Coronavirus spike protein-specific antibodies indicate frequent infections and reinfections in infancy and among BNT162b2-vaccinated healthcare workers.

The prevalence of seasonal human coronavirus (HCoV) infections in early childhood and adults has not been well analyzed in longitudinal serological studies. Here we analyzed the changes in HCoV (229E, HKU1, NL63, OC43, MERS, and SARS-CoV-2) spike-specific antibody levels in follow-up serum specimens of 140 children at the age of 1, 2, and 3 years, and of 113 healthcare workers vaccinated for Covid-19 with BNT162b2-vaccine. IgG antibody levels against six recombinant HCoV spike subunit 1 (S1) proteins were measured by enzyme immunoassay. We show that by the age of three years the cumulative seropositivity for seasonal HCoVs increased to 38-81% depending on virus type. BNT162b2 vaccinations increased anti-SARS-CoV-2 S1 antibodies, but no increase in seasonal coronavirus antibodies associated with vaccinations. In healthcare workers (HCWs), during a 1-year follow-up, diagnostic antibody rises were seen in 5, 4 and 14% of the cases against 229E, NL63 and OC43 viruses, respectively, correlating well with the circulating HCoVs. In 6% of the HCWs, a diagnostic antibody rise was seen against S1 of HKU1, however, these rises coincided with anti-OC43 S1 antibody rises. Rabbit and guinea pig immune sera against HCoV S1 proteins indicated immunological cross-reactivity within alpha-CoV (229E and NL63) and beta-CoV (HKU1 and OC43) genera.

Computational and Enzymatic Studies of Sartans in SARS-CoV-2 Spike RBD-ACE2 Binding: The Role of Tetrazole and Perspectives as Antihypertensive and COVID-19 Therapeutics.

This study is an extension of current research into a novel class of synthetic antihypertensive drugs referred to as "bisartans", which are bis-alkylated imidazole derivatives bearing two symmetric anionic biphenyltetrazoles. Research to date indicates that bisartans are superior to commercially available hypertension drugs, since the former undergo stronger docking to angiotensin-converting enzyme 2 (ACE2). ACE2 is the key receptor involved in SARS-CoV-2 entry, thus initiating COVID-19 infection and in regulating levels of vasoactive peptides such as angiotensin II and beneficial heptapeptides A(1-7) and Alamandine in the renin-angiotensin system (RAS). In previous studies using in vivo rabbit-iliac arterial models, we showed that Na+ or K+ salts of selected Bisartans initiate a potent dose-response inhibition of vasoconstriction. Furthermore, computational studies revealed that bisartans undergo stable binding to the vital interfacial region between ACE2 and the SARS-CoV-2 "receptor binding domain" (i.e., the viral RBD). Thus, bisartan homologs are expected to interfere with SARS-CoV-2 infection and/or suppress disease expression in humans. The primary goal of this study was to investigate the role of tetrazole in binding and the network of amino acids of SARS-CoV-2 Spike RBD-ACE2 complex involved in interactions with sartans. This study would, furthermore, allow the expansion of the synthetic space to create a diverse suite of new bisartans in conjunction with detailed computational and in vitro antiviral studies. A critical role for tetrazole was uncovered in this study, shedding light on the vital importance of this group in the binding of sartans and bisartans to the ACE2/Spike complex. The in silico data predicting an interaction of tetrazole-containing sartans with ACE2 were experimentally validated by the results of surface plasmon resonance (SPR) analyses performed with a recombinant human ACE2 protein.

A rabbit model of tracheal collapse for optimal self-expanding metal stents.

