SARS-CoV-2 501Y.V2 variants lack higher infectivity but do have immune escape.

The 501Y.V2 variants of SARS-CoV-2 containing multiple mutations in spike are now dominant in South Africa and are rapidly spreading to other countries. Here, experiments with 18 pseudotyped viruses showed that the 501Y.V2 variants do not confer increased infectivity in multiple cell types except for murine ACE2-overexpressing cells, where a substantial increase in infectivity was observed. Notably, the susceptibility of the 501Y.V2 variants to 12 of 17 neutralizing monoclonal antibodies was substantially diminished, and the neutralization ability of the sera from convalescent patients and immunized mice was also reduced for these variants. The neutralization resistance was mainly caused by E484K and N501Y mutations in the receptor-binding domain of spike. The enhanced infectivity in murine ACE2-overexpressing cells suggests the possibility of spillover of the 501Y.V2 variants to mice. Moreover, the neutralization resistance we detected for the 501Y.V2 variants suggests the potential for compromised efficacy of monoclonal antibodies and vaccines.

The Impact of Mutations in SARS-CoV-2 Spike on Viral Infectivity and Antigenicity.

The spike protein of SARS-CoV-2 has been undergoing mutations and is highly glycosylated. It is critically important to investigate the biological significance of these mutations. Here, we investigated 80 variants and 26 glycosylation site modifications for the infectivity and reactivity to a panel of neutralizing antibodies and sera from convalescent patients. D614G, along with several variants containing both D614G and another amino acid change, were significantly more infectious. Most variants with amino acid change at receptor binding domain were less infectious, but variants including A475V, L452R, V483A, and F490L became resistant to some neutralizing antibodies. Moreover, the majority of glycosylation deletions were less infectious, whereas deletion of both N331 and N343 glycosylation drastically reduced infectivity, revealing the importance of glycosylation for viral infectivity. Interestingly, N234Q was markedly resistant to neutralizing antibodies, whereas N165Q became more sensitive. These findings could be of value in the development of vaccine and therapeutic antibodies.

Structurally Resolved SARS-CoV-2 Antibody Shows High Efficacy in Severely Infected Hamsters and Provides a Potent Cocktail Pairing Strategy.

Understanding how potent neutralizing antibodies (NAbs) inhibit SARS-CoV-2 is critical for effective therapeutic development. We previously described BD-368-2, a SARS-CoV-2 NAb with high potency; however, its neutralization mechanism is largely unknown. Here, we report the 3.5-Å cryo-EM structure of BD-368-2/trimeric-spike complex, revealing that BD-368-2 fully blocks ACE2 recognition by occupying all three receptor-binding domains (RBDs) simultaneously, regardless of their "up" or "down" conformations. Also, BD-368-2 treats infected adult hamsters at low dosages and at various administering windows, in contrast to placebo hamsters that manifested severe interstitial pneumonia. Moreover, BD-368-2's epitope completely avoids the common binding site of VH3-53/VH3-66 recurrent NAbs, evidenced by tripartite co-crystal structures with RBDs. Pairing BD-368-2 with a potent recurrent NAb neutralizes SARS-CoV-2 pseudovirus at pM level and rescues mutation-induced neutralization escapes. Together, our results rationalized a new RBD epitope that leads to high neutralization potency and demonstrated BD-368-2's therapeutic potential in treating COVID-19.

A Thermostable mRNA Vaccine against COVID-19.

There is an urgent need for vaccines against coronavirus disease 2019 (COVID-19) because of the ongoing SARS-CoV-2 pandemic. Among all approaches, a messenger RNA (mRNA)-based vaccine has emerged as a rapid and versatile platform to quickly respond to this challenge. Here, we developed a lipid nanoparticle-encapsulated mRNA (mRNA-LNP) encoding the receptor binding domain (RBD) of SARS-CoV-2 as a vaccine candidate (called ARCoV). Intramuscular immunization of ARCoV mRNA-LNP elicited robust neutralizing antibodies against SARS-CoV-2 as well as a Th1-biased cellular response in mice and non-human primates. Two doses of ARCoV immunization in mice conferred complete protection against the challenge of a SARS-CoV-2 mouse-adapted strain. Additionally, ARCoV is manufactured as a liquid formulation and can be stored at room temperature for at least 1 week. ARCoV is currently being evaluated in phase 1 clinical trials.

Rational prediction of immunogenicity clustering through cross-reactivity analysis of thirteen SARS-CoV-2 variants.

