Antigenic analysis of the influenza B virus hemagglutinin protein.

Influenza B viruses (IBVs) primarily infect humans and are a common cause of respiratory infections in humans. Here, to systematically analyze the antigenicity of the IBVs Hemagglutinin (HA) protein, 31 B/Victoria and 19 B/Yamagata representative circulating strains were selected from Global Initiative of Sharing All Influenza Data (GISAID), and pseudotyped viruses were constructed with the vesicular stomatitis virus system. Guinea pigs were immunized with three doses of vaccines (one dose of DNA vaccines following two doses of pseudotyped virus vaccines) of the seven IBV vaccine strains, and neutralizing antibodies against the pseudotyped viruses were tested. By comparing differences between various vaccine strains, we constructed several pseudotyped viruses that contained various mutations based on vaccine strain BV-21. The vaccine strains showed good neutralization levels against the epidemic virus strains of the same year, with neutralization titers ranging from 370 to 840, while the level of neutralization against viruses prevalent in previous years decreased 1-10-fold. Each of the high-frequency epidemic strains of B/Victoria and B/Yamagata not only induced high neutralizing titers, but also had broadly neutralizing effects against virus strains of different years, with neutralizing titers ranging from 1000 to 7200. R141G, D197N, and R203K were identified as affecting the antigenicity of IBV. In this study, pseudotyped virus system was used to monitor the cross-neutralizing efficacy of high-frequency epidemic strains and vaccine strains recommended by the World Health Organization. Additionally, we identified three mutation sites that can seriously affect the antigenicity of B/Victoria vaccine strains. These mutation sites provide valuable references for the selection and design of a universal IBV vaccine strain in the future.

WHO International Standards for antibodies to HPV6 HPV11 HPV31 HPV33 HPV45 HPV52 and HPV58.

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.

Afucosylated anti-EBOV antibody MIL77-3 engages sGP to elicit NK cytotoxicity.

MIL77-3 is one component of antibody cocktail that is produced in our lab and represents an effective regimen for animals suffering from Zaire Ebolavirus (EBOV) infection. MIL77-3 is engineered to increase its affinity for the FcγRIIIa (CD16a) by deleting the fucose in the framework region. The potential effects of this modification on host immune responses, however, remain largely unknown. Herein, we demonstrated that MIL77-3 recognized secreted glycoproptein (sGP), produced by EBOV, and formed the immunocomplex to potently augment antibody-dependent cytotoxicity of human peripheral blood-derived natural killer cells (pNKs), including CD56dim and CD56bright subpopulations, in contrast to the counterparts (Mab114, rEBOV548, fucosylated MIL77-3). Intriguingly, this effect was not observed when NK92-CD16a cell line was utilized and restored by the addition of beads-coupled or membrane-anchored sGP in combination with MIL77-3. Furthermore, sGP bound to unrecognized receptors on T cells contaminated in pNKs rather than NK92-CD16a cells. Administration of beads-coupled sGP/MIL77-3 complex in mice elicited NK activation. Overall, this work reveals an immune-stimulating function of sGP/MIL77-3 complex by triggering cytotoxic activity of NK cells, highlighting the necessity to evaluate the potential impact of MIL77-3 on host immune reaction in clinical trials. IMPORTANCE: Zaire Ebolavirus (EBOV) is highly lethal and causes sporadic outbreaks. The passive administration of monoclonal antibodies (mAbs) represents a promising treatment regimen against EBOV. Mounting evidence has shown that the efficacy of a subset of therapeutic mAbs in vivo is intimately associated with its capacity to trigger NK activity, supporting glycomodification of Fc region of anti-EBOV mAbs as a putative strategy to enhance Fc-mediated immune effector function as well as protection in vivo. Our work here uncovers the potential harmful influence of this modification on host immune responses, especially for mAbs with cross-reactivity to secreted glycoproptein (sGP) (e.g., MIL77-3), and highlights it is necessary to evaluate the NK-stimulating activity of a fucosylated mAb engaged with sGP when a new candidate is developed.

Neutralization potency of the 2023-24 seasonal influenza vaccine against circulating influenza H3N2 strains.