Investigating the characteristics of tracheas can help the understanding of diseases related to the trachea, particularly tracheal collapse (TC) in dogs. This study aimed to compare the mechanical properties of tracheas from New Zealand White (NZW) rabbits and dogs and to introduce a method for inducing a model of TC in the normal trachea. Tracheal samples were obtained from NZW rabbit cadavers (n=5) weighing 3.62-3.92 kg and from dog cadavers (n=5) weighing 2.97-3.28 kg. Three live NZW rabbits weighing 3.5-4.0 kg were used to establish the model. The radial forces of both sample sets were measured using a digital force gauge and statistically compared. Subsequently, TC was surgically induced in three female NZW rabbits by physically weakening their tracheal cartilage under general anesthesia. Their clinical signs were monitored for 3 months, and radiographic examinations were performed monthly for 3 months. The mean radial forces of the two sample sets were comparable (P>0.05). The clinical signs, radiographic examinations, and macroscopic examinations were all comparable to those of dogs with TC. The cadaveric study between the rabbits and dogs demonstrated that the surgically induced rabbit model of TC is an excellent candidate for the experimental study of dogs with TC. This study also provides a reference of tracheal radial force values to enable selection of appropriate mesh types and wire diameters of self-expanding metal stents.

Potent immunogenicity and broad-spectrum protection potential of microneedle array patch-based COVID-19 DNA vaccine candidates encoding dimeric RBD chimera of SARS-CoV and SARS-CoV-2 variants.

Breakthrough infections by SARS-CoV-2 variants pose a global challenge to COVID-19 pandemic control, and the development of more effective vaccines of broad-spectrum protection is needed. In this study, we constructed pVAX1-based plasmids encoding receptor-binding domain (RBD) chimera of SARS-CoV-1 and SARS-CoV-2 variants, including pAD1002 (encoding RBDSARS/BA1), pAD1003 (encoding RBDSARS/Beta) and pAD131 (encoding RBDBA1/Beta). Plasmids pAD1002 and pAD131 were far more immunogenic than pAD1003 in terms of eliciting RBD-specific IgG when intramuscularly administered without electroporation. Furthermore, dissolvable microneedle array patches (MAP) greatly enhanced the immunogenicity of these DNA constructs in mice and rabbits. MAP laden with pAD1002 (MAP-1002) significantly outperformed inactivated SARS-CoV-2 virus vaccine in inducing RBD-specific IFN-γ+ effector and memory T cells, and generated T lymphocytes of different homing patterns compared to that induced by electroporated DNA in mice. In consistence with the high titer neutralization results of MAP-1002 antisera against SARS-CoV-2 pseudoviruses, MAP-1002 protected human ACE2-transgenic mice from Omicron BA.1 challenge. Collectively, MAP-based DNA constructs encoding chimeric RBDs of SARS-CoV-1 and SARS-CoV-2 variants, as represented by MAP-1002, are potential COVID-19 vaccine candidates worthy further translational study.

Exosome-Based Multivalent Vaccine: Achieving Potent Immunization, Broadened Reactivity, and Strong T-Cell Responses with Nanograms of Proteins.

Currently approved vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have focused solely on the spike protein to provide immunity. The first vaccines were developed rapidly using spike mRNA delivered by lipid nanoparticles but required ultralow-temperature storage and have had limited immunity against variations in spike. Subsequently, protein-based vaccines were developed, which offer broader immunity but require significant time for development and the use of an adjuvant to boost the immune response. Here, exosomes were used to deliver a bivalent protein-based vaccine in which two independent viral proteins were used. Exosomes were engineered to express either SARS-CoV-2 delta spike (Stealth X-Spike [STX-S]) or the more conserved nucleocapsid (Stealth X-Nucleocapsid [STX-N]) protein on the surface. When administered as a single product (STX-S or STX-N) or in combination (STX-S+N), both STX-S and STX-N induced strong immunization with the production of potent humoral and cellular immune responses. Interestingly, these results were obtained with the administration of only nanograms of protein and without an adjuvant. In two independent animal models (mouse and rabbit), the administration of nanograms of the STX-S+N vaccine resulted in increased antibody production, potent neutralizing antibodies with cross-reactivity to other variants of spike, and strong T-cell responses. Importantly, no competition of immune responses was observed, allowing the delivery of nucleocapsid with spike to offer improved SARS-CoV-2 immunity. These data show that the StealthX exosome platform has the enormous potential to revolutionize vaccinology by combining the advantages of mRNA and recombinant protein vaccines into a superior, rapidly generated, low-dose vaccine resulting in potent, broader immunity. IMPORTANCE The pandemic emergency has brought to light the need for a new generation of rapidly developed vaccines that induce longer-lasting, potent, and broader immune responses. While the mRNA vaccines played a critical role during the emergency in reducing SARS-CoV-2 hospitalization rates and deaths, more efficient approaches are needed. A multivalent, protein-based vaccine delivered by exosomes could meet this urgent need due to the high speed of development, manufacturability, and the ability to produce a strong antibody response, with neutralizing antibodies and a strong T-cell response able to broadly combat viral infection with a minimum number of injections.

