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.

Hybrid response to SARS-CoV-2 and Neisseria meningitidis C after an OMV-adjuvanted immunization in mice and their offspring.

COVID-19, caused by SARS-CoV-2, and meningococcal disease, caused by Neisseria meningitidis, are relevant infectious diseases, preventable through vaccination. Outer membrane vesicles (OMVs), released from Gram-negative bacteria, such as N. meningitidis, present adjuvant characteristics and may confer protection against meningococcal disease. Here, we evaluated in mice the humoral and cellular immune response to different doses of receptor binding domain (RBD) of SARS-CoV-2 adjuvanted by N. meningitidis C:2a:P1.5 OMVs and aluminum hydroxide, as a combined preparation for these pathogens. The immunization induced IgG antibodies of high avidity for RBD and OMVs, besides IgG that recognized the Omicron BA.2 variant of SARS-CoV-2 with intermediary avidity. Cellular immunity showed IFN-γ and IL-4 secretion in response to RBD and OMV stimuli, demonstrating immunologic memory and a mixed Th1/Th2 response. Offspring presented transferred IgG of similar levels and avidity as their mothers. Humoral immunity did not point to the superiority of any RBD dose, but the group immunized with a lower antigenic dose (0.5 µg) had the better cellular response. Overall, OMVs enhanced RBD immunogenicity and conferred an immune response directed to N. meningitidis too.

Disposable graphene-oxide screen-printed electrode integrated with portable device for detection of SARS-CoV-2 in clinical samples.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnosis is the need of the hour, as cases are persistently increasing, and new variants are constantly emerging. The ever-changing nature of the virus leading to multiple variants, has brought an imminent need for early, accurate and rapid detection methods. Herein, we have reported the design and fabrication of Screen-Printed Electrodes (SPEs) with graphene oxide (GO) as working electrode and modified with specific antibodies for SARS-CoV-2 Receptor Binding Domain (RBD). Flexibility of design, and portable nature has made SPEs the superior choice for electrochemical analysis. The developed immunosensor can detect RBD as low as 0.83 fM with long-term storage capacity. The fabricated SPEs immunosensor was tested using a miniaturized portable device and potentiostat on 100 patient nasopharyngeal samples and corroborated with RT-PCR data, displayed 94 % sensitivity. Additionally, the in-house developed polyclonal antibodies detected RBD antigen of the mutated Omicron variant of SARS-CoV-2 successfully. We have not observed any cross-reactivity/binding of the fabricated immunosensor with MERS (cross-reactive antigen) and Influenza A H1N1 (antigen sharing common symptoms). Hence, the developed SPEs sensor may be applied for bedside point-of-care diagnosis of SARS-CoV-2 using miniaturized portable device, in clinical samples.

Engineered Multivalent Nanobodies Efficiently Neutralize SARS-CoV-2 Omicron Subvariants BA.1, BA.4/5, XBB.1 and BQ.1.1.

Most available neutralizing antibodies are ineffective against highly mutated SARS-CoV-2 Omicron subvariants. Therefore, it is crucial to develop potent and broad-spectrum alternatives to effectively manage Omicron subvariants. Here, we constructed a high-diversity nanobody phage display library and identified nine nanobodies specific to the SARS-CoV-2 receptor-binding domain (RBD). Five of them exhibited cross-neutralization activity against the SARS-CoV-2 wild-type (WT) strain and the Omicron subvariants BA.1 and BA.4/5, and one nanobody demonstrated marked efficacy even against the Omicron subvariants BQ.1.1 and XBB.1. To enhance the therapeutic potential, we engineered a panel of multivalent nanobodies with increased neutralizing potency and breadth. The most potent multivalent nanobody, B13-B13-B13, cross-neutralized all tested pseudoviruses, with a geometric mean of the 50% inhibitory concentration (GM IC50) value of 20.83 ng/mL. An analysis of the mechanism underlying the enhancement of neutralization breadth by representative multivalent nanobodies demonstrated that the strategic engineering approach of combining two or three nanobodies into a multivalent molecule could improve the affinity between a single nanobody and spike, and could enhance tolerance toward escape mutations such as R346T and N460K. Our engineered multivalent nanobodies may be promising drug candidates for treating and preventing infection with Omicron subvariants and even future variants.

