SARS-CoV-2 Reverse Genetics Reveals a Variable Infection Gradient in the Respiratory Tract.

The mode of acquisition and causes for the variable clinical spectrum of coronavirus disease 2019 (COVID-19) remain unknown. We utilized a reverse genetics system to generate a GFP reporter virus to explore severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis and a luciferase reporter virus to demonstrate sera collected from SARS and COVID-19 patients exhibited limited cross-CoV neutralization. High-sensitivity RNA in situ mapping revealed the highest angiotensin-converting enzyme 2 (ACE2) expression in the nose with decreasing expression throughout the lower respiratory tract, paralleled by a striking gradient of SARS-CoV-2 infection in proximal (high) versus distal (low) pulmonary epithelial cultures. COVID-19 autopsied lung studies identified focal disease and, congruent with culture data, SARS-CoV-2-infected ciliated and type 2 pneumocyte cells in airway and alveolar regions, respectively. These findings highlight the nasal susceptibility to SARS-CoV-2 with likely subsequent aspiration-mediated virus seeding to the lung in SARS-CoV-2 pathogenesis. These reagents provide a foundation for investigations into virus-host interactions in protective immunity, host susceptibility, and virus pathogenesis.

An Expeditious Neutralization Assay for Porcine Reproductive and Respiratory Syndrome Virus Based on a Recombinant Virus Expressing Green Fluorescent Protein.

Due to the extensive genetic and antigenic variation in Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), as well as its rapid mutability and evolution, PRRS prevention and control can be challenging. An expeditious and sensitive neutralization assay for PRRSV is presented to monitor neutralizing antibodies (NAbs) in serum during vaccine research. Here, a PRRSV expressing eGFP was successfully rescued with reverse genetics based on the infectious clone HuN4-F112-eGFP which we constructed. The fluorescent protein expressions of the reporter viruses remained stable for at least five passages. Based on this reporter virus, the neutralization assay can be easily used to evaluate the level of NAbs by counting cells with green fluorescence. Compared with the classical CPE assay, the newly developed assay increases sensitivity by one- to four-fold at the early antibody response stage, thus saving 2 days of assay waiting time. By using this assay to unveil the dynamics of neutralizing antibodies against PRRSV, priming immunity through either a single virulent challenge or only vaccination could produce limited NAbs, but re-infection with PRRSV would induce a faster and stronger NAb response. Overall, the novel HuN4-F112-eGFP-based neutralization assay holds the potential to provide a highly efficient platform for evaluating the next generation of PRRS vaccines.

Validation of a triple-color pseudovirion-based neutralization assay for immunogenicity assessment of a 14-valent recombinant human papillomavirus vaccine.

Validation of bioanalytical methods is crucial, especially in the pharmaceutical industry, to determine their suitability for specific purposes and the accuracy of analytical results. The pseudovirion-based neutralization assay (PBNA) is considered the gold standard for detecting and quantifying neutralizing antibodies against human papillomavirus in vaccine development for disease prevention. This paper introduces an improved triple-color PBNA method, capable of simultaneous detection of two or three human papillomavirus (HPV types for use in the development of a 14-valent HPV vaccine candidate. The primary objective was to comprehensively validate the triple-color PBNA method for general vaccine immunogenicity assays. Results show that the method has good specificity, accuracy, precision, linearity, robustness, and applicability. This innovative triple-color PBNA offers an improved approach for large-scale immunogenicity assessment in vaccine development. This study lays a solid foundation that can serve as a guiding paradigm for assessing vaccine responses in preclinical and clinical phases, providing valuable insights to the field.

Serum Neutralizing Antibody Titers 12 Months After Coronavirus Disease 2019 Messenger RNA Vaccination: Correlation to Clinical Variables in an Adult, US Population.