SARS-CoV-2 breakthrough infections in vaccinated individuals underscore the threat posed by continuous mutating variants, such as Omicron, to vaccine-induced immunity. This necessitates the search for broad-spectrum immunogens capable of countering infections from such variants. This study evaluates the immunogenicity relationship among SARS-CoV-2 variants, from D614G to XBB, through Guinea pig vaccination, covering D614G, Alpha, Beta, Gamma, Delta, BA.1, BA.2, BA.2.75, BA.2.75.2, BA.5, BF.7, BQ.1.1, and XBB, employing three immunization strategies: three-dose monovalent immunogens, three-dose bivalent immunogens, and a two-dose vaccination with D614G followed by a booster immunization with a variant strain immunogen. Three distinct immunogenicity clusters were identified: D614G, Alpha, Beta, Gamma, and Delta as cluster 1, BA.1, BA.2, and BA.2.75 as cluster 2, BA.2.75.2, BA.5, BF.7, BQ.1.1, and XBB as cluster 3. Broad-spectrum protection could be achieved through a combined immunization strategy using bivalent immunogens or D614G and XBB, or two initial D614G vaccinations followed by two XBB boosters. A comparison of neutralizing antibody levels induced by XBB boosting and equivalent dosing of D614G and XBB revealed that the XBB booster produced higher antibody levels. The study suggests that vaccine antigen selection should focus on the antigenic alterations among variants, eliminating the need for updating vaccine components for each variant.

LaserNet: a method of laser stripe center extraction under non-ideal conditions.

The extraction of the center of a laser stripe is a key step in line-structure measurement, where noise interference and changes in the surface color of an object are the main factors affecting extraction accuracy. To obtain sub-pixel level center coordinates under such non-ideal conditions, we propose LaserNet, a novel deep learning-based algorithm, to the best of our knowledge, which consists of a laser region detection sub-network and a laser position optimization sub-network. The laser region detection sub-network is used to determine potential stripe regions, and the laser position optimization sub-network uses the local image of these regions to obtain the accurate center position of the laser stripe. The experimental results show that LaserNet can eliminate noise interference, handle color changes, and give accurate results under non-ideal conditions. The three-dimensional reconstruction experiments further demonstrate the effectiveness of the proposed method.

Assays Based on Pseudotyped Viruses.

Pseudotyped viruses are more and more widely used in virus research and the evaluation of antiviral products because of their high safety, simple operation, high accessibility, ease in achieving standardization, and high throughput. The development of measures based on pseudotyped virus is closely related to the characteristics of viruses, and it is also necessary to follow the principles of assay development. Only in the process of method development, where the key parameters that affect the results are systematically optimized and the preliminary established method is fully validated, can the accuracy, reliability, and repeatability of the test results be ensured. Only the method established on this basis can be transferred to different laboratories and make the results of different laboratories comparable. This paper summarizes the specific aspects and general principles in the development of assays based on pseudotyped virus, which is of reference value for the development of similar methods.

Pseudotyped Virus for Papillomavirus.

Papillomavirus is difficult to culture in vitro, which limits its related research. The development of pseudotyped virus technology provides a valuable research tool for virus infectivity research, vaccine evaluation, infection inhibitor evaluation, and so on. Depending on the application fields, different measures have been developed to generate various kinds of pseudotyped papillomavirus. L1-based and L2-based HPV vaccines should be evaluated using different pseudotyped virus system. Pseudotyped papillomavirus animal models need high-titer pseudotyped virus and unique handling procedure to generate robust results. This paper reviewed the development, optimization, standardization, and application of various pseudotyped papillomavirus methods.

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.

Pseudotyped Viruses.

Pseudotyped viruses have been constructed for many viruses. They can mimic the authentic virus and have many advantages compared to authentic viruses. Thus, they have been widely used as a surrogate of authentic virus for viral function analysis, detection of neutralizing antibodies, screening viral entry inhibitors, and others. This chapter reviewed the progress in the field of pseudotyped viruses in general, including the definition and the advantages of pseudotyped viruses, their potential usage, different strategies or vectors used for the construction of pseudotyped viruses, and factors that affect the construction of pseudotyped viruses.

Aggregation of high-frequency RBD mutations of SARS-CoV-2 with three VOCs did not cause significant antigenic drift.