Molecular basis of hippopotamus ACE2 binding to SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a wide range of hosts, including hippopotami, which are semi-aquatic mammals and phylogenetically closely related to Cetacea. In this study, we characterized the binding properties of hippopotamus angiotensin-converting enzyme 2 (hiACE2) to the spike (S) protein receptor binding domains (RBDs) of the SARS-CoV-2 prototype (PT) and variants of concern (VOCs). Furthermore, the cryo-electron microscopy (cryo-EM) structure of the SARS-CoV-2 PT S protein complexed with hiACE2 was resolved. Structural and mutational analyses revealed that L30 and F83, which are specific to hiACE2, played a crucial role in the hiACE2/SARS-CoV-2 RBD interaction. In addition, comparative and structural analysis of ACE2 orthologs suggested that the cetaceans may have the potential to be infected by SARS-CoV-2. These results provide crucial molecular insights into the susceptibility of hippopotami to SARS-CoV-2 and suggest the potential risk of SARS-CoV-2 VOCs spillover and the necessity for surveillance. IMPORTANCE: The hippopotami are the first semi-aquatic artiodactyl mammals wherein SARS-CoV-2 infection has been reported. Exploration of the invasion mechanism of SARS-CoV-2 will provide important information for the surveillance of SARS-CoV-2 in hippopotami, as well as other semi-aquatic mammals and cetaceans. Here, we found that hippopotamus ACE2 (hiACE2) could efficiently bind to the RBDs of the SARS-CoV-2 prototype (PT) and variants of concern (VOCs) and facilitate the transduction of SARS-CoV-2 PT and VOCs pseudoviruses into hiACE2-expressing cells. The cryo-EM structure of the SARS-CoV-2 PT S protein complexed with hiACE2 elucidated a few critical residues in the RBD/hiACE2 interface, especially L30 and F83 of hiACE2 which are unique to hiACE2 and contributed to the decreased binding affinity to PT RBD compared to human ACE2. Our work provides insight into cross-species transmission and highlights the necessity for monitoring host jumps and spillover events on SARS-CoV-2 in semi-aquatic/aquatic mammals.

Specific features of epitope-MIPs and whole-protein MIPs as illustrated for AFP and RBD of SARS-CoV-2.

Molecularly imprinted polymer (MIP) nanofilms for alpha-fetoprotein (AFP) and the receptor binding domain (RBD) of the spike protein of SARS-CoV-2 using either a peptide (epitope-MIP) or the whole protein (protein-MIP) as the template were prepared by electropolymerization of scopoletin. Conducting atomic force microscopy revealed after template removal and electrochemical deposition of gold a larger surface density of imprinted cavities for the epitope-imprinted polymers than when using the whole protein as template. However, comparable affinities towards the respective target protein (AFP and RBD) were obtained for both types of MIPs as expressed by the KD values in the lower nanomolar range. On the other hand, while the cross reactivity of both protein-MIPs towards human serum albumin (HSA) amounts to around 50% in the saturation region, the nonspecific binding to the respective epitope-MIPs is as low as that for the non-imprinted polymer (NIP). This effect might be caused by the different sizes of the imprinted cavities. Thus, in addition to the lower costs the reduced nonspecific binding is an advantage of epitope-imprinted polymers for the recognition of proteins.

Efficient Expression in Leishmania tarentolae (LEXSY) of the Receptor-Binding Domain of the SARS-CoV-2 S-Protein and the Acetylcholine-Binding Protein from Lymnaea stagnalis.