BACKGROUND: We studied whether comorbid conditions affect strength and duration of immune responses after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) messenger RNA vaccination in a US-based, adult population. METHODS: Sera (before and after BNT162b2 vaccination) were tested serially up to 12 months after 2 doses of vaccine for SARS-CoV-2-anti-Spike neutralizing capacity by pseudotyping assay in 124 individuals; neutralizing titers were correlated to clinical variables with multivariate regression. Postbooster (third dose) effect was measured at 1 and 3 months in 72 and 88 subjects, respectively. RESULTS: After completion of primary vaccine series, neutralizing antibody half maximal inhibitory concentration (IC50) values were high at 1 month (14-fold increase from prevaccination), declined at 6 months (3.3-fold increase), and increased at 1 month postbooster (41.5-fold increase). Three months postbooster, IC50 decreased in coronavirus disease (COVID)-naïve individuals (18-fold increase) and increased in prior COVID 2019 (COVID-19+) individuals (132-fold increase). Age >65 years (ß = -0.94, P = .001) and malignancy (ß = -0.88, P = .002) reduced strength of response at 1 month. Both neutralization strength and durability at 6 months, respectively, were negatively affected by end-stage renal disease ([ß = -1.10, P = .004]; [ß = -0.66, P = .014]), diabetes mellitus ([ß = -0.57, P = .032]; [ß = -0.44, P = .028]), and systemic steroid use ([ß = -0.066, P = .032]; [ß = -0.55, P = .037]). Postbooster IC50 was robust against WA-1 and B.1.617.2. Postbooster neutralization increased with prior COVID-19 (ß = 2.9, P < .0001), and malignancy reduced neutralization response (ß = -0.68, P = .03), regardless of infection status. CONCLUSIONS: Multiple clinical factors affect the strength and duration of neutralization response after primary series vaccination, but not the postbooster dose strength. Malignancy was associated with lower booster-dose response regardless of prior COVID infection, suggesting a need for clinically guided vaccine regimens.

SARS-CoV-2 Seroprevalence and Cross-Variant Antibody Neutralization in Cats, United Kingdom.

Anthropogenic transmission of SARS-CoV-2 to pet cats highlights the importance of monitoring felids for exposure to circulating variants. We tested cats in the United Kingdom for SARS-CoV-2 antibodies; seroprevalence peaked during September 2021-February 2022. The variant-specific response in cats trailed circulating variants in humans, indicating multiple human-to-cat transmissions over a prolonged period.

Simple prediction of COVID-19 convalescent plasma units with high levels of neutralization antibodies.

BACKGROUND: Hyperimmune convalescent COVID-19 plasma (CCP) containing anti-SARS-CoV-2 neutralizing antibodies (NAbs) was proposed as a therapeutic option for patients early in the new coronavirus disease pandemic. The efficacy of this therapy depends on the quantity of neutralizing antibodies (NAbs) in the CCP units, with titers ≥ 1:160 being recommended. The standard neutralizing tests (NTs) used for determining appropriate CCP donors are technically demanding and expensive and take several days. We explored whether they could be replaced by high-throughput serology tests and a set of available clinical data. METHODS: Our study included 1302 CCP donors after PCR-confirmed COVID-19 infection. To predict donors with high NAb titers, we built four (4) multiple logistic regression models evaluating the relationships of demographic data, COVID-19 symptoms, results of various serological testing, the period between disease and donation, and COVID-19 vaccination status. RESULTS: The analysis of the four models showed that the chemiluminescent microparticle assay (CMIA) for the quantitative determination of IgG Abs to the RBD of the S1 subunit of the SARS-CoV-2 spike protein was enough to predict the CCP units with a high NAb titer. CCP donors with respective results > 850 BAU/ml SARS-CoV-2 IgG had a high probability of attaining sufficient NAb titers. Including additional variables such as donor demographics, clinical symptoms, or time of donation into a particular predictive model did not significantly increase its sensitivity and specificity. CONCLUSION: A simple quantitative serological determination of anti-SARS-CoV-2 antibodies alone is satisfactory for recruiting CCP donors with high titer NAbs.

Antigenic comparison of H3N8 equine influenza viruses belonging to Florida sublineage clade 1 between vaccine strains and North American strains isolated in 2021-2022.

