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Abstract Previously established World Health Organization (WHO) International Standards (IS) for anti-HPV16 and HPV18 antibodies are used to harmonize results across human papillomavirus (HPV) serology assays. Here, we present an international collaborative study to establish ISs for antibodies against HPV6 (NIBSC code 19/298), HPV11 (20/174), HPV31 (20/176), HPV33 (19/290), HPV45 (20/178), HPV52 (19/296) and HPV58 (19/300). The candidate standards were prepared using sera from naturally infected individuals. Each candidate was shown to be monospecific for reactivity against its indicated HPV type except for the HPV11 candidate, which was also reactive against other types. Expression of antibody levels relative to the relevant candidate IS reduced inter-laboratory variation allowing greater comparability between laboratories. Based on these results, the WHO Expert Committee on Biological Standardization established each of the 7 candidates as the 1st IS for antiserum to its indicated HPV type for use in the standardization of HPV pseudovirion-based neutralization and antibody-binding assays.

ABSTRACTTo summarize the distribution of types of human papillomavirus (HPV) associated with HPV-related diseases and investigate the potential causes of high prevalence of HPV 52 and 58 by summarizing the prevalence of lineages, sub-lineages, and mutations among Chinese women. We searched PubMed, EMBASE, CNKI, and WanFang from January, 2012 to June, 2023 to identify all the eligible studies. We excluded patients who had received HPV vaccinations. Data were summarized in tables and cloud/rain maps. A total of 102 studies reporting HPV distribution and 15 studies reporting HPV52/HPV58 variants were extracted. Among Chinese women, the top five prevalent HPV types associated with cervical cancer (CC) were HPV16, 18, 58, 52, and 33. In patients with vaginal cancers and precancerous lesions, the most common HPV types were 16 and 52 followed by 58. For women with condyloma acuminatum (CA), the most common HPV types were 11 and 6. In Chinese women with HPV infection, lineage B was the most prominently identified for HPV52, and lineage A was the most common for HPV58. In addition to HPV types 16, which is prevalent worldwide, our findings revealed the unique high prevalence of HPV 52/58 among Chinese women with HPV-related diseases. HPV 52 variants were predominantly biased toward lineage B and sub-lineage B2, and HPV 58 variants were strongly biased toward lineage A and sub-lineage A1. Further investigations on the association between the high prevalent lineage and sub-lineage in HPV 52/58 and the risk of cancer risk are needed. Our findings underscore the importance of vaccination with the nine-valent HPV vaccine in China.

Abstract Spike‐protein‐based pseudotyped viruses were used to evaluate vaccines during the COVID‐19 pandemic. However, they cannot be used to evaluate the envelope (E), membrane (M), and nucleocapsid (N) proteins. The first generation of virus‐like particle (VLP) pseudotyped viruses contains these four structural proteins, but their titers for wild‐type severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) are relatively low, even lower for the omicron variant, rendering them unsuitable for neutralizing antibody detection. By optimizing the spike glycoprotein signal peptide, substituting the complexed M and E proteins with SARS‐COV‐1, optimizing the N protein with specific mutations (P199L, S202R, and R203M), and truncating the packaging signal, PS9, we increased the titer of the wild‐type VLP pseudotyped virus over 100‐fold, and successfully packaged the omicron VLP pseudotyped virus. The SARS‐CoV‐2 VLP pseudotyped viruses maintained stable titers, even through 10 freeze–thaw cycles. The key neutralization assay parameters were optimized, including cell type, cell number, and viral inoculum. The assay demonstrated minimal variation in both intra‐ and interassay results, at 11.5% and 11.1%, respectively. The correlation between the VLP pseudotyped virus and the authentic virus was strong (r = 0.9). Suitable for high‐throughput detection of various mutant strains in clinical serum. In summary, we have developed a reliable neutralization assay for SARS‐CoV‐2 based on VLP pseudotyped virus.

Summary: The intact proviral DNA assay (IPDA) based on droplet digital PCR was developed to identify intact proviral DNA and quantify HIV-1 latency reservoirs in patients infected with HIV-1. However, the genetic characteristics of different HIV-1 subtypes are non-consistent due to their high mutation and recombination rates. Here, we identified that the IPDA based on the sequences features of an HIV-1 subtype could not effectively detect different HIV-1 subtypes due to the high diversity of HIV-1. Furthermore, we demonstrated that mutations in env gene outside the probe binding site affect the detection efficiency of IPDA. Since mutations in env gene outside the probe binding site may also lead to the formation of stop codons, thereby preventing the formation of viruses and ultimately overestimating the number of HIV-1 latency reservoirs, it is important to address the effect of mutations on the IPDA.