Species differences in plasma protein binding of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease inhibitor nirmatrelvir.

Plasma protein binding (PPB) studies on the SARS-CoV-2 main protease inhibitor nirmatrelvir revealed considerable species differences primarily in dog and rabbit, which prompted further investigations into the biochemical basis for these differences.The unbound fraction (fu) of nirmatrelvir in dog and rabbit plasma was concentration (2-200 µM)-dependent (dog fu,p 0.024-0.69, rabbit fu,p 0.010-0.82). Concentration (0.1-100 µM)-dependent binding in serum albumin (SA) (fu,SA 0.040-0.82) and alpha-1-acid glycoprotein (AAG) (fu,AAG 0.050-0.64) was observed in dogs. Nirmatrelvir showed minimal binding to rabbit SA (1-100 µM: fu,SA 0.70-0.79), while binding to rabbit AAG was concentration-dependent (0.1-100 µM: fu,AAG 0.024-0.66). In contrast, nirmatrelvir (2 µM) revealed minimal binding (fu,AAG 0.79-0.88) to AAG from rat and monkeys. Nirmatrelvir showed minimal-to-moderate binding to SA (1-100 µM; fu,SA 0.70-1.0) and AAG (0.1-100 µM; fu,AAG 0.48-0.58) from humans across tested concentrations.Nirmatrelvir molecular docking studies using published crystal structures and homology models of human and preclinical species SA and AAG were used to rationalise the species differences to plasma proteins. This suggested that species differences in PPB are primarily driven by molecular differences in albumin and AAG resulting in differences in binding affinity.

Enhancement of immunogenicity and neutralizing responses against SARS-CoV-2 spike protein using the Fc fusion fragment.

AIMS: Vaccination has played an important role in protecting against death and the severity of COVID-19. The recombinant protein vaccine platform has a long track record of safety and efficacy. Here, we fused the SARS-CoV-2 spike S1 subunit to the Fc region of IgG and investigated immunogenicity, reactivity to human vaccinated sera, and neutralizing activity as a candidate protein vaccine. MATERIALS AND METHOD: We evaluated the immunogenicity of CHO-expressed S1-Fc fusion protein and tag-free S1 protein in rabbits via the production of S1-specific polyclonal antibodies. We subsequently compared the neutralizing activities of sera from immunized rabbits and human-vaccinated individuals using a surrogate Virus Neutralization Test (sVNT). KEY FINDINGS: The results indicate that S1-specific polyclonal antibodies were induced in all groups; however, antibody levels were higher in rabbits immunized with S1-Fc fusion protein than tag-free S1 protein. Moreover, the reactivity of human vaccinated sera against S1-Fc fusion protein was significantly higher than tag-free S1 protein. Lastly, the results of the virus-neutralizing activity revealed that vaccination with S1-Fc fusion protein induced the highest level of neutralizing antibody response against SARS-CoV-2. SIGNIFICANCE: Our results demonstrate that the S1 protein accompanied by the Fc fragment significantly enhances the immunogenicity and neutralizing responses against SARS-CoV-2. It is hoped that this platform can be used for human vaccination.

Immunogenicity of adjuvanted plant-produced SARS-CoV-2 Beta spike VLP vaccine in New Zealand white rabbits.