Leishmania tarentolae (LEXSY) system is an inexpensive and effective expression approach for various research and medical purposes. The stated advantages of this system are the possibility of obtaining the soluble product in the cytoplasm, a high probability of correct protein folding with a full range of post-translational modifications (including uniform glycosylation), and the possibility of expressing multi-subunit proteins. In this paper, a LEXSY expression system has been employed for obtaining the receptor binding domain (RBD) of the spike-protein of the SARS-CoV-2 virus and the homopentameric acetylcholine-binding protein (AChBP) from Lymnaea stagnalis. RBD is actively used to obtain antibodies against the virus and in various scientific studies on the molecular mechanisms of the interaction of the virus with host cell targets. AChBP represents an excellent structural model of the ligand-binding extracellular domain of all subtypes of nicotinic acetylcholine receptors (nAChRs). Both products were obtained in a soluble glycosylated form, and their structural and functional characteristics were compared with those previously described.

Functional dissection of the spike glycoprotein S1 subunit and identification of cellular cofactors for regulation of swine acute diarrhea syndrome coronavirus entry.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a novel porcine enteric coronavirus, and the broad interspecies infection of SADS-CoV poses a potential threat to human health. This study provides experimental evidence to dissect the roles of distinct domains within the SADS-CoV spike S1 subunit in cellular entry. Specifically, we expressed the S1 and its subdomains, S1A and S1B. Cell binding and invasion inhibition assays revealed a preference for the S1B subdomain in binding to the receptors on the cell surface, and this unknown receptor is not utilized by the porcine epidemic diarrhea virus. Nanoparticle display demonstrated hemagglutination of erythrocytes from pigs, humans, and mice, linking the S1A subdomain to the binding of sialic acid (Sia) involved in virus attachment. We successfully rescued GFP-labeled SADS-CoV (rSADS-GFP) from a recombinant cDNA clone to track viral infection. Antisera raised against S1, S1A, or S1B contained highly potent neutralizing antibodies, with anti-S1B showing better efficiency in neutralizing rSADS-GFP infection compared to anti-S1A. Furthermore, depletion of heparan sulfate (HS) by heparinase treatment or pre-incubation of rSADS-GFP with HS or constituent monosaccharides could inhibit SADS-CoV entry. Finally, we demonstrated that active furin cleavage of S glycoprotein and the presence of type II transmembrane serine protease (TMPRSS2) are essential for SADS-CoV infection. These combined observations suggest that the wide cell tropism of SADS-CoV may be related to the distribution of Sia or HS on the cell surface, whereas the S1B contains the main protein receptor binding site. Specific host proteases also play important roles in facilitating SADS-CoV entry.IMPORTANCESwine acute diarrhea syndrome coronavirus (SADS-CoV) is a novel pathogen infecting piglet, and its unique genetic evolution characteristics and broad species tropism suggest the potential for cross-species transmission. The virus enters cells through its spike (S) glycoprotein. In this study, we identify the receptor binding domain on the C-terminal part of the S1 subunit (S1B) of SADS-CoV, whereas the sugar-binding domain located at the S1 N-terminal part of S1 (S1A). Sialic acid, heparan sulfate, and specific host proteases play essential roles in viral attachment and entry. The dissection of SADS-CoV S1 subunit's functional domains and identification of cellular entry cofactors will help to explore the receptors used by SADS-CoV, which may contribute to exploring the mechanisms behind cross-species transmission and host tropism.

A Biosensor Assay Based on Coiled-Coil-Mediated Human ACE2 Receptor Capture for the Analysis of Its Interactions with the SARS-CoV-2 Receptor Binding Domain.

Surface plasmon resonance (SPR)-based biosensing enables the characterization of protein-protein interactions. Several SPR-based approaches have been designed to evaluate the binding mechanism between the angiotensin-converting enzyme 2 (ACE2) receptor and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein leading to a large range of kinetic and thermodynamic constants. This chapter describes a robust SPR assay based on the K5/E5 coiled-coil capture strategy that reduces artifacts. In this method, ACE2 receptors were produced with an E5-tag and immobilized as ligands in the SPR assay. This chapter details methods for high-yield production and purification of the studied proteins, functionalization of the sensor chip, conduction of the SPR assay, and data analysis.

tANCHOR cell-based ELISA approach as a surrogate for antigen-coated plates to monitor specific IgG directed to the SARS-CoV-2 receptor-binding domain.