Abstract Regarding the extensive global attention to severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) that constitutes an international public health emergency, pseudovirus neutralization assays have been widely applied due to their advantages of being able to be conducted in biosafety level 2 laboratories and having a high safety factor. In this study, by adding a blue fluorescent protein (AmCyan) gene to the HIV system pSG3‐△env backbone plasmid HpaI and truncating the C‐terminal 21 amino acids of the SARS‐CoV‐2 spike protein (S), high‐titer SARS‐CoV‐2‐Sdel21‐AmCyan fluorescent pseudovirus was successfully packaged. The fluorescent pseudovirus was used to establish a neutralization assay in a 96‐well plate using 293T cells stably transfected with the AF cells. Then, parameters such as the ratio of backbone and membrane plasmid, sensitive cells, inoculation of cells and virus, as well as incubation and detection time were optimized. The pseudovirus neutralization assay demonstrated high accuracy, sensitivity, repeatability, and a strong correlation with the luminescent pseudovirus neutralization assay. Additionally, we scaled up the neutralizing antibody determination method by increasing the plate size from 96 wells to 384 wells. We have established a robust fluorescent pseudotyped virus neutralization assay for SARS‐CoV‐2 using the HIV system, providing a foundation for serum neutralization antibody detection, monoclonal antibody screening, and vaccine development.

IntroductionHepatitis E virus (HEV), with heightened virulence in immunocompromised individuals and pregnant women, is a pervasive threat in developing countries. A globaly available vaccine against HEV is currently lacking.MethodsWe designed a multi-epitope vaccine based on protein ORF2 and ORF3 of HEV using immunoinformatics.ResultsThe vaccine comprised 23 nontoxic, nonallergenic, soluble peptides. The stability of the docked peptide vaccine-TLR3 complex was validated by molecular dynamic simulations. The induction of effective cellular and humoral immune responses by the multi-peptide vaccine was verified by simulated immunization.DiscussionThese findings provide a foundation for future HEV vaccine studies.

The global outbreak of COVID-19 has caused a severe threat to human health; therefore, simple, high-throughput neutralization assays are desirable for developing vaccines and drugs against COVID-19. In this study, a high-titre SARS-CoV-2 pseudovirus was successfully packaged by truncating the C-terminus of the SARS-CoV-2 spike protein by 21 amino acids and infecting 293 T cells that had been stably transfected with the angiotensin-converting enzyme 2 (ACE2) receptor and furin (named AF cells), to establish a simple, high-throughput, and automated 384-well plate neutralization assay. The method was optimized for cell amount, virus inoculation, incubation time, and detection time. The automated assay showed good sensitivity, accuracy, reproducibility, Z’ factor, and a good correlation with the live virus neutralization assay. The high-throughput approach would make it available for the SARS-CoV-2 neutralization test in large-scale clinical trials and seroepidemiological surveys which would aid the accelerated vaccine development and evaluation.

From December 2022 to January 2023, SARS-CoV-2 infections caused by BA.5 and BF.7 subvariants of B.1.1.529 (Omicron) spread in China. It is urgently needed to evaluate the protective immune responses in the infected individuals against the current circulating variants to predict the future potential infection waves, such as the BQ.1.1, XBB.1.5, and CH1.1 variants. In this study, we constructed a panel of pseudotyped viruses for SARS-CoV-2 for the past and current circulating variants, including D614G, Delta, BA.1, BA.5, BF.7, BQ.1.1, XBB.1.5 and CH.1.1. We investigated the neutralization sensitivity of these pseudotyped viruses to sera from individuals who had BA.5 or BF.7 breakthrough infections in the infection wave of last December in China. The mean neutralization ID50 against infected variants BA.5 and BF.7 are 533 and 444, respectively. The highest neutralizing antibody level was observed when tested against the D614G strain, with the ID50 of 742, which is about 1.52-folds higher than that against the BA.5/BF.7 variant. The ID50 for BA.1, Delta, and BQ.1.1 pseudotyped viruses were about 2–3 folds lower when compared to BA.5/BF.7. The neutralization activities of these serum samples against XBB.1.5 and CH.1.1 decreased 7.39-folds and 15.25-folds when compared to that against BA.5/BF.7. The immune escape capacity of these two variants might predict new infection waves in future when the neutralizing antibody levels decrease furtherly.

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.

The efficacy of many coronavirus disease 2019 (COVID-19) vaccines has been shown to decrease to varying extents against new severe acute respiratory syndrome coronavirus 2 variants, which are responsible for the continuing COVID-19 pandemic. Combining intramuscular and intranasal vaccination routes is a promising approach for achieving more potent immune responses. We evaluated the immunogenicity of prime-boost protocols with a chimpanzee adenovirus serotype 68 vector-based vaccine, ChAdTS-S, administered via both intranasal and intramuscular routes in BALB/c mice. Intramuscular priming followed by an intranasal booster elicited the highest levels of IgG, IgA, and pseudovirus neutralizing antibody titres among all the protocols tested at day 42 after prime immunization compared with the intranasal priming/intramuscular booster and prime-boost protocols using only one route. In addition, intramuscular priming followed by an intranasal booster induced high T-cell responses, measured using the IFN-γ ELISpot assay, that were similar to those observed upon intramuscular vaccination. All ChAdTS-S vaccination groups induced Th1-skewing of the T-cell response according to intracellular cytokine staining and Meso Scale Discovery cytokine profiling assays on day 56 after priming. This study provides reference data for assessing vaccination schemes of adenovirus-based COVID-19 vaccines with high immune efficacy.