The outbreak of the SARS-CoV-2 global pandemic heightened the pace of vaccine development with various vaccines being approved for human use in a span of 24 months. The SARS-CoV-2 trimeric spike (S) surface glycoprotein, which mediates viral entry by binding to ACE2, is a key target for vaccines and therapeutic antibodies. Plant biopharming is recognized for its scalability, speed, versatility, and low production costs and is an increasingly promising molecular pharming vaccine platform for human health. We developed Nicotiana benthamiana-produced SARS-CoV-2 virus-like particle (VLP) vaccine candidates displaying the S-protein of the Beta (B.1.351) variant of concern (VOC), which triggered cross-reactive neutralising antibodies against Delta (B.1.617.2) and Omicron (B.1.1.529) VOCs. In this study, immunogenicity of the VLPs (5 µg per dose) adjuvanted with three independent adjuvants i.e. oil-in-water based adjuvants SEPIVAC SWETM (Seppic, France) and "AS IS" (Afrigen, South Africa) as well as a slow-release synthetic oligodeoxynucleotide (ODN) adjuvant designated NADA (Disease Control Africa, South Africa) were evaluated in New Zealand white rabbits and resulted in robust neutralising antibody responses after booster vaccination, ranging from 1:5341 to as high as 1:18204. Serum neutralising antibodies elicited by the Beta variant VLP vaccine also showed cross-neutralisation against the Delta and Omicron variants with neutralising titres ranging from 1:1702 and 1:971, respectively. Collectively, these data provide support for the development of a plant-produced VLP based candidate vaccine against SARS-CoV-2 based on circulating variants of concern.

Proximity to livestock farms and COVID-19 in the Netherlands, 2020-2021.

OBJECTIVES: In the Netherlands, during the first phase of the COVID-19 epidemic, the hotspot of COVID-19 overlapped with the country's main livestock area, while in subsequent phases this distinct spatial pattern disappeared. Previous studies show that living near livestock farms influence human respiratory health and immunological responses. This study aimed to explore whether proximity to livestock was associated with SARS-CoV-2 infection. METHODS: The study population was the population of the Netherlands excluding the very strongly urbanised areas and border areas, on January 1, 2019 (12, 628, 244 individuals). The cases are the individuals reported with a laboratory-confirmed positive SARS-CoV-2 test with onset before January 1, 2022 (2, 223, 692 individuals). For each individual, we calculated distance to nearest livestock farm (cattle, goat, sheep, pig, poultry, horse, rabbit, mink). The associations between residential (6-digit postal-code) distance to the nearest livestock farm and individuals' SARS-CoV-2 status was studied with multilevel logistic regression models. Models were adjusted for individuals' age categories, the social status of the postal code area, particulate matter (PM10)- and nitrogen dioxide (NO2)-concentrations. We analysed data for the entire period and population as well as separately for eight time periods (Jan-Mar, Apr-Jun, Jul-Sep and Oct-Dec in 2020 and 2021), four geographic areas of the Netherlands (north, east, west and south), and for five age categories (0-14, 15-24, 25-44, 45-64 and > 65 years). RESULTS: Over the period 2020-2021, individuals' SARS-CoV-2 status was associated with living closer to livestock farms. This association increased from an Odds Ratio (OR) of 1.01 (95% Confidence Interval [CI] 1.01-1.02) for patients living at a distance of 751-1000 m to a farm to an OR of 1.04 (95% CI 1.04-1.04), 1.07 (95% CI 1.06-1.07) and 1.11 (95% CI 1.10-1.12) for patients living in the more proximate 501-750 m, 251-500m and 0-250 m zones around farms, all relative to patients living further than 1000 m around farms. This association was observed in three out of four quarters of the year in both 2020 and 2021, and in all studied geographic areas and age groups. CONCLUSIONS: In this exploratory study with individual SARS-CoV-2 notification data and high-resolution spatial data associations were found between living near livestock farms and individuals' SARS-CoV-2 status in the Netherlands. Verification of the results in other countries is warranted, as well as investigations into possible underlying exposures and mechanisms.