Variants of SARS-CoV-2 continue to emerge, posing great challenges in outbreak prevention and control. It is important to understand in advance the impact of possible variants of concern (VOCs) on infectivity and antigenicity. Here, we constructed one or more of the 15 high-frequency naturally occurring amino acid changes in the receptor-binding domain (RBD) of Alpha, Beta, and Gamma variants. A single mutant of A520S, V367F, and S494P in the above three VOCs enhanced infectivity in ACE2-overexpressing 293T cells of different species, LLC-MK2 and Vero cells. Aggregation of multiple RBD mutations significantly reduces the infectivity of the possible three VOCs. Regarding neutralization, it is noteworthy that E484K, N501Y, K417N, and N439K predispose to monoclonal antibodies (mAbs) protection failure in the 15 high-frequency mutations. Most importantly, almost all possible VOCs (single RBD mutation or aggregation of multiple mutations) showed no more than a fourfold decrease in neutralizing activity with convalescent sera, vaccine sera, and immune sera of guinea pigs with different immunogens, and no significant antigenic drift was formed. In conclusion, our pseudovirus results could reduce the concern that the aggregation of multiple high-frequency mutations in the RBD of the spike protein of the three VOCs would lead to severe antigenic drift, and this would provide value for vaccine development strategies.

A platform method for plasmid isoforms analysis by capillary gel electrophoresis.

In the production of novel biological products, plasmids are often engineered into delivery vectors for target genes, which can be used directly as vaccines or as intermediate products for gene/cell therapy. Plasmid DNA exists in several topological forms such as supercoiled, linear, and open circular. As supercoiled plasmid shows the highest efficiency in transfecting eukaryotic cells, the content of supercoiled plasmids becomes an important indicator of plasmid quality. CGE is an effective analysis method for separating different topological structures of plasmids. For the purpose of providing plasmid manufacturers and regulatory agencies with an efficient and readily used tool for monitoring the quality of plasmids, this article identifies the optimal separation and detection conditions of CGE, presents a platform-based plasmid analytical method, and uses plasmid of different sizes to verify the feasibility of this method. In terms of detector, the LIF detector has obvious advantages over the ultraviolet detector in sensitivity and resolution. Using the optimal CE condition (10× gel buffer), baseline separation of different topological forms and impurities can be achieved for different plasmid sizes (5.9, 7.8, 15.4 kb). In addition, 6.5 kb plasmid was used to compare the different separation technologies such as CGE-LIF, ion exchange chromatography and agarose gel electrophoresis. The result shows that CGE-LIF can provide better resolution and quantitation accuracy than ion exchange chromatography and agarose gel electrophoresis. CGE-LIF, as a quick and convenient method to separate and quantify plasmids, has the advantages of high sensitivity, high resolution, and high quantitative accuracy. Therefore, it is ideal for analysis of plasmids with different sizes, and it can also be used as a platform method for manufacturers and regulatory agencies to monitor the purity and stability of plasmids.

Infectivity and antigenicity of pseudoviruses with high-frequency mutations of SARS-CoV-2 identified in Portugal.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had a major impact on global human health. During the spread of SARS-CoV-2, weakened host immunity and the use of vaccines with low efficacy may result in the development of more-virulent strains or strains with resistance to existing vaccines and antibodies. The prevalence of SARS-CoV-2 mutant strains differs between regions, and this variation may have an impact on the effectiveness of vaccines. In this study, an epidemiological investigation of SARS-CoV-2 in Portugal was performed, and the VSV-ΔG-G* pseudovirus system was used to construct 12 spike protein epidemic mutants, D614G, A222V+D614G, B.1.1.7, S477N+D614G, P1162R+D614G+A222V, D839Y+D614G, L176F+D614G, B.1.1.7+L216F, B.1.1.7+M740V, B.1.258, B.1.258+L1063F, and B.1.258+N751Y. The mutant pseudoviruses were used to infect four susceptible cell lines (Huh7, hACE2-293T-293T, Vero, and LLC-MK2) and 14 cell lines overexpressing ACE2 from different species. Mutant strains did not show increased infectivity or cross-species transmission. Neutralization activity against these pseudoviruses was evaluated using mouse serum and 11 monoclonal antibodies. The neutralizing activity of immunized mouse serum was not significantly reduced with the mutant strains, but the mutant strains from Portugal could evade nine of the 11 monoclonal antibodies tested. Neutralization resistance was mainly caused by the mutations S477N, N439K, and N501Y in the spike-receptor binding domain. These findings emphasize the importance of SARS-CoV-2 mutation tracking in different regions for epidemic prevention and control.

Simultaneous determination of capsid proteins in nine-valent human papilloma virus vaccines by liquid chromatography tandem mass spectrometry.