Enzyme-linked immunosorbent assay (ELISA) systems use plates coated with peptides or expressed and purified proteins to monitor immunoglobulins derived from patient serum. However, there is currently no easy, flexible, and fast adaptive ELISA-based system for testing antibodies directed against new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. In this study, we utilized the tANCHOR protein display system that provides a cell surface decorated with the receptor-binding domain (RBD) to monitor specific antibodies derived from SARS-CoV-2 convalescent and vaccinated individuals directed against it. To test sera from vaccinees or convalescent individuals, only the RBD coding sequence needs to be cloned in the tANCHOR vector system and transfected into HeLa cells. Time-consuming protein expression, isolation, and purification followed by coating assay plates are not necessary. With this technique, the immune evasion of new SARS-CoV-2 variants from current vaccination regimes can be examined quickly and reliably.

Caveats of chimpanzee ChAdOx1 adenovirus-vectored vaccines to boost anti-SARS-CoV-2 protective immunity in mice.

Several COVID-19 vaccines use adenovirus vectors to deliver the SARS-CoV-2 spike (S) protein. Immunization with these vaccines promotes immunity against the S protein, but against also the adenovirus itself. This could interfere with the entry of the vaccine into the cell, reducing its efficacy. Herein, we evaluate the efficiency of an adenovirus-vectored vaccine (chimpanzee ChAdOx1 adenovirus, AZD1222) in boosting the specific immunity compared to that induced by a recombinant receptor-binding domain (RBD)-based vaccine without viral vector. Mice immunized with the AZD1222 human vaccine were given a booster 6 months later, with either the homologous vaccine or a recombinant vaccine based on RBD of the delta variant, which was prevalent at the start of this study. A significant increase in anti-RBD antibody levels was observed in rRBD-boosted mice (31-61%) compared to those receiving two doses of AZD1222 (0%). Significantly higher rates of PepMix™- or RBD-elicited proliferation were also observed in IFNγ-producing CD4 and CD8 cells from mice boosted with one or two doses of RBD, respectively. The lower efficiency of the ChAdOx1-S vaccine in boosting specific immunity could be the result of a pre-existing anti-vector immunity, induced by increased levels of anti-adenovirus antibodies found both in mice and humans. Taken together, these results point to the importance of avoiding the recurrent use of the same adenovirus vector in individuals with immunity and memory against them. It also illustrates the disadvantages of ChAdOx1 adenovirus-vectored vaccine with respect to recombinant protein vaccines, which can be used without restriction in vaccine-booster programs. KEY POINTS: • ChAdOx1 adenovirus vaccine (AZD1222) may not be effective in boosting anti-SARS-CoV-2 immunity • A recombinant RBD protein vaccine is effective in boosting anti-SARS-CoV-2 immunity in mice • Antibodies elicited by the rRBD-delta vaccine persisted for up to 3 months in mice.

Inhibition potential of natural flavonoids against selected omicron (B.1.19) mutations in the spike receptor binding domain of SARS-CoV-2: a molecular modeling approach.