Severe acute respiratory syndrome coronavirus 2 variants have continued to emerge in diverse geographic locations with a temporal distribution. The Lambda variant containing multiple mutations in the spike protein, has thus far appeared mainly in South America. The variant harbours two mutations in the receptor binding domain, L452Q and F490S, which may change its infectivity and antigenicity to neutralizing antibodies. In this study, we constructed 10 pseudoviruses to study the Lambda variant and each individual amino acid mutation's effect on viral function, and used eight cell lines to study variant infectivity. In total, 12 monoclonal antibodies, 14 convalescent sera, and 23 immunized sera induced by mRNA vaccines, inactivated vaccine, and adenovirus type 5 vector vaccine were used to study the antigenicity of the Lambda variant. We found that compared with the D614G reference strain, Lambda demonstrated enhanced infectivity of Calu-3 and LLC-MK2 cells by 3.3-fold and 1.6-fold, respectively. Notably, the sensitivity of the Lambda variant to 5 of 12 neutralizing monoclonal antibodies, 9G11, AM180, R126, X593, and AbG3, was substantially diminished. Furthermore, convalescent- and vaccine-immunized sera showed on average 1.3–2.5-fold lower neutralizing titres against the Lambda variant. Single mutation analysis revealed that this reduction in neutralization was caused by L452Q and F490S mutations. Collectively, the reduced neutralization ability of the Lambda variant suggests that the efficacy of monoclonal antibodies and vaccines may be compromised during the current pandemic.

The ubiquitously-expressed proteolytic enzyme furin is closely related to the pathogenesis of SARS-CoV-2 and therefore represents a key target for antiviral therapy. Based on bioinformatic analysis and pseudovirus tests, we discovered a second functional furin site located in the spike protein. Furin still increased the infectivity of mutated SARS-CoV-2 pseudovirus in 293T-ACE2 cells when the canonical polybasic cleavage site (682–686) was deleted. However, K814A mutation eliminated the enhancing effect of furin on virus infection. Furin inhibitor prevented infection by 682–686-deleted SARS-CoV-2 in 293T-ACE2-furin cells, but not the K814A mutant. K814A mutation did not affect the activity of TMPRSS2 and cathepsin L but did impact the cleavage of S2 into S2′ and cell–cell fusion. Additionally, we showed that this functional furin site exists in RaTG13 from bat and PCoV-GD/GX from pangolin. Therefore, we discovered a new functional furin site that is pivotal in promoting SARS-CoV-2 infection.

The emergence of Omicron/BA.1 has brought new challenges to fight against SARS-CoV-2. A large number of mutations in the Spike protein suggest that its susceptibility to immune protection elicited by the existing COVID-19 infection and vaccines may be altered. In this study, we constructed the pseudotyped SARS-CoV-2 variant Omicron. The sensitivity of 28 serum samples from COVID-19 convalescent patients infected with SARS-CoV-2 original strain was tested against pseudotyped Omicron as well as the other variants of concern (VOCs, Alpha, Beta, Gamma, Delta) and variants of interest (VOIs, Lambda, Mu). Our results indicated that the mean neutralization ED50 of these sera against Omicron decreased to 66, which is about 8.4-folds compared to the D614G reference strain (ED50 = 556), whereas the neutralization activity of other VOC and VOI pseudotyped viruses decreased only about 1.2–4.5-folds. The finding from our in vitro assay suggest that Omicron variant may lead to more significant escape from immune protection elicited by previous SARS-CoV-2 infection and perhaps even by existing COVID-19 vaccines.

SARS-CoV-2 has caused the COVID-19 pandemic. B.1.617 variants (including Kappa and Delta) have been transmitted rapidly in India. The transmissibility, pathogenicity, and neutralization characteristics of these variants have received considerable interest. In this study, 22 pseudotyped viruses were constructed for B.1.617 variants and their corresponding single amino acid mutations. B.1.617 variants did not exhibit significant enhanced infectivity in human cells, but mutations T478K and E484Q in the receptor binding domain led to enhanced infectivity in mouse ACE2-overexpressing cells. Furin activities were slightly increased against B.1.617 variants and cell–cell fusion after infection of B.1.617 variants were enhanced. Furthermore, B.1.617 variants escaped neutralization by several mAbs, mainly because of mutations L452R, T478K, and E484Q in the receptor binding domain. The neutralization activities of sera from convalescent patients, inactivated vaccine-immunized volunteers, adenovirus vaccine-immunized volunteers, and SARS-CoV-2 immunized animals against pseudotyped B.1.617 variants were reduced by approximately twofold, compared with the D614G variant.