Seasonal influenza is a severe disease that significantly impacts public health, causing millions of infections and hundreds of thousands of deaths each year. Seasonal influenza viruses, particularly the H3N2 subtype, exhibit high antigenic variability, often leading to mismatch between vaccine strains and circulating strains. Therefore, rapidly assessing the alignment between existing seasonal influenza vaccine and circulating strains is crucial for enhancing vaccine efficacy. This study, based on a pseudovirus platform, evaluated the match between current influenza H3N2 vaccine strains and circulating strains through cross-neutralization assays using clinical human immune sera against globally circulating influenza virus strains. The research results show that although mutations are present in the circulating strains, the current H3N2 vaccine strain still imparting effective protection, providing a scientific basis for encouraging influenza vaccination. This research methodology can be sustainably applied for the neutralization potency assessment of subsequent circulating strains, establishing a persistent methodological framework.

Optimization and validation of a virus-like particle pseudotyped virus neutralization assay for SARS-CoV-2.

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.

APOBEC3-related mutations in the spike protein-encoding region facilitate SARS-CoV-2 evolution.

SARS-CoV-2 evolves gradually to cause COVID-19 epidemic. One of driving forces of SARS-CoV-2 evolution might be activation of apolipoprotein B mRNA editing catalytic subunit-like protein 3 (APOBEC3) by inflammatory factors. Here, we aimed to elucidate the effect of the APOBEC3-related viral mutations on the infectivity and immune evasion of SARS-CoV-2. The APOBEC3-related C > U mutations ranked as the second most common mutation types in the SARS-CoV-2 genome. mRNA expression of APOBEC3A (A3A), APOBEC3B (A3B), and APOBEC3G (A3G) in peripheral blood cells increased with disease severity. A3B, a critical member of the APOBEC3 family, was significantly upregulated in both severe and moderate COVID-19 patients and positively associated with neutrophil proportion and COVID-19 severity. We identified USP18 protein, a key molecule centralizing the protein-protein interaction network of key APOBEC3 proteins. Furthermore, mRNA expression of USP18 was significantly correlated to ACE2 and TMPRSS2 expression in the tissue of upper airways. Knockdown of USP18 mRNA significantly decreased A3B expression. Ectopic expression of A3B gene increased SARS-CoV-2 infectivity. C > U mutations at S371F, S373L, and S375F significantly conferred with the immune escape of SARS-CoV-2. Thus, APOBEC3, whose expression are upregulated by inflammatory factors, might promote SARS-CoV-2 evolution and spread via upregulating USP18 level and facilitating the immune escape. A3B and USP18 might be therapeutic targets for interfering with SARS-CoV-2 evolution.

Distribution of HPV types among women with HPV-related diseases and exploration of lineages and variants of HPV 52 and 58 among HPV-infected patients in China: A systematic literature review.

To 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.

COVID-19 Serum Drives Spike-Mediated SARS-CoV-2 Variation.

Neutralizing antibodies targeting the spike (S) protein of SARS-CoV-2, elicited either by natural infection or vaccination, are crucial for protection against the virus. Nonetheless, the emergence of viral escape mutants presents ongoing challenges by contributing to breakthrough infections. To define the evolution trajectory of SARS-CoV-2 within the immune population, we co-incubated replication-competent rVSV/SARS-CoV-2/GFP chimeric viruses with sera from COVID-19 convalescents. Our findings revealed that the E484D mutation contributes to increased viral resistant against both convalescent and vaccinated sera, while the L1265R/H1271Y double mutation enhanced viral infectivity in 293T-hACE2 and Vero cells. These findings suggest that under the selective pressure of polyclonal antibodies, SARS-CoV-2 has the potential to accumulate mutations that facilitate either immune evasion or greater infectivity, facilitating its adaption to neutralizing antibody responses. Although the mutations identified in this study currently exhibit low prevalence in the circulating SARS-CoV-2 populations, the continuous and meticulous surveillance of viral mutations remains crucial. Moreover, there is an urgent necessity to develop next-generation antibody therapeutics and vaccines that target diverse, less mutation-prone antigenic sites to ensure more comprehensive and durable immune protection against SARS-CoV-2.

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.

HIV-1 env gene mutations outside the targeting probe affects IPDA efficiency.

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.

Establishment of the First National Standard for Neutralizing Antibodies against SARS-CoV-2 XBB Variants.