Equine influenza virus strains of Florida sublineage clade 1 (Fc1) have been circulating in North America. In this study, virus neutralization assays were performed to evaluate antigenic differences between Fc1 vaccine strains and North American Fc1 strains isolated in 2021-2022, using equine antisera against A/equine/South Africa/4/2003 (a vaccine strain recommended by the World Organisation for Animal Health) and A/equine/Ibaraki/1/2007 (a Japanese vaccine strain). Antibody titers against four North American Fc1 strains isolated in 2021-2022 were comparable to those against the homologous vaccine strains. These results suggest that current Fc1 vaccine strains are effective against North American strains from 2021-2022.

Assessment of the impact of pathogen reduction technologies on the neutralizing activity of COVID-19 convalescent plasma.

COVID-19 convalescent plasma (CCP) could improve the clinical outcome of COVID-19 patients when high-titer CCP is administered in early stages of disease. However, CCP donors have a risk profile like first-time donors, pathogen reduction treatment (PRT) may mitigate such risk but should not impact CCP quality. The current study aims to assess the impact of PRT-technologies available in Saudi Arabia on the neutralizing activity of CCP. STUDY DESIGN: and Methods: CCP was collected from eligible donors by plasmapheresis. The neutralization titer was determined with an in-house microneutralization assay (MNA) using a local SARS-CoV-2 clinical isolate. Selected units were split and subject to PRT with amotosalen/UVA (AS) or Riboflavin/UVB (RB) (pairwise side-by-side comparison) followed by a second MNA analysis. 51 CCP units were collected, 27 were included in the analysis reaching the minimum MNA titer of 1:40 (4 reached high titer (≥1:250)). 27 CCP units were treated with AS and 14 with RB, the median MNA pre-treatment titer was 1:80 (1:40-640). The impact of AS and RB PRT on CCP neutralizing activity was not significantly different, nor in the total analysis neither in the pairwise comparison (94.6 vs 96.4 % retention, p > 0.05). No correlation of titer and blood group was observed, but a trend for increasing MNA titer with donor age, choosing donors with an age > 45 years would increase the number of high-titer CCP donors. The difference in impact of AS and RB on CCP MNA titer was below the limit of detection of the assay (0.5-fold).

Establishment of a neutralization assay for Nipah virus using a high-titer pseudovirus system.

OBJECTIVE: To construct a high-titer Nipah pseudovirus packaging system using the HIV lentivirus backbone vector and establish a safe neutralization assay for Nipah pseudovirus in biosafety level 2 facilities. METHODS: Nipah virus (NiV) fusion protein (F) and glycoprotein (G) recombinant expression plasmids, psPAX2, and pLenti CMV Puro LUC (w168-1) were transiently transfected into 293T cells for 72 h for the generation of a NiV pseudovirus. The neutralization ability of Nipah virus F and G protein antibodies was assessed using the pseudovirus. RESULTS: A NiV pseudovirus was constructed using 293T cells. The ideal mass ratio of plasmid psPAX2: w168-1: F: G for transfection was determined to be 4:4:1:1. The specificity of recombinant F and G protein expression was indicated by indirect immunofluorescence and western blotting. The pseudovirus particles showed obvious spikes under a transmission electron microscope. The NiV pseudovirus titer was 4.73 × 105 median tissue culture infective dose per mL, and the pseudovirus could be effectively neutralized by polyclonal antibodies specifically targeting the F and G proteins respectively. CONCLUSIONS: A NiV pseudovirus was successfully generated using HIV vector systems, and was used as a platform for a safe and reliable pseudovirus-based neutralizing assay that can be performed in biosafety level 2 facilities.

Pseudotyped Viruses for Coronaviruses.