Abstract A steep rise in Omicron reinfection cases suggests that this variant has increased immune evasion ability. To evaluate its antigenicity relationship with other variants, antisera from guinea pigs immunized with spike protein of SARS-CoV-2 variants of concern (VOCs) and variants of interest (VOIs) were cross-tested against pseudotyped variants. The neutralization activity against Omicron was markedly reduced when other VOCs or VOIs were used as immunogens, and Omicron (BA.1)-elicited sera did not efficiently neutralize the other variants. However, a Beta or Omicron booster, when administered as the 4th dose 3-months after the 3rd dose of any of the variants, could elicit broad neutralizing antibodies against all of the current variants including Omicron BA.1. Further analysis with 280 available antigen–antibody structures and quantification of immune escape from 715 reported neutralizing antibodies provide explanations for the observed differential immunogenicity. Three distinct clades predicted using an in silico algorithm for clustering of sarbecoviruses based on immune escape provide key information for rational design of vaccines.

The ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 or 2019-nCoV) variants has been associated with the transmission and pathogenicity of COVID-19. Therefore, exploring the optimal immunisation strategy to improve the broad-spectrum cross-protection ability of COVID-19 vaccines is of great significance. Herein, we assessed different heterologous prime-boost strategies with chimpanzee adenovirus vector-based COVID-19 vaccines plus Wuhan-Hu-1 (WH-1) strain (AdW) and Beta variant (AdB) and mRNA-based COVID-19 vaccines plus WH-1 strain (ARW) and Omicron (B.1.1.529) variant (ARO) in 6-week-old female BALB/c mice. AdW and AdB were administered intramuscularly or intranasally, while ARW and ARO were administered intramuscularly. Intranasal or intramuscular vaccination with AdB followed by ARO booster exhibited the highest levels of cross-reactive IgG, pseudovirus-neutralising antibody (PNAb) responses, and angiotensin-converting enzyme-2 (ACE2)-binding inhibition rates against different 2019-nCoV variants among all vaccination groups. Moreover, intranasal AdB vaccination followed by ARO induced higher levels of IgA and neutralising antibody responses against live 2019-nCoV than intramuscular AdB vaccination followed by ARO. A single dose of AdB administered intranasally or intramuscularly induced broader cross-NAb responses than AdW. Th1-biased cellular immune response was induced in all vaccination groups. Intramuscular vaccination-only groups exhibited higher levels of Th1 cytokines than intranasal vaccination-only and intranasal vaccination-containing groups. However, no obvious differences were found in the levels of Th2 cytokines between the control and all vaccination groups. Our findings provide a basis for exploring vaccination strategies against different 2019-nCoV variants to achieve high broad-spectrum immune efficacy.

Abstract Emerging SARS-CoV-2 variants are the most serious problem for COVID-19 prophylaxis and treatment. To determine whether the SARS-CoV-2 vaccine strain should be updated following variant emergence like seasonal flu vaccine, the changed degree on antigenicity of SARS-CoV-2 variants and H3N2 flu vaccine strains was compared. The neutralization activities of Alpha, Beta and Gamma variants’ spike protein-immunized sera were analysed against the eight current epidemic variants and 20 possible variants combining the top 10 prevalent RBD mutations based on the Delta variant, which were constructed using pseudotyped viruses. Meanwhile, the neutralization activities of convalescent sera and current inactivated and recombinant protein vaccine-elicited sera were also examined against all possible Delta variants. Eight HA protein-expressing DNAs elicited-animal sera were also tested against eight pseudotyped viruses of H3N2 flu vaccine strains from 2011–2019. Our results indicate that the antigenicity changes of possible Delta variants were mostly within four folds, whereas the antigenicity changes among different H3N2 vaccine strains were approximately 10–100-fold. Structural analysis of the antigenic characterization of the SARS-CoV-2 and H3N2 mutations supports the neutralization results. This study indicates that the antigenicity changes of the current SARS-CoV-2 may not be sufficient to require replacement of the current vaccine strain.

Li Zhang, Zhimin Cui, and Qianqian Li et al. compare the infectivity, host tropism, and antigenicity of 10 SARS-CoV-2 variants using a VSV-based pseudovirus system. Their results suggest that variants carrying E484K display the most significant reduction in sensitivity to neutralization, and may provide further insight into the development of relevant therapeutics for SARS-CoV-2 infection.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains widely pandemic around the world. Animal models that are sensitive to the virus are therefore urgently needed to evaluate potential vaccines and antiviral agents; however, SARS-CoV-2 requires biosafety level 3 containment. To overcome this, we developed an animal model using the intranasal administration of SARS-CoV-2 pseudovirus. As the pseudovirus contains the firefly luciferase reporter gene, infected tissues and the viral load could be monitored by in vivo bioluminescent imaging. We used the model to evaluate the protective efficacy of monoclonal antibodies and the tissue tropism of different variants. The model may also be a useful tool for the safe and convenient preliminary evaluation of the protective efficacy of vaccine candidates against SARS-CoV-2, as well as the treatment efficacy of anti-viral drugs.