Development of a novel double-antibody sandwich quantitative ELISA for detecting SADS-CoV infection.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an emerging swine enteric alphacoronavirus that can cause acute diarrhea, vomiting, dehydration, and death of newborn piglets. In this study, we developed a double-antibody sandwich quantitative enzyme-linked immunosorbent assay (DAS-qELISA) for detection of SADS-CoV by using an anti-SADS-CoV N protein rabbit polyclonal antibody (PAb) and a specific monoclonal antibody (MAb) 6E8 against the SADS-CoV N protein. The PAb was used as the capture antibodies and HRP-labeled 6E8 as the detector antibody. The detection limit of the developed DAS-qELISA assay was 1 ng/mL of purified antigen and 101.08TCID50/mL of SADS-CoV, respectively. Specificity assays showed that the developed DAS-qELISA has no cross-reactivity with other swine enteric coronaviruses, such as porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine deltacoronavirus (PDCoV). Three-day-old piglets were challenged with SADS-CoV and collected anal swab samples which were screened for the presence of SADS-CoV by using DAS-qELISA and reverse transcriptase PCR (RT-PCR). The coincidence rate of the DAS-qELISA and RT-PCR was 93.93%, and the kappa value was 0.85, indicating that DAS-qELISA is a reliable method for applying antigen detection of clinical samples. KEY POINTS: • The first double-antibody sandwich quantitative enzyme-linked immunosorbent assay for detection SADS-CoV infection. • The custom ELISA is useful for controlling the SADS-CoV spread.

RBD and Spike DNA-Based Immunization in Rabbits Elicited IgG Avidity Maturation and High Neutralizing Antibody Responses against SARS-CoV-2.

Neutralizing antibodies (nAbs) are a critical part of coronavirus disease 2019 (COVID-19) research as they are used to gain insight into the immune response to severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infections. Among the technologies available for generating nAbs, DNA-based immunization methods are an alternative to conventional protocols. In this pilot study, we investigated whether DNA-based immunization by needle injection in rabbits was a viable approach to produce a functional antibody response. We demonstrated that three doses of DNA plasmid carrying the gene encoding the full-length spike protein (S) or the receptor binding domain (RBD) of SARS-CoV-2 induced a time-dependent increase in IgG antibody avidity maturation. Moreover, the IgG antibodies displayed high cross neutralization by live SARS-CoV-2 and pseudoviruses neutralization assays. Thus, we established a simple, low cost and feasible DNA-based immunization protocol in rabbits that elicited high IgG avidity maturation and nAbs production against SARS-CoV-2, highlighting the importance of DNA-based platforms for developing new immunization strategies against SARS-CoV-2 and future emerging epidemics.

Zebrafish-based platform for emerging bio-contaminants and virus inactivation research.

Emerging bio-contaminants such as viruses have affected health and environment settings of every country. Viruses are the minuscule entities resulting in severe contagious diseases like SARS, MERS, Ebola, and avian influenza. Recent epidemic like the SARS-CoV-2, the virus has undergone mutations strengthen them and allowing to escape from the remedies. Comprehensive knowledge of viruses is essential for the development of targeted therapeutic and vaccination treatments. Animal models mimicking human biology like non-human primates, rats, mice, and rabbits offer competitive advantage to assess risk of viral infections, chemical toxins, nanoparticles, and microbes. However, their economic maintenance has always been an issue. Furthermore, the redundancy of experimental results due to aforementioned aspects is also in examine. Hence, exploration for the alternative animal models is crucial for risk assessments. The current review examines zebrafish traits and explores the possibilities to monitor emerging bio-contaminants. Additionally, a comprehensive picture of the bio contaminant and virus particle invasion and abatement mechanisms in zebrafish and human cells is presented. Moreover, a zebrafish model to investigate the emerging viruses such as coronaviridae and poxviridae has been suggested.

Changes in the vitreous body after experimental vitreous hemorrhage in rabbit: An interdisciplinary study.