Seven coronaviruses have been identified that can infect humans, four of which usually cause mild symptoms, including HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1, three of which are lethal coronaviruses, named severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus, and severe acute respiratory syndrome coronavirus 2. Pseudotyped virus is an important tool in the field of human coronavirus research because it is safe, easy to prepare, easy to detect, and highly modifiable. In addition to the application of pseudotyped viruses in the study of virus infection mechanism, vaccine, and candidate antiviral drug or antibody evaluation and screening, pseudotyped viruses can also be used as an important platform for further application in the prediction of immunogenicity and antigenicity after virus mutation, cross-species transmission prediction, screening, and preparation of vaccine strains with better broad spectrum and antigenicity. Meanwhile, as clinical trials of various types of vaccines and post-clinical studies are also being carried out one after another, the establishment of a high-throughput and fully automated detection platform based on SARS-CoV-2 pseudotyped virus to further reduce the cost of detection and manual intervention and improve the efficiency of large-scale detection is also a demand for the development of SARS-CoV-2 pseudotyped virus.

Validation of a SARS-CoV-2 Surrogate Neutralization Test Detecting Neutralizing Antibodies against the Major Variants of Concern.

SARS-CoV-2 infection and/or vaccination elicit a broad range of neutralizing antibody responses against the different variants of concern (VOC). We established a new variant-adapted surrogate virus neutralization test (sVNT) and assessed the neutralization activity against the ancestral B.1 (WT) and VOC Delta, Omicron BA.1, BA.2, and BA.5. Analytical performances were compared against the respective VOC to the reference virus neutralization test (VNT) and two CE-IVD labeled kits using three different cohorts collected during the COVID-19 waves. Correlation analyses showed moderate to strong correlation for Omicron sub-variants (Spearman's r = 0.7081 for BA.1, r = 0.7205 for BA.2, and r = 0.6042 for BA.5), and for WT (r = 0.8458) and Delta-sVNT (r = 0.8158), respectively. Comparison of the WT-sVNT performance with two CE-IVD kits, the "Icosagen SARS-CoV-2 Neutralizing Antibody ELISA kit" and the "Genscript cPass, kit" revealed an overall good correlation ranging from 0.8673 to -0.8773 and a midway profile between both commercial kits with 87.76% sensitivity and 90.48% clinical specificity. The BA.2-sVNT performance was similar to the BA.2 Genscript test. Finally, a correlation analysis revealed a strong association (r = 0.8583) between BA.5-sVNT and VNT sVNT using a double-vaccinated cohort (n = 100) and an Omicron-breakthrough infection cohort (n = 91). In conclusion, the sVNT allows for the efficient prediction of immune protection against the various VOCs.

Monoclonal Antibodies as SARS-CoV-2 Serology Standards: Experimental Validation and Broader Implications for Correlates of Protection.

COVID-19 has highlighted challenges in the measurement quality and comparability of serological binding and neutralization assays. Due to many different assay formats and reagents, these measurements are known to be highly variable with large uncertainties. The development of the WHO international standard (WHO IS) and other pool standards have facilitated assay comparability through normalization to a common material but does not provide assay harmonization nor uncertainty quantification. In this paper, we present the results from an interlaboratory study that led to the development of (1) a novel hierarchy of data analyses based on the thermodynamics of antibody binding and (2) a modeling framework that quantifies the probability of neutralization potential for a given binding measurement. Importantly, we introduced a precise, mathematical definition of harmonization that separates the sources of quantitative uncertainties, some of which can be corrected to enable, for the first time, assay comparability. Both the theory and experimental data confirmed that mAbs and WHO IS performed identically as a primary standard for establishing traceability and bridging across different assay platforms. The metrological anchoring of complex serological binding and neuralization assays and fast turn-around production of an mAb reference control can enable the unprecedented comparability and traceability of serological binding assay results for new variants of SARS-CoV-2 and immune responses to other viruses.

Anti-SARS-CoV-2 (COVID-19) vaccination efficacy in patients with severe neuromuscular diseases.