The omicron (B.1.19) variant of contagious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is considered a variant of concern (VOC) due to its increased transmissibility and highly infectious nature. The spike receptor-binding domain (RBD) is a hotspot of mutations and is regarded as a prominent target for screening drug candidates owing to its crucial role in viral entry and immune evasion. To date, no effective therapy or antivirals have been reported; therefore, there is an urgent need for rapid screening of antivirals. An extensive molecular modelling study has been performed with the primary goal to assess the inhibition potential of natural flavonoids as inhibitors against RBD from a manually curated library. Out of 40 natural flavonoids, five natural flavonoids, namely tomentin A (-8.7 kcal/mol), tomentin C (-8.6 kcal/mol), hyperoside (-8.4 kcal/mol), catechin gallate (-8.3 kcal/mol), and corylifol A (-8.2 kcal/mol), have been considered as the top-ranked compounds based on their binding affinity and molecular interaction profiling. The state-of-the-art molecular dynamics (MD) simulations of these top-ranked compounds in complex with RBD exhibited stable dynamics and structural compactness patterns on 200 nanoseconds. Additionally, complexes of these molecules demonstrated favorable free binding energies and affirmed the docking and simulation results. Moreover, the post-simulation validation of these interacted flavonoids using principal component analysis (PCA) revealed stable interaction patterns with RBD. The integrated results suggest that tomentin A, tomentin C, hyperoside, catechin gallate, and corylifol A might be effective against the emerging variants of SARS-CoV-2 and should be further evaluated using in-vitro and in-vivo experiments.Communicated by Ramaswamy H. Sarma.

A CTB-SARS-CoV-2-ACE-2 RBD Mucosal Vaccine Protects Against Coronavirus Infection.

Mucosal vaccines protect against respiratory virus infection by stimulating the production of IgA antibodies that protect against virus invasion of the mucosal epithelium. In this study, a novel protein subunit mucosal vaccine was constructed for protection against infection by the beta coronavirus SARS-CoV-2. The vaccine was assembled by linking a gene encoding the SARS-CoV-2 virus S1 angiotensin converting enzyme receptor binding domain (ACE-2-RBD) downstream from a DNA fragment encoding the cholera toxin B subunit (CTB), a mucosal adjuvant known to stimulate vaccine immunogenicity. A 42 kDa vaccine fusion protein was identified in homogenates of transformed E. coli BL-21 cells by acrylamide gel electrophoresis and by immunoblotting against anti-CTB and anti-ACE-2-RBD primary antibodies. The chimeric CTB-SARS-CoV-2-ACE-2-RBD vaccine fusion protein was partially purified from clarified bacterial homogenates by nickel affinity column chromatography. Further vaccine purification was accomplished by polyacrylamide gel electrophoresis and electro-elution of the 42 kDa chimeric vaccine protein. Vaccine protection against SARS-CoV-2 infection was assessed by oral, nasal, and parenteral immunization of BALB/c mice with the CTB-SARS-CoV-2-ACE-2-RBD protein. Vaccine-induced SARS-CoV-2 specific antibodies were quantified in immunized mouse serum by ELISA analysis. Serum from immunized mice contained IgG and IgA antibodies that neutralized SARS-CoV-2 infection in Vero E6 cell cultures. In contrast to unimmunized mice, cytological examination of cell necrosis in lung tissues excised from immunized mice revealed no detectable cellular abnormalities. Mouse behavior following vaccine immunization remained normal throughout the duration of the experiments. Together, our data show that a CTB-adjuvant-stimulated CTB-SARS-CoV-2-ACE-2-RBD chimeric mucosal vaccine protein synthesized in bacteria can produce durable and persistent IgA antibodies in mice that neutralize the SARS-CoV-2 subvariant Omicron BA.1.1.

Classification of five SARS-CoV-2 serotypes based on RBD antigenicities.

The continuous evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in a significant number of variants, particularly with the emergence of Omicron with many sub-variants. These variants have exhibited increased immune escape, leading to reduced efficacy of existing vaccines and therapeutic antibodies. Given the diminished cross-neutralization observed among these variants, it is plausible that SARS-CoV-2 has developed multiple serotypes. As the major antigenic site, the receptor-binding domain (RBD) of viral spike (S) protein was chosen for serotyping. We selected 23 representative variants, including pre-Omicron variants and Omicron sub-variants, and classified them into five serotypes based on systematic evaluation of the antigenicities of their RBDs. Each serotype includes several genetically distinct variants. Serotype-I encompasses all pre-Omicron variants (with two subtypes), while the remaining four serotypes are all comprised of Omicron sub-variants at different stages of evolution. We propose that these serotypes can serve as a foundation for rapid classification of newly emerging SARS-CoV-2 variants, and guide the development of future broad-spectrum vaccines and neutralizing antibodies against the coronavirus disease 2019 (COVID-19).