Abstract Coronavirus disease 2019 (COVID-19), a pandemic disease caused by the newly emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused more than 3.8 million deaths to date. Neutralizing antibodies are effective therapeutic measures. However, many naturally occurring mutations at the receptor-binding domain (RBD) have emerged, and some of them can evade existing neutralizing antibodies. Here, we utilized RenMab, a novel mouse carrying the entire human antibody variable region, for neutralizing antibody discovery. We obtained several potent RBD-blocking antibodies and categorized them into four distinct groups by epitope mapping. We determined the involved residues of the epitope of three representative antibodies by cryo-electron microscopy (Cryo-EM) studies. Moreover, we performed neutralizing experiments with 50 variant strains with single or combined mutations and found that the mixing of three epitope-distinct antibodies almost eliminated the mutant escape. Our study provides a sound basis for the rational design of fully human antibody cocktails against SARS-CoV-2 and pre-emergent coronaviral threats.

Abstract The severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) variants, particularly those with multiple mutations in receptor‐binding domain (RBD), pose a critical challenge to the efficacy of coronavirus disease 2019 (COVID‐19) vaccines and therapeutic neutralizing monoclonal antibodies (mAbs). Omicron sublineages BA.1, BA.2, BA.3, as well as the recent emergence of C.1.2, B.1.630, B.1.640.1, and B.1.640.2, have multiple mutations in RBD and may lead to severe neutralizing antibody evasion. It is urgent to evaluate the antigenic change of the above seven variants against mAbs and sera from guinea pigs immunized with variants of concern (VOCs) (Alpha, Beta, Gamma, Delta, Omicron) and variants of interest (VOIs) (Lambda, Mu) immunogens. Only seven out of the 24 mAbs showed no reduction in neutralizing activity against BA.1, BA.2, and BA.3. However, among these seven mAbs, the neutralization activity of XGv337 and XGv338 against C.1.2, B.1.630, B.1.640.1, and B.1.640.2 were decreased. Therefore, only five neutralizing mAbs showed no significant change against these seven variants. Using VOCs and VOIs as immunogens, we found that the antigenicity of variants could be divided into three clusters, and each cluster showed similar antigenicity to different immunogens. Among them, D614G, B.1.640.1, and B.1.630 formed a cluster, C.1.2 and B.1.640.2 formed a cluster, and BA.1, BA.2, and BA.3 formed a cluster.

Abstract The origin and intermediate host for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is yet to be determined. Coronaviruses found to be closely related to SARS-CoV-2 include RaTG13 derived from bat and two clusters (PCoV-GD and PCoV-GX) of coronaviruses identified in pangolin. Here, we studied the infectivity and antigenicity patterns of SARS-CoV-2 and the three related coronaviruses. Compared with the other three viruses, RaTG13 showed almost no infectivity to a variety of cell lines. The two pangolin coronaviruses and SARS-CoV-2 showed similar infectious activity. However, in SARS-CoV-2-susceptible cell lines, the pangolin coronaviruses presented even higher infectivity. The striking difference between the SARS-CoV-2 and pangolin coronaviruses is that the latter can infect porcine cells, which could be partially attributed to an amino acid difference at the position of 498 of the spike protein. The infection by SARS-CoV-2 was mainly mediated by Furin and TMPRSS2, while PCoV-GD and PCoV-GX mainly depend on Cathepsin L. Extensive cross-neutralization was found between SARS-CoV-2 and PCoV-GD. However, almost no cross-neutralization was observed between PCoV-GX and SARS-CoV-2 or PCoV-GD. More attention should be paid to pangolin coronaviruses and to investigate the possibility of these coronaviruses spreading across species to become zoonoses among pigs or humans.

Hand-foot-and-mouth disease is a contagious disease common among children under 5 years old worldwide. It is caused by strains of enterovirus, especially EV-A71, which can lead to severe disease. Vaccines are the only way to fight this disease. Accordingly, it is necessary to establish an efficient and accurate methodology to evaluate vaccine efficacy in vivo. Here, we established a practical method using a hSCARB2 knock-in mouse model, which was susceptible to EV-A71 infection at 5–6 weeks of age, to directly determine the efficacy of vaccines. Unlike traditional approaches, one-week-old hSCARB2 mice were immunized twice with a licensed vaccine, with an interval of a week. The titre of antibodies was measured after 1 week. Mice at 4 weeks of age were challenged with EV-A71 intraperitoneally and intracranially, respectively. The unimmunized hSCARB2 mice displayed systemic clinical symptoms and succumbed to the disease at a rate of approximately 50%. High viral loads were detected in the lungs, brain, and muscles, accompanied by clear pathological changes. The expression of IL-1β, IL-13, IL-17, and TNF-α was significantly upregulated. By contrast, the immunized group was practically normal and indistinguishable from the control mice. These results indicate that the hSCARB2 knock-in mouse is susceptible to infection in adulthood, and the in vivo efficacy of EV-A71 vaccine could be directly evaluated in this mouse model. The method developed here may be used in the development of new vaccines against HFMD or quality control of licensed vaccines.