PURPOSE: To explore the changes in vitreous body after vitreous hemorrhage and assess its prognosis from the perspective of vitreoretinal interface. METHODS: The experiment was performed on 32 New Zealand rabbits (64 eyes), weighing 2500-3000 g for 4 months and unlimited gender, which was injected with 0.2 mL of autologous blood into the center of vitreous cavity-the study group (right eyes), and the control one was treated in the same manner with equal volumes of saline. The rabbits were randomly and equally divided into the following four batches according to the days of observation: Days 3, 7, 14, and 30 after injection. IOP and severity grading were evaluated before rabbits' execution and eyeballs were enucleated. The anterior segment was separated to flow out the vitreous body naturally to detect the liquefaction degree and viscosity. Then, chemical composition of electrolytes, PCT and bFGF were determined by colorimetry and enzyme-linked immunosorbent assay (ELISA). Finally, the incidence of posterior vitreous detachment (PVD) was observed after vitreous sampled. The studies were double-blind. RESULTS: After injection, the extent of vitreous opacity and coagulum size decreased over time. Both the degree of liquefaction and the length of tow differed significantly between two groups at different time points (all p < 0.001). The liquefaction degree in the study group rose obviously from the Day 14, which the viscosity declined significantly on the initial time. Biochemical markers fluctuated temporarily, except for basic fibroblast growth factor (bFGF), which continued to rise and was correlated with the liquefaction degree (r = 0.658, p < 0.001). Besides, the incidence of PVD increased from the 14th day (p < 0.05), and it was highly positively correlated with the number of macrophages (r = 0.934; p < 0.001). CONCLUSION: After vitreous hemorrhage, the changes of the vitreous body are relatively minor earlier (2-4 weeks), but irreversible later. Specifically, the degree of liquefaction increases with a decrease in viscosity, and the chemotaxis of macrophages and bFGF induce incomplete PVD.

Molecular phylogeny of coronaviruses and host receptors among domestic and close-contact animals reveals subgenome-level conservation, crossover, and divergence.

BACKGROUND: Coronaviruses have the potential to cross species barriers. To learn the molecular intersections among the most common coronaviruses of domestic and close-contact animals, we analyzed representative coronavirus genera infecting mouse, rat, rabbit, dog, cat, cattle, white-tailed deer, swine, ferret, mink, alpaca, Rhinolophus bat, dolphin, whale, chicken, duck and turkey hosts; reference or complete genome sequences were available for most of these coronavirus genera. Protein sequence alignments and phylogenetic trees were built for the spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins. The host receptors and enzymes aminopeptidase N (APN), angiotensin converting enzyme 2 (ACE2), sialic acid synthase (SAS), transmembrane serine protease 2 (TMPRSS2), dipeptidyl peptidase 4 (DPP4), cathepsin L (and its analogs) and furin were also compared. RESULTS: Overall, the S, E, M, and N proteins segregated according to their viral genera (α, ß, or γ), but the S proteins of alphacoronaviruses lacked conservation of phylogeny. Interestingly, the unique polybasic furin cleavage motif found in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) but not in severe acute respiratory syndrome coronavirus (SARS-CoV) or Middle East respiratory syndrome coronavirus (MERS-CoV) exists in several ß-coronaviruses and a few α- or γ-coronaviruses. Receptors and enzymes retained host species-dependent relationships with one another. Among the hosts, critical ACE2 residues essential for SARS-CoV-2 spike protein binding were most conserved in white-tailed deer and cattle. CONCLUSION: The polybasic furin cleavage motif found in several ß- and other coronaviruses of animals points to the existence of an intermediate host for SARS-CoV-2, and it also offers a counternarrative to the theory of a laboratory-engineered virus. Generally, the S proteins of coronaviruses show crossovers of phylogenies indicative of recombination events. Additionally, the consistency in the segregation of viral proteins of the MERS-like coronavirus (NC_034440.1) from pipistrelle bat supports its classification as a ß-coronavirus. Finally, similarities in host enzymes and receptors did not always explain natural cross-infections. More studies are therefore needed to identify factors that determine the cross-species infectivity of coronaviruses.

Characterization of two linear epitopes SARS CoV-2 spike protein formulated in tandem repeat.