A bivalent form of a RBD-specific synthetic antibody effectively neutralizes SARS-CoV-2 variants.

Coronavirus Disease 2019 (COVID-19) pandemic is severely impacting the world, and tremendous efforts have been made to deal with it. Despite many advances in vaccines and therapeutics, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants remains an intractable challenge. We present a bivalent Receptor Binding Domain (RBD)-specific synthetic antibody, specific for the RBD of wild-type (lineage A), developed from a non-antibody protein scaffold composed of LRR (Leucine-rich repeat) modules through phage display. We further reinforced the unique feature of the synthetic antibody by constructing a tandem dimeric form. The resulting bivalent form showed a broader neutralizing activity against the variants. The in vivo neutralizing efficacy of the bivalent synthetic antibody was confirmed using a human ACE2-expressing mouse model that significantly alleviated viral titer and lung infection. The present approach can be used to develop a synthetic antibody showing a broader neutralizing activity against a multitude of SARS-CoV-2 variants.

Dynamics of Serum-Neutralizing Antibody Responses in Vaccinees through Multiple Doses of the BNT162b2 Vaccine.

SARS-CoV-2 mRNA vaccines are administered as effective prophylactic measures for reducing virus transmission rates and disease severity. To enhance the durability of post-vaccination immunity and combat SARS-CoV-2 variants, boosters have been administered to two-dose vaccinees. However, long-term humoral responses following booster vaccination are not well characterized. A 16-member cohort of healthy SARS-CoV-2 naïve participants were enrolled in this study during a three-dose BNT162b2 vaccine series. Serum samples were collected from vaccinees over 420 days and screened for antigen (Ag)-specific antibody titers, IgG subclass distribution, and neutralizing antibody (nAb) responses. Vaccine boosting restored peak Ag-specific titers with sustained α-RBD IgG and IgA antibody responses when measured at six months post-boost. RBD- and spike-specific IgG4 antibody levels were markedly elevated in three-dose but not two-dose immune sera. Although strong neutralization responses were detected in two- and three-dose vaccine sera, these rapidly decayed to pre-immune levels by four and six months, respectively. While boosters enhanced serum IgG Ab reactivity and nAb responses against variant strains, all variants tested showed resistance to two- and three-dose immune sera. Our data reflect the poor durability of vaccine-induced nAb responses which are a strong predictor of protection from symptomatic SARS-CoV-2 infection. The induction of IgG4-switched humoral responses may permit extended viral persistence via the downregulation of Fc-mediated effector functions.

Structural insights into ACE2 interactions and immune activation of SARS-CoV-2 and its variants: an in-silico study.

The initial interaction between COVID-19 and the human body involves the receptor-binding domain (RBD) of the viral spike protein with the angiotensin-converting enzyme 2 (ACE2) receptor. Likewise, the spike protein can engage with immune-related proteins, such as toll-like receptors (TLRs) and pulmonary surfactant proteins A (SP-A) and D (SP-D), thereby triggering immune responses. In this study, we utilize computational methods to investigate the interactions between the spike protein and TLRs (specifically TLR2 and TLR4), as well as (SP-A) and (SP-D). The study is conducted on four variants of concern (VOC) to differentiate and identify common virus behaviours. An assessment of the structural stability of various variants indicates slight changes attributed to mutations, yet overall structural integrity remains preserved. Our findings reveal the spike protein's ability to bind with TLR4 and TLR2, prompting immune activation. In addition, our in-silico results reveal almost similar docking scores and therefore affinity for both ACE2-spike and TLR4-spike complexes. We demonstrate that even minor changes due to mutations in all variants, surfactant A and D proteins can function as inhibitors against the spike in all variants, hindering the ACE2-RBD interaction.Communicated by Ramaswamy H. Sarma.