INTRODUCTION: Patients with severe neuromuscular disease (sNMD) are considered at high risk of severe COVID-19. Muscle tissue is often replaced by fibroadipose tissue in these diseases whereas the new mRNA-based vaccines are injected intramuscularly. We aimed at evaluating the efficacy of two injections associated with a booster injection of mRNA vaccine in these patients. METHODS: We performed an observational, prospective, single-centre study to investigate the level of anti-S antibodies (Abs) and their neutralization activity at weeks 6 (W6) and 24 (W24) after two injections of mRNA-1273 vaccine and at weeks 12 (BW12) and 29 (BW29) after a booster injection of BNT162b2 vaccine in patients with sNMD. RESULTS: Thirty-three patients with sNMD were included. At W6, 30 patients (90.1%) showed a protective serum level of specific anti-S Abs with a strong neutralization capacity. We observed a decline over time: only 12 patients (36.3%) retained anti-S Abs levels considered as protective at W24. The neutralization activity remained above the cut off in 23 (69.7%). The booster vaccination restored robust neutralization activity for all analysed 22 patients (100%) at BW12, which was maintained without any significant drop at BW29 (16). No severe adverse event was reported in this cohort and none of the 33 patients developed symptomatic COVID-19 over one year. CONCLUSIONS: This study provides evidence that most sNMD patients receiving two injections of COVID-19 mRNA-based vaccines develop a strong humoral response after vaccination. A decline over time was observed but a single booster injection restores a long-term immunity. Moreover, no safety issues were observed.

Neutralizing Type I Interferon Autoantibodies in Japanese Patients with Severe COVID-19.

PURPOSE: Autoantibodies (aAbs) to type I interferons (IFNs) have been found in less than 1% of individuals under the age of 60 in the general population, with the prevalence increasing among those over 65. Neutralizing autoantibodies (naAbs) to type I IFNs have been found in at least 15% of patients with life-threatening COVID-19 pneumonia in several cohorts of primarily European descent. We aimed to evaluate the prevalence of aAbs and naAbs to IFN-α2 or IFN-ω in Japanese patients who suffered from COVID-19 as well as in the general population. METHODS: Patients who suffered from COVID-19 (n = 622, aged 0-104) and an uninfected healthy control population (n = 3,456, aged 20-91) were enrolled in this study. The severities of the COVID-19 patients were as follows: critical (n = 170), severe (n = 235), moderate (n = 112), and mild (n = 105). ELISA and ISRE reporter assays were used to detect aAbs and naAbs to IFN-α2 and IFN-ω using E. coli-produced IFNs. RESULTS: In an uninfected general Japanese population aged 20-91, aAbs to IFNs were detected in 0.087% of individuals. By contrast, naAbs to type I IFNs (IFN-α2 and/or IFN-ω, 100 pg/mL) were detected in 10.6% of patients with critical infections, 2.6% of patients with severe infections, and 1% of patients with mild infections. The presence of naAbs to IFNs was significantly associated with critical disease (P = 0.0012), age over 50 (P = 0.0002), and male sex (P = 0.137). A significant but not strong correlation between aAbs and naAbs to IFN-α2 existed (r = - 0.307, p value < 0.0001) reinforced the importance of measuring naAbs in COVID-19 patients, including those of Japanese ancestry. CONCLUSION: In this study, we revealed that patients with pre-existing naAbs have a much higher risk of life-threatening COVID-19 pneumonia in Japanese population.

Development of a Rapid Live SARS-CoV-2 Neutralization Assay Based on a qPCR Readout.

Measuring SARS-CoV-2 neutralizing antibodies after vaccination or natural infection remains a priority in the ongoing COVID-19 pandemic to determine immunity, especially against newly emerging variants. The gold standard for assessing antibody-mediated immunity against SARS-CoV-2 are cell-based live virus neutralization assays. These assays usually take several days, thereby limiting test capacities and the availability of rapid results. In this study, therefore, we developed a faster live virus assay, which detects neutralizing antibodies through the early measurement of antibody-mediated intracellular virus reduction by SARS-CoV-2 qRT-PCR. In our assay, Vero E6 cells are infected with virus isolates preincubated with patient sera and controls. After 24 h, the intracellular viral load is determined by qRT-PCR using a standard curve to calculate percent neutralization. Utilizing COVID-19 convalescent-phase sera, we show that our novel assay generates results with high sensitivity and specificity as we detected antiviral activity for all tested convalescent-phase sera, but no antiviral activity in prepandemic sera. The assay showed a strong correlation with a conventional virus neutralization assay (rS = 0.8910), a receptor-binding domain ELISA (rS = 0.8485), and a surrogate neutralization assay (rS = 0.8373), proving that quantifying intracellular viral RNA can be used to measure seroneutralization. Our assay can be adapted easily to new variants, as demonstrated by our cross-neutralization experiments. This characteristic is key for rapidly determining immunity against newly emerging variants. Taken together, the novel assay presented here reduces turnaround time significantly while making use of a highly standardized and sensitive SARS-CoV-2 qRT-PCR method as a readout.