The vital roles of diagnostic tools and vaccines are prominent in controlling COVID-19. Spike protein of the SARS CoV-2, specifically the epitopes in that protein, are the critical components of the vaccines and immunological diagnostic tools. Two epitopes in the spike protein, the S14P5 and S21P2, identified previously are of great interest because they are linear and elicit neutralizing antibodies. The present study formulated each epitope in the tandem-repeat structure to increase their immunogenicity and facilitate their production. The tandem repeats (TR) were expressed efficiently in E. coli, yielding 58 mg and 46 mg per liter culture for TR-S14P5 and TR-S212, respectively. ELISA using either one of the repeating epitopes can be used as a serological test to identify individuals infected by the SARS-CoV-2 virus. The area under curves (AUC), based on testing 157 serum samples from COVID-19 patients and 26 from COVID-19-free individuals, were 0.806 and 0.889 for TR-S14P5 and TR-S21P2-based ELISAs, respectively. For 100% diagnostic specificity, the sensitivity was only 70%. The low sensitivity supposedly resulted from some samples being from early infection prior to antibody conversion. Both recombinant epitopes were highly immunogenic in rabbits, and the immune sera recognized inactivated SARS CoV-2 virus in dot-blot assays. These antibodies should be useful as a reagent for detecting SARS-CoV-2 antigens. Furthermore, the TR-S14P5 and TR-S21P2, being conserved and denaturation-resistant, are envisaged to be ideal for intra-nasal vaccines, which are required to complement current COVID-19 to overcome rapidly mutated SARS CoV-2.

Preclinical safety assessment of the suction-assisted intradermal injection of the SARS-CoV-2 DNA vaccine candidate pGO-1002 in white rabbit.

pGO-1002, a non-viral DNA vaccine that expresses both spike and ORF3a antigens of SARS-CoV-2, is undergoing phase 1 and phase 2a clinical trials in Korea and the US. A preclinical repeated-dose toxicity study in New Zealand white rabbits in compliance with Good Laboratory Practice (GLP) was conducted to assess the potential toxicity, local tolerance, and immunogenicity of the vaccine and GeneDerm suction device. The dose rate was 1.2 mg/head pGO-1002, and this was administered intradermally to a group of animals (eight animals/sex/group) three times at 2-week intervals, followed by a 4-week recovery period. After each administration, suction was applied to the injection site using the GeneDerm device. Mortality, clinical signs, body weight, food consumption, skin irritation, ophthalmology, body temperature, urinalysis, and clinical pathology were also monitored. Gross observations and histopathological evaluation were performed. Overall, pGO-1002 administration-related changes were confined to minor damage or changes at the injection site, increased spleen weight and minimal increased cellularity in white pulp. All changes of injection site were considered local inflammatory changes or pharmacological actions due to the vaccine with the changes in spleen considered consistent with vaccine-induced immune activation. All findings showed reversibility during the 4-week recovery period. Animals vaccinated with pGO-1002, administered by intradermal injection and followed by application of suction with GeneDerm, developed humoral and cellular responses against the SARS-CoV-2 antigens consistent with prior studies in rats. Collectively, it was concluded that the pGO-1002 vaccine was safe and effective under these experimental conditions and these data supported future human study of the vaccine, now known as GLS-5310, for clinical trial use.

SARS-CoV-2-encoded ORF8 protein possesses complement inhibitory properties.

Hyperactivation of the complement system, a major component of innate immunity, has been recognized as one of the core clinical features in severe covid-19 patients. However, how the virus escapes the targeted elimination by the network of activated complement pathways still remains an enigma. Here, we identified SARS-CoV-2-encoded ORF8 protein as one of the major binding partners of human complement C3/C3b components and their metabolites. Our results demonstrated that preincubation of ORF8 with C3/C3b in the fluid phase has two immediate functional consequences in the alternative pathway; this preincubation inhibits factor I-mediated proteolysis and blocks factor B zymogen activation into active Bb. ORF8 binding results in the occlusion of both factor H and factor B from C3b, rendering the complexes resistant to factor I-mediated proteolysis and inhibition of pro-C3-convertase (C3bB) formation, respectively. We also confirmed the complement inhibitory activity of ORF8 in our hemolysis-based assay, where ORF8 prevented human serum-induced lysis of rabbit erythrocytes with an IC50 value of about 2.3 µM. This inhibitory characteristic of ORF8 was also supported by in-silico protein-protein docking analysis, as it appeared to establish primary interactions with the ß-chain of C3b, orienting itself near the C3b CUB (C1r/C1s, Uegf, Bmp1) domain like a peptidomimetic compound, sterically hindering the binding of essential cofactors required for complement amplification. Thus, ORF8 has characteristics to act as an inhibitor of critical regulatory steps in the alternative pathway, converging to hasten the decay of C3-convertase and thereby, attenuating the complement amplification loop.