ABSTRACTPseudoviruses are useful virological tools because of their safety and versatility, especially for emerging and re-emerging viruses. Due to its high pathogenicity and infectivity and the lack of effective vaccines and therapeutics, live SARS-CoV-2 has to be handled under biosafety level 3 conditions, which has hindered the development of vaccines and therapeutics. Based on a VSV pseudovirus production system, a pseudovirus-based neutralization assay has been developed for evaluating neutralizing antibodies against SARS-CoV-2 in biosafety level 2 facilities. The key parameters for this assay were optimized, including cell types, cell numbers, virus inoculum. When tested against the SARS-CoV-2 pseudovirus, SARS-CoV-2 convalescent patient sera showed high neutralizing potency, which underscore its potential as therapeutics. The limit of detection for this assay was determined as 22.1 and 43.2 for human and mouse serum samples respectively using a panel of 120 negative samples. The cutoff values were set as 30 and 50 for human and mouse serum samples, respectively. This assay showed relatively low coefficient of variations with 15.9% and 16.2% for the intra- and inter-assay analyses respectively. Taken together, we established a robust pseudovirus-based neutralization assay for SARS-CoV-2 and are glad to share pseudoviruses and related protocols with the developers of vaccines or therapeutics to fight against this lethal virus.

ABSTRACTTo better evaluate HIV-1 vaccines and therapeutics, the National Institutes for Food and Drug Control of China developed a panel of HIV-1 pseudoviruses including 462 viral strains derived from China, covering the majority of contemporaneous subtypes and circulating recombinant forms. Compared with the standard pseudovirus panels derived from other countries/regions, the Chinese isolates are more susceptible to neutralization by the sera obtained in China, revealing the strain/subtype specificity. Some of these pseudoviruses have already been used for the evaluation of HIV vaccines and drug candidates in Chinese clinical trials. The pseudoviruses panel is widely shared with interested scientists involved in the research and development of vaccines and antiviral drugs against HIV-1 strains prevalent in China.

To determine whether the neutralization activity of monoclonal antibodies, convalescent sera and vaccine-elicited sera was affected by the top five epidemic SARS-CoV-2 variants in the UK, including D614G+L18F+A222V, D614G+A222V, D614G+S477N, VOC-202012/01(B.1.1.7) and D614G+69-70del+N439K, a pseudovirus-neutralization assay was performed to evaluate the relative neutralization titers against the five SARS-CoV-2 variants and 12 single deconvolution mutants based on the variants. In this study, 18 monoclonal antibodies, 10 sera from convalescent COVID-19 patients, 10 inactivated-virus vaccine-elicited sera, 14 mRNA vaccine-elicited sera, nine RBD-immunized mouse sera, four RBD-immunized horse sera, and four spike-encoding DNA-immunized guinea pig sera were tested and analyzed. The N501Y, N439K, and S477N mutations caused immune escape from nine of 18 mAbs. However, the convalescent sera, inactivated virus vaccine-elicited sera, mRNA vaccine-elicited sera, spike DNA-elicited sera, and recombinant RBD protein-elicited sera could still neutralize these variants (within three-fold changes compared to the reference D614G variant). The neutralizing antibody responses to different types of vaccines were different, whereby the response to inactivated-virus vaccine was similar to the convalescent sera.

Rift Valley fever virus (RVFV), a recognized category A priority pathogen, causes large outbreaks of Rift Valley fever with some fatalities in humans in humans and huge economic losses in livestock. As wild-type RVFV must be handled in BSL-3 or BSL-4 laboratories, we constructed a high-titer vesicular stomatitis virus (VSV) pseudotype bearing RVFV envelope glycoproteins to detect neutralizing antibodies in vitro under BSL-2 conditions. The neutralizing properties of 39 amino acid mutant sites that have occurred naturally over time in the RVFV envelope glycoproteins were analyzed with their corresponding pseudoviral mutants separately. Compared with the results in the primary strain, the variants showed no statistically significant differences. We next established a Balb/c mouse pseudovirus infection model for detecting neutralizing antibodies against pseudovirus. Five immunizations with pseudoviral DNA protected the mice from infection with the pseudovirus. Bioluminescence imaging, which we used to evaluate viral dissemination and distribution in the mice, showed a good relationship between the neutralizing antibodies titers in vitro. These pseudovirus methods will allow for the safe determination of neutralizing antibodies in vivo and in vitro, and will assist with studies on vaccines and drugs against RVFV with the long term objective of Rift Valley fever prevention.

