to monosialylated ganglioside co-receptors is highly dependent on N343 glycosylation in the WHu-1, but not in the delta RBD, and that affinity changes significantly across VoCs. Ultimately, the molecular and functional insight we provide in this work reinforces our understanding of the role of glycosylation in protein structure and function and it also allows us to identify the structural constraints within which the glycosylation site at N343 can become a hotspot for mutations in the SARS-CoV-2 S glycan shield.