Seroprevalence of Pteropine orthoreovirus in humans remain similar after nearly two decades (2001-2002 vs. 2017) in Tioman Island, Malaysia.

Pteropine orthoreovirus (PRV) is an emerging zoonotic respiratory virus that can be transmitted from bats to humans. In Malaysia, aside from PRV2P (Pulau virus) being isolated from Pteropus hypomelanus sampled in Tioman Island, PRV3M (Melaka virus), PRV4K (Kampar virus), and PRV7S (Sikamat virus) were all isolated from samples of patients who reported having a disease spectrum from acute respiratory distress to influenza-like illness and sometimes even with enteric symptoms such as diarrhea and abdominal pain. Screening of sera collected from human volunteers on Tioman Island in 2001-2002 demonstrated that 12.8% (14/109) were positive for PRV2P and PRV3M. Taking all these together, we aim to investigate the serological prevalence of PRV (including PRV4K and PRV7S) among Tioman Island inhabitants again with the assumption that the seroprevalence rate will remain nearly similar to the above reported if human exposure to bats is still happening in the island. Using sera collected from human volunteers on the same island in 2017, we demonstrated seroprevalence of 17.8% (28/157) against PRV2P and PRV3M, respectively. Seropositivity of 11.4% among Tioman Island inhabitants against PRV4K and PRV7S, respectively, was described in this study. In addition, the seroprevalence of 89.5% (17/19), 73.6% (14/19), 63.0% (12/19), and 73.6% (14/19) against PRV2P, PRV3M, PRV4K, and PRV7S, respectively, were observed among pteropid bats in the island. We revealed that the seroprevalence of PRV among island inhabitants remains nearly similar after nearly two decades, suggesting that potential spill-over events in bat-human interface areas in the Tioman Island. We are unclear whether such spillover was directly from bats to humans, as suspected for the PRV3M human cases, or from an intermediate host(s) yet to be identified. There is a high possibility of the viruses circulating among the bats as demonstrated by high seroprevalence against PRV in the bats.

Clinical efficacy and in vitro neutralization capacity of monoclonal antibodies for severe acute respiratory syndrome coronavirus 2 delta and omicron variants.

We aimed to provide in vitro data on the neutralization capacity of different monoclonal antibody (mAb) preparations against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) delta and omicron variant, respectively, and describe the in vivo RNA kinetics of coronavirus disease 2019 (COVID-19) patients treated with the respective mAbs. Virus neutralization assays were performed to assess the neutralizing effect of the mAb formulations casirivimab/imdevimab and sotrovimab on the SARS-CoV-2 delta and omicron variant. Additionally, respiratory tract SARS-CoV-2 RNA kinetics are provided for 25 COVID-19 patients infected with either delta variant (n = 18) or omicron variant (n = 7) treated with the respective mAb formulations during their hospital stay. In the virus neutralization assay, sotrovimab exhibits neutralizing capacity at therapeutically achievable concentrations against the SARS-CoV-2 delta and omicron variant. In contrast, casivirimab/imdevimab had neutralizing capacity against the delta variant but failed neutralization against the omicron variant except for a very high concentration above the currently recommended therapeutic dosage. In patients with delta variant infections treated with casivirimab/imdevimab, we observed a rapid decrease of respiratory viral RNA at day 3 after mAb therapy. In contrast, no such prompt decline was observed in patients with delta variant or omicron variant infections receiving sotrovimab.