ABSTRACTBecause of its high infectivity in humans and the lack of effective vaccines, Nipah virus is classified as a category C agent and handling has to be performed under biosafety level 4 conditions in non-endemic countries, which has hindered the development of vaccines. Based on a highly efficient pseudovirus production system using a modified HIV backbone vector, a pseudovirus-based mouse model has been developed for evaluating the efficacy of Nipah vaccines in biosafety level 2 facilities. For the first time, the correlates of protection have been identified in a mouse model. The limited levels of neutralizing antibodies against immunogens fusion protein (F), glycoprotein (G), and combination of F and G (FG) were found to be 148, 275, and 115, respectively, in passive immunization. Relatively lower limited levels of protection of 52, and 170 were observed for immunogens F, and G, respectively, in an active immunization model. Although the minimal levels for protection of neutralizing antibody in passive immunization were slightly higher than those in active immunization, neutralizing antibody played a key role in protection against Nipah virus infection. The immunogens F and G provided similar protection, and the combination of these immunogens did not provide better outcomes. Either immunogen F or G would provide sufficient protection for Nipah vaccine. The Nipah pseudovirus mouse model, which does not involve highly pathogenic virus, has the potential to greatly facilitate the standardization and implementation of an assay to propel the development of NiV vaccines.

ABSTRACTThe genetic and/or antigenic differences between street rabies virus (RABV) and vaccine strains could potentially affect effectiveness of rabies vaccines. As such, it is important to continue monitoring the glycoprotein (G) of the street isolates. All RABVG sequences in public database were retrieved and analysed. Using a pseudovirus system, we investigated 99 naturally occurring mutants for their reactivities to well-characterized neutralizing monoclonal antibodies (mAbs) and vaccine-induced antisera. A divergence in G sequences was found between vaccine strains and recent street isolates, with mutants demonstrating resistance to neutralizing mAbs and vaccine-induced antibodies. Moreover, antigenic variants were observed in a wide range of animal hosts and geographic locations, with most of them emerging since 2010. As the number of antigenic variants has increased in recent years, close monitoring on street isolates should be strengthened.

Chronic hepatitis E virus (HEV) infection is frequently reported in immunocompromised patients, but has also been increasingly reported in non-immunocompromised individuals. We characterized the course of chronic HEV infection in immunocompetent rabbits. In two independent experiments, 40 specific-pathogen-free rabbits were infected with a rabbit HEV genotype 3 strain in serial diluted titers (108 to 104 copies/mL). Serum and fecal samples were collected weekly and were tested for HEV RNA, antigen, anti-HEV and liver enzymes. Rabbits that spontaneously cleared the infection before 10 weeks post-inoculation (wpi) were kept to the end of the study as recovery control. Liver tissues were collected from HEV-infected rabbits at 5, 10 and 26 wpi for histopathological analysis. Nineteen rabbits (47.5%) developed chronic HEV infection with persistent viraemia and fecal HEV shedding for >6 months. Seroconversion to anti-HEV was observed in 84.2% (16/19) of the chronically infected rabbits. Serum levels of aminotransferase were persistently elevated in most of the rabbits. Characterizations of chronic HEV infection in immunocompetent settings could be recapitulated in rabbits, which can serve as a valuable tool for future studies on pathogenesis.

Sensitive assays for HIV-1 neutralizing antibody detection are urgently needed for vaccine immunogen optimization and identification of protective immune response levels. In this study, we developed an easy-to-use HIV-1 pseudovirus neutralization assay based on a human CD4+ lymphoblastoid cell line A3R5 by employing a high efficient pseudovirus production system. Optimal conditions for cell counts, infection time, virus dose and concentration of DEAE-dextran were tested and identified. For T-cell line-adapted tier 1 virus strains, significantly higher inhibitory efficiency was observed for both monoclonal neutralizing antibody (4 fold) and plasma (2 fold) samples in A3R5 than those in TZM-bl assay. For circulating tier 2 strains, the A3R5 pseudovirus assay showed even much higher sensitivity for both neutralizing antibody (10 fold) and plasma (9 fold) samples. When sequential neutralizing antibody seroconverting samples were tested in both A3R5 and TZM-bl assays, the seroconverting points could be detected earlier for tier 1 (15.7 weeks) and tier 2 (68.3 weeks) strains in A3R5 assay respectively. The high sensitive pseudovirus assay using more physiological target cells could serve as an alternative to the TZM-bl assay for evaluation of vaccine-induced neutralizing antibodies and identification of the correlates of protection.

Marburg virus (MARV) can cause lethal hemorrhagic fever in humans. Handling of MARV is restricted to high-containment biosafety level 4 (BSL-4) facilities, which greatly impedes research into this virus. In this study, a high titer of MARV pseudovirus was generated through optimization of the HIV backbone vectors, the ratio of backbone vector to MARV glycoprotein expression vector, and the transfection reagents. An in vitro neutralization assay and an in vivo bioluminescent imaging mouse model for MARV were developed based on the pseudovirus. Protective serum against MARV was successfully induced in guinea pigs, which showed high neutralization activity in vitro and could also protect Balb/c mice from MARV pseudovirus infection in vivo. This system could be a convenient tool to enable the evaluation of vaccines and therapeutic drugs against MARV in non-BSL-4 laboratories.