Large-Scale Purification and Characterization of Recombinant Receptor-Binding Domain (RBD) of SARS-CoV-2 Spike Protein Expressed in Yeast.

SARS-CoV-2 spike protein is an essential component of numerous protein-based vaccines for COVID-19. The receptor-binding domain of this spike protein is a promising antigen with ease of expression in microbial hosts and scalability at comparatively low production costs. This study describes the production, purification, and characterization of RBD of SARS-CoV-2 protein, which is currently in clinical trials, from a commercialization perspective. The protein was expressed in Pichia pastoris in a large-scale bioreactor of 1200 L capacity. Protein capture and purification are conducted through mixed-mode chromatography followed by hydrophobic interaction chromatography. This two-step purification process produced RBD with an overall productivity of ~21 mg/L at >99% purity. The protein's primary, secondary, and tertiary structures were also verified using LCMS-based peptide mapping, circular dichroism, and fluorescence spectroscopy, respectively. The glycoprotein was further characterized for quality attributes such as glycosylation, molecular weight, purity, di-sulfide bonding, etc. Through structural analysis, it was confirmed that the product maintained a consistent quality across different batches during the large-scale production process. The binding capacity of RBD of spike protein was also assessed using human angiotensin-converting enzyme 2 receptor. A low binding constant range of KD values, ranging between 3.63 × 10-8 to 6.67 × 10-8, demonstrated a high affinity for the ACE2 receptor, revealing this protein as a promising candidate to prevent the entry of COVID-19 virus.

Distinct anti-NP, anti-RBD and anti-Spike antibody profiles discriminate death from survival in COVID-19.

Introduction: Infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces rapid production of IgM, IgA, and IgG antibodies directed to multiple viral antigens that may have impact diverse clinical outcomes. Methods: We evaluated IgM, IgA, and IgG antibodies directed to the nucleocapsid (NP), IgA and IgG to the Spike protein and to the receptor-binding domain (RBD), and the presence of neutralizing antibodies (nAb), in a cohort of unvaccinated SARS-CoV-2 infected individuals, in the first 30 days of post-symptom onset (PSO) (T1). Results: This study included 193 coronavirus disease 2019 (COVID-19) participants classified as mild, moderate, severe, critical, and fatal and 27 uninfected controls. In T1, we identified differential antibody profiles associated with distinct clinical presentation. The mild group presented lower levels of anti-NP IgG, and IgA (vs moderate and severe), anti-NP IgM (vs severe, critical and fatal), anti-Spike IgA (vs severe and fatal), and anti-RBD IgG (vs severe). The moderate group presented higher levels of anti-RBD IgA, comparing with severe group. The severe group presented higher levels of anti-NP IgA (vs mild and fatal) and anti-RBD IgG (vs mild and moderate). The fatal group presented higher levels of anti-NP IgM and anti-Spike IgA (vs mild), but lower levels of anti-NP IgA (vs severe). The levels of nAb was lower just in mild group compared to severe, critical, and fatal groups, moreover, no difference was observed among the more severe groups. In addition, we studied 82 convalescent individuals, between 31 days to 6 months (T2) or more than 6 months (T3), PSO, those: 12 mild, 26 moderate, and 46 severe plus critical. The longitudinal analyzes, for the severe plus critical group showed lower levels of anti-NP IgG, IgA and IgM, anti-Spike IgA in relation T3. The follow-up in the fatal group, reveals that the levels of anti-spike IgG increased, while anti-NP IgM levels was decreased along the time in severe/critical and fatal as well as anti-NP IgG and IgA in several/critical groups. Discussion: In summary, the anti-NP IgA and IgG lower levels and the higher levels of anti-RBD and anti-Spike IgA in fatal compared to survival group of individuals admitted to the intensive care unit (ICU). Collectively, our data discriminate death from survival, suggesting that anti-RBD IgA and anti-Spike IgA may play some deleterious effect, in contrast with the potentially protective effect of anti-NP IgA and IgG in the survival group.