Immunogenicity correlation in cynomolgus monkeys between Luminex-based total IgG immunoassay and pseudovirion-based neutralization assay for a 14-valent recombinant human papillomavirus vaccine.

A new virus-like particle based vaccine covering 14 types of high-risk and disease-inducing human papillomavirus (HPV) can offer better coverage against HPV-induced diseases, particularly cervical cancers. However, the assessment of immunogenicity of the vaccine is an important task, representing not only its significant clinical characteristics, but also a major challenge, in terms of both the suitability of methods and the clinical sample testing throughput supporting clinical development. This work covers the development and evaluation of a method based on Luminex technology (a coded-bead and flow-cytometric approach) to assess the HPV-type specific total immunoglobulin G (IgG). This method can evaluate the antibodies in sera post immunization against multiple types of HPV simultaneously (i.e., with multiplexing capability), save time and cost, and improve test throughput with higher sensitivity and precision than the classical, plate-based enzyme-linked immunoassay and competitive Luminex-based immunoassays. Using cynomolgus monkeys as model, we demonstrated the good correlation between the results from the pseudovirion-based neutralization assay (PBNA), and the Luminex-based total IgG assay, supporting that the latter method can be considered as a viable, dependable replacement method for the PBNA supporting immunogenicity evaluation of HPV vaccine in preclinical development and clinical investigation.

Construction of SARS-CoV-2 spike-pseudotyped retroviral vector inducing syncytia formation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is handled in biosafety level 3 (BSL-3) facilities, whereas the antiviral screening of pseudotype virus is conducted in BSL-2 facilities. In this study, we developed a SARS-CoV-2 spike-pseudotyped virus based on a semi-replication-competent retroviral (s-RCR) vector system. The s-RCR vector system was divided into two packageable vectors, each with gag-pol and env genes. For env vector construction, SARS-CoV-2 SΔ19 env was inserted into the pCLXSN-IRES-EGFP retroviral vector to generate pCLXSN-SΔ19 env-EGFP. When pCLXSN-gag-pol and pCLXSN-SΔ19env-EGFP were co-transfected into HEK293 T cells to generate an s-RCR virus, titers of the s-RCR virus were consistently low in this transient transfection system (1 × 104 TU/mL). However, a three-fold higher amounts of MLV-based SARS-CoV-2 pseudotyped viruses (3 × 104 TU/mL) were released from stable producer cells, and the spike proteins induced syncytia formation in HEK293-hACE2 cells. Furthermore, s-RCR stocks collected from stable producer cells induced more substantial syncytia formation in the Vero E6-TMPRSS2 cell line than in the Vero E6 cell line. Therefore, a combination of the s-RCR vector and the two cell lines (HEK293-hACE2 or Vero E6-TMPRSS2) that induce syncytia formation can be useful for the rapid screening of novel fusion inhibitor drugs.

Virtual screening and in vitro validation of natural compound inhibitors against SARS-CoV-2 spike protein.

The COVID-19 pandemic caused by the SARS-CoV-2 virus has led to a major public health burden and has resulted in millions of deaths worldwide. As effective treatments are limited, there is a significant requirement for high-throughput, low resource methods for the discovery of novel antivirals. The SARS-CoV-2 spike protein plays a key role in viral entry and has been identified as a therapeutic target. Using the available spike crystal structure, we performed a virtual screen with a library of 527 209 natural compounds against the receptor binding domain of this protein. Top hits from this screen were subjected to a second, more comprehensive molecular docking experiment and filtered for favourable ADMET properties. The in vitro activity of 10 highly ranked compounds was assessed using a virus neutralisation assay designed to facilitate viral entry in a physiologically relevant manner via the plasma membrane route. Subsequently, four compounds ZINC02111387, ZINC02122196, SN00074072 and ZINC04090608 were identified to possess antiviral activity in the µM range. These findings validate the virtual screening method as a tool for identifying novel antivirals and provide a basis for future drug development against SARS-CoV-2.