Pseudovirion-based neutralization assay is considered the gold standard method for evaluating the immune response to human papillomavirus (HPV) vaccines. In this study, we developed a multicolor neutralization assay to simultaneously detect the neutralizing antibodies against different HPV types. FluoroSpot was used to interpret the fluorescent protein expression instead of flow cytometry. The results of FluoroSpot and flow cytometry showed good consistency, with R2 > 0.98 for the log-transformed IC50 values. Regardless of the reporter color, the single-, dual-, and triple-color neutralization assays reported identical results for the same samples. In low-titer samples from naturally HPV-infected individuals, there was strong agreement between the single- and triple-color assays, with kappa scores of 0.92, 0.89, and 0.96 for HPV16, HPV18, and HPV58, respectively. Good reproducibility was observed for the triple-color assay, with coefficients of variation of 2.0%–41.5% within the assays and 8.3%–36.2% between the assays. Three triple-color systems, HPV16-18-58, HPV6-33-45, and HPV11-31-52, were developed that could evaluate the immunogenicity of a nonavalent vaccine in three rounds of the assay. With the advantages of an easy-to-use procedure and less sample consumption, the multiple-color assay is more suitable than classical assays for large sero-epidemiological studies and clinical trials and is more amenable to automation.

BACKGROUND: Pre-existing immunity to Vaccinia Tian Tan virus (VTT) resulting from a large vaccination campaign against smallpox prior to the early 1980s in China, has been a major issue for application of VTT-vector based vaccines. It is essential to establish a sensitive and high-throughput neutralization assay to understand the epidemiology of Vaccinia-specific immunity in current populations in China. METHODOLOGY/PRINCIPAL FINDINGS: A new anti-Vaccinia virus (VACV) neutralization assay that used the attenuated replication-competent VTT carrying the firefly luciferase gene of Photinus pyralis (rTV-Fluc) was established and standardized for critical parameters that included the choice of cell line, viral infection dose, and the infection time. The current study evaluated the maintenance of virus-specific immunity after smallpox vaccination by conducting a non-randomized, cross-sectional analysis of antiviral antibody-mediated immune responses in volunteers examined 30-55 years after vaccination. The rTV-Fluc neutralization assay was able to detect neutralizing antibodies (NAbs) against Vaccinia virus without the ability to differentiate strains of Vaccinia virus. We showed that the neutralizing titers measured by our assay were similar to those obtained by the traditional plaque reduction neutralization test (PRNT). Using this assay, we found a low prevalence of NAb to VTT (7.6%) in individuals born before 1980 from Beijing and Anhui provinces in China, and when present, anti-VTT NAb titers were low. No NAbs were detected in all 222 samples from individuals born after 1980. There was no significant difference observed for titer or prevalence by gender, age range and geographic origin. CONCLUSION: A simplified, sensitive, standardized, reproducible, and high-throughput assay was developed for the quantitation of NAbs against different Vaccinia strains. The current study provides useful insights for the future development of VTT-based vaccination in Beijing and Anhui provinces of China.

BACKGROUND: The presence of various levels of Adenovirus serotype 5 neutralizing antibodies (Ad5NAb) is thought to contribute to the inconsistent clinical results obtained from vaccination and gene therapy studies. Currently, two platforms based on high-throughput technology are available for Ad5NAb quantification, chemiluminescence- and fluorescence-based assays. The aim of this study was to compare the results of two assays in the seroepidemiology of Ad5NAb in a local population of donors. METHODOLOGY/PRINCIPAL FINDINGS: The fluorescence-based neutralizing antibody detection test (FRNT) using recombinant Ad5-EGFP virus and the chemiluminescence-based neutralizing antibody test (CLNT) using Ad5-Fluc were developed and standardized for detecting the presence of Ad5NAb in serum samples from the population of donors in Beijing and Anhui provinces, China. First, the overall percentage of people positive for Ad5NAb performed by CLNT was higher than that obtained by FRNT (85.4 vs 69.9%, p

BACKGROUND: The vaccinia virus Guang9 strain (VG9), derived from the vaccinia virus Tian Tan strain (VTT) has been found to be less virulent than VTT. METHODOLOGY/PRINCIPAL FINDINGS: To investigate whether VG9 could be a potential replicating virus vector, the TK genes in VG9 and VTT were replaced with the HIV-1 envelope gene via homologous recombination, resulting in the recombinant viruses, VG9-E and VTT-E. The biology, virulence, humoral and cellular immunological responses of VG9-E and VTT-E were evaluated. Our results indicated no obvious difference in range of host cells and diffusion between two recombinant viruses. Neurovirulence for VG9-E in weanling and suckling mice, and skin virulence in rabbits, were lower than that of VTT-E. The humoral immune responses, including binding antibody and neutralizing antibody responses, induced by VG9-E were not significantly different from those for VTT-E whilst IFN-γ response which represented cellular immune response induced by VG9-E was significantly higher than that did by VTT-E. CONCLUSIONS/SIGNIFICANCE: Our results indicated that VG9-E was less virulent, yet induced higher cellular immune response than VTT-E. Therefore, it could be an ideal replicating vaccinia vector for HIV vaccine research and development.