A liquid chromatography-tandem mass spectrometry method was developed to determine nine types of capsid proteins simultaneously in nine-valent human papillomavirus vaccines. Signature peptides were optimized in terms of specificity, repeatability, determination accuracy and sensitivity. As a result, three signature peptides per capsid protein were obtained. The linear calibration curves were achieved in the range of 11.6-373.6 nmol/L (R2  > 0.998). Compared to our previous liquid chromatography-tandem mass spectrometry method, the current method was more sensitive (3.18-fold) and it can be used for quality evaluation of nine-valent human papillomavirus vaccines, unlike the previous method, which could only be used for bivalent human papillomavirus vaccines. Then, they were utilized to determine nine types of capsid proteins in nine-valent human papillomavirus vaccines from four different manufactures. Intraday and interday precision values for the determination of capsid proteins in nine-valent human papillomavirus vaccines were less than 6.8 and 9.1%, respectively. Recovery rates of all capsid proteins investigated were in the range of 80-120%. In addition, the current assay was used for determination of free capsid protein in nine-valent human papilloma virus vaccines, and the results were used to evaluate the adsorption rate of the adjuvant.

Antigenic Drift of Influenza A(H7N9) Virus Hemagglutinin.

Background: Since the emergence of influenza A(H7N9) virus in 2013, there have been 5 waves of influenza A(H7N9) epidemics in China. However, evolution of the hemagglutinin (HA) protein antigenicity has not been systematically investigated. Methods: To better understand how antigenic drift in HA proteins of influenza (A)H7N9 virus occurs, 902 influenza A(H7N9) virus HA protein sequences from a public database were retrieved and analyzed. Fifty-three mutants with single amino acid substitutions in HA protein were introduced into pseudoviruses, and their antigenic characteristics were analyzed using pseudovirus-based assays. Results: The frequencies of 9 mutations incrementally increased over the past 5 years, with mutations identified at multiple sites. While mean neutralization titers of most variants remained unchanged, 3 mutations, A143V, A143T, and R148K, displayed a median 4-fold lower susceptibility to neutralization by antisera against influenza A/Anhui/1/2013(H7N9) virus. Notably, A143V and A143T were located outside the previously reported antigenic sites. The most dominant variant (A143V/R148K) in the most recent season constituted 74.11% of all mutations and demonstrated a 10-fold reduction in its reactivity to influenza A/Anhui/1/2013(H7N9) virus antisera. Importantly, compared with the DNA construct without the corresponding HA protein mutation, DNA vaccine encoding the A143V/R148K mutant induced a 5-fold increase in the neutralizing activity against this circulating virus. Conclusions: An appropriate vaccine strain should be considered in response to increasing antigenic drift in influenza A(H7N9) virus HA protein.

Naturally Occurring Single Amino Acid Substitution in the L1 Major Capsid Protein of Human Papillomavirus Type 16: Alteration of Susceptibility to Antibody-Mediated Neutralization.

Background: Each vaccine for human papillomavirus type 16 (HPV16) has been developed on the basis of a single variant, and whether these vaccines can prevent infection due to naturally occurring variants was not clear. Methods: To examine this question, constructs of 39 naturally occurring single amino acid substitutions in L1 were generated for pseudovirion production, based on the analysis of 1204 HPV16 L1 protein sequences from the National Center for Biotechnology Information and Papilloma Virus Episteme. Results: Thirty-one of 39 HPV16 L1 mutants produced infectious pseudovirions that exhibited similar particle-to-infectivity ratios, compared with reference pseudovirions. Twenty-one of 31 pseudovirion-producing mutants showed different susceptibilities to monoclonal antibodies, with 6 resulting in complete loss of reactivity to some of the tested monoclonal antibodies. The vaccinated sera neutralized all 31 variants. Mean neutralization titers of most variants changed by approximately 4-fold, compared with the reference pseudovirions, with the C428W and K430Q mutations displaying 9-fold and 11-fold lower susceptibilities, respectively, to neutralization by the sera than the reference pseudovirions. Conclusions: These results suggest that the current HPV vaccines may not offer equal protection against all of the naturally occurring HPV16 variants discovered so far.

Multiple human papillomavirus infections and type-competition in women from a clinic attendee population in China.