Preclinical evaluation of safety and immunogenicity of a primary series intranasal COVID-19 vaccine candidate (BBV154) and humoral immunogenicity evaluation of a heterologous prime-boost strategy with COVAXIN (BBV152).

Most if not all vaccine candidates developed to combat COVID-19 due to SARS-CoV-2 infection are administered parenterally. As SARS-CoV-2 is transmitted through infectious respiratory fluids, vaccine-induced mucosal immunity could provide an important contribution to control this pandemic. ChAd-SARS-CoV-2-S (BBV154), a replication-defective chimpanzee adenovirus (ChAd)-vectored intranasal (IN) COVID-19 vaccine candidate, encodes a prefusion-stabilized version of the SARS-CoV-2 spike protein containing two proline substitutions in the S2 subunit. We performed preclinical evaluations of BBV154 in mice, rats, hamsters and rabbits. Repeated dose toxicity studies presented excellent safety profiles in terms of pathology and biochemical analysis. IN administration of BBV154 elicited robust mucosal and systemic humoral immune responses coupled with Th1 cell-mediated immune responses. BBV154 IN vaccination also elicited potent variant (omicron) cross neutralization antibodies. Assessment of anti-vector (ChAd36) neutralizing antibodies following repeated doses of BBV154 IN administration showed insignificant titers of ChAd36 neutralizing antibodies. However, the immune sera derived from the same animals displayed significantly higher levels of SARS-CoV-2 virus neutralization (p<0.003). We also evaluated the safety and immunogenicity of heterologous prime-boost vaccination with intramuscular (IM) COVAXIN-prime followed by BBV154 IN administration. COVAXIN priming followed by BBV154 IN-booster showed an acceptable reactogenicity profile comparable to the homologous COVAXIN/COVAXIN or BBV154/BBV154 vaccination model. Heterologous vaccination of COVAXIN-prime and BBV154 booster also elicited superior (p<0.005) and cross variant (omicron) protective immune responses (p<0.013) compared with the homologous COVAXIN/COVAXIN schedule. BBV154 has successfully completed both homologous and heterologous combination schedules of human phase 3 clinical trials and received the restricted emergency use approval (in those aged above 18 years) from the Drugs Controller General of India (DCGI).

Gastrointestinal helminths increase Bordetella bronchiseptica shedding and host variation in supershedding.

Co-infected hosts, individuals that carry more than one infectious agent at any one time, have been suggested to facilitate pathogen transmission, including the emergence of supershedding events. However, how the host immune response mediates the interactions between co-infecting pathogens and how these affect the dynamics of shedding remains largely unclear. We used laboratory experiments and a modeling approach to examine temporal changes in the shedding of the respiratory bacterium Bordetella bronchiseptica in rabbits with one or two gastrointestinal helminth species. Experimental data showed that rabbits co-infected with one or both helminths shed significantly more B. bronchiseptica, by direct contact with an agar petri dish, than rabbits with bacteria alone. Co-infected hosts generated supershedding events of higher intensity and more frequently than hosts with no helminths. To explain this variation in shedding an infection-immune model was developed and fitted to rabbits of each group. Simulations suggested that differences in the magnitude and duration of shedding could be explained by the effect of the two helminths on the relative contribution of neutrophils and specific IgA and IgG to B. bronchiseptica neutralization in the respiratory tract. However, the interactions between infection and immune response at the scale of analysis that we used could not capture the rapid variation in the intensity of shedding of every rabbit. We suggest that fast and local changes at the level of respiratory tissue probably played a more important role. This study indicates that co-infected hosts are important source of variation in shedding, and provides a quantitative explanation into the role of helminths to the dynamics of respiratory bacterial infections.