To investigate the multi-infection patterns and type competition for human papillomavirus (HPV) in Chinese clinic attending women and evaluate the association between the infection status and cancer risk. Three hundred and thirty-two HPV-DNA-positive samples were genotyped for 21 HPVs and tested for 13 types of HPV neutralizing antibody using pseudoviron-based assay. Odds ratios (ORs) were calculated to evaluate the coinfection patterns for both DNA and neutralizing antibody (NAb), and the associations of HSIL+ with HPV DNA and NAb. Of the 332 HPV-DNA-positive subjects, 279 (84.0%) were detected as NAb positive. Multi-positive results were identified in 23.2% (77/332) HPV DNA tests and 60.2% (168/279) HPV NAb assays. The NAb titers for the multiple-positive samples (geometric mean 214) were significantly higher than the single-positive samples (geometric mean 114) (P < 0.01). HPV16-HPV52 was identified as a type-competition pair for both HPV DNA (OR = 0.09; 95%CI = 0.02-0.42) and NAb (OR = 0.27; 95%CI = 0.11-0.69) data. Compared to other types, HPV16 DNA was associated significantly with high risk of HSIL+ (OR = 2.57; 95%CI = 1.23-5.38). HPV16-HPV52 was identified as a potential type-competition pair, which might result in the type modulation with the implementation of the approved bi- or quadri-valent vaccines. J. Med. Virol. 88:1989-1998, 2016. © 2016 Wiley Periodicals, Inc.

Regulation and quality evaluation system for HIV diagnostics in China.

A sophisticated regulatory framework has been constructed for Human immunodeficiency virus (HIV) diagnostics in China, which have developed over the past 30 years. China National Institutes for Food and Drug Control acts as the legal institution in this regulatory framework, launching important activities to ensure the quality of HIV diagnostics. These include the analysis of the main problems faced in developing domestic HIV diagnostics, by investigating the quality of HIV diagnostics and their development; exploring the key factors affecting the quality of HIV diagnostics, to determine the criteria for screening national reference samples; the development of new technologies and methods for preparing reference samples; and the establishment of nine types of national reference panels and nine national standards to evaluate the quality of HIV diagnostics. Based on these researches, a quality evaluation system was established, including nine types of national reference panels, nine national standards for HIV diagnostics, and five sample banks (HIV-positive sample bank, HIV-negative sample bank, common international genotype sample bank, seroconversion series sample bank, HIV virus bank) to evaluate the quality of HIV diagnostics in China. The regulatory framework and the quality evaluation system are pivotal in ensuring the quality of the HIV diagnostics licensed in China.

Optimization and validation of a high throughput method for detecting neutralizing antibodies against human papillomavirus (HPV) based on pseudovirons.

The pseudoviron-based neutralization assay is accepted as the gold standard to evaluate the functional humoral immune response against HPV. The goal of this study was to develop and optimize a human papillomavirus (HPV) neutralization assay using HPV pseudovirons with Gaussia luciferase (Gluc) as the reporter gene. For this purpose, high-titers Gluc pseudovirons were generated by cotransfecting 293TT cells with HPV structural genes and Gluc expressing plasmids. Six types of neutralizing monoclonal antibodies, vaccines immunized serum samples and WHO international antibody standard were used to validate the new developed assay. The ideal circumstances of the assay were identified for cell counts (30,000/well for 96-well plate), pseudoviron inoculating size (100 times RLU above background) and incubation time (72 hr). The sensitivity of the Gluc assay was comparable to secreted alkaline phosphatase (SEAP) assay and higher than the green florescent protein (GFP) assay. The non-specific background for different types of sample was significantly different (rabbit sera > human sera > mouse sera, P < 0.01). The non-specific neutralization effects were not attributed to IgG antibody. The cutoff value for this assay was determined as 50% inhibition at a dilution of 1:40. Without requirements of sample dilution and different incubation times at different temperature before processing, the detection time was shortened from more than 90 min to less than 5 min for a 96-well plate compared with the SEAP-based assay. With the advantages of short detection time and easy-to-use procedure, the newly developed assay is more suitable for large sero-epidemiological studies or clinical trials and more amenable to automation.

SARS-CoV-2 Neutralization Assays Used in Clinical Trials: A Narrative Review.

Since the emergence of COVID-19, extensive research efforts have been undertaken to accelerate the development of multiple types of vaccines to combat the pandemic. These include inactivated, recombinant subunit, viral vector, and nucleic acid vaccines. In the development of these diverse vaccines, appropriate methods to assess vaccine immunogenicity are essential in both preclinical and clinical studies. Among the biomarkers used in vaccine evaluation, the neutralizing antibody level serves as a pivotal indicator for assessing vaccine efficacy. Neutralizing antibody detection methods can mainly be classified into three types: the conventional virus neutralization test, pseudovirus neutralization test, and surrogate virus neutralization test. Importantly, standardization of these assays is critical for their application to yield results that are comparable across different laboratories. The development and use of international or regional standards would facilitate assay standardization and facilitate comparisons of the immune responses induced by different vaccines. In this comprehensive review, we discuss the principles, advantages, limitations, and application of different SARS-CoV-2 neutralization assays in vaccine clinical trials. This will provide guidance for the development and evaluation of COVID-19 vaccines.