Neutralizing antibodies (NtAbs) against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are indicators of vaccine efficacy that enable immunity surveillance. However, the rapid mutation of SARS-CoV-2 variants prevents the timely establishment of standards required for effective XBB vaccine evaluation. Therefore, we prepared four candidate standards (No. 11, No. 44, No. 22, and No. 33) using plasma, purified immunoglobulin, and a broad-spectrum neutralizing monoclonal antibody. Collaborative calibration was conducted across nine Chinese laboratories using neutralization methods against 11 strains containing the XBB and BA.2.86 sublineages. This study demonstrated the reduced neutralization potency of the first International Standard antibodies to SARS-CoV-2 variants of concern against XBB variants. No. 44 displayed broad-spectrum neutralizing activity against XBB sublineages, effectively reduced interlaboratory variability for nearly all XBB variants, and effectively minimized the geometric mean titer (GMT) difference between the live and pseudotyped virus. No. 22 showed a broader spectrum and higher neutralizing activity against all strains but failed to reduce interlaboratory variability. Thus, No. 44 was approved as a National Standard for NtAbs against XBB variants, providing a unified NtAb measurement standard for XBB variants for the first time. Moreover, No. 22 was approved as a national reference reagent for NtAbs against SARS-CoV-2, offering a broad-spectrum activity reference for current and potentially emerging variants.

Multi-epitope vaccine design for hepatitis E virus based on protein ORF2 and ORF3.

Introduction: Hepatitis 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. Methods: We designed a multi-epitope vaccine based on protein ORF2 and ORF3 of HEV using immunoinformatics. Results: The 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. Discussion: These findings provide a foundation for future HEV vaccine studies.

Development of a SARS-CoV-2 neutralization assay based on a pseudotyped virus using a HIV system.

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.

A novel MARV glycoprotein-specific antibody with potentials of broad-spectrum neutralization to filovirus.

Marburg virus (MARV) is one of the filovirus species that cause deadly hemorrhagic fever in humans, with mortality rates up to 90%. Neutralizing antibodies represent ideal candidates to prevent or treat virus disease. However, no antibody has been approved for MARV treatment to date. In this study, we identified a novel human antibody named AF-03 that targeted MARV glycoprotein (GP). AF-03 possessed a high binding affinity to MARV GP and showed neutralizing and protective activities against the pseudotyped MARV in vitro and in vivo. Epitope identification, including molecular docking and experiment-based analysis of mutated species, revealed that AF-03 recognized the Niemann-Pick C1 (NPC1) binding domain within GP1. Interestingly, we found the neutralizing activity of AF-03 to pseudotyped Ebola viruses (EBOV, SUDV, and BDBV) harboring cleaved GP instead of full-length GP. Furthermore, NPC2-fused AF-03 exhibited neutralizing activity to several filovirus species and EBOV mutants via binding to CI-MPR. In conclusion, this work demonstrates that AF-03 represents a promising therapeutic cargo for filovirus-caused disease.

Immune escape of BA.2.86 is comparable to XBB subvariants from the plasma of BA.5- and BA.5-XBB-convalescent subpopulations.

The EG.5.1 variant of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been prevalent since mid-July 2023 in the United States and China. The variant BA.2.86 has become a major concern because it is 34 mutations away from the parental variant BA.2 and >30 mutations from XBB.1.5. There is an urgent need to evaluate whether the immunity of the population and current vaccines are protective against EG.5.1 and BA.2.86. Based on a cohort of two breakthrough-infected groups, the levels of neutralizing antibodies (NAbs) against different subvariants were measured using pseudovirus-based neutralization assays. XBB.1.5, EG.5.1, and BA.2.86 are comparably immune-evasive from neutralization by the plasma of individuals recovered from BA.5 infection (BA.5-convalescent) or XBB.1.9.2/XBB.1.5 infection following BA.5 infection (BA.5-XBB-convalescent). NAb levels against EG.5.1 and BA.2.86 subvariants remained >120 geometric mean titers (GMTs) in BA.5-XBB-convalescent individuals 2 months postinfection but were <40 GMTs in BA.5-convalescent individuals. Furthermore, the XBB-targeting messenger RNA (mRNA) vaccine RQ3033 induced higher levels of NAbs against XBB.1.5, EG.5.1, and BA.2.86 than against BA.5-XBB infection. The results suggest that BA.2.86 and EG.5.1 are unlikely to cause more severe concerns than the currently circulating XBB subvariants and that the XBB.1.5-targeting mRNA vaccine tested has promising protection against EG.5.1 and BA.2.86.

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.

Functional characterization of AF-04, an afucosylated anti-MARV GP antibody.

Marburg virus (MARV), one member of the Filoviridae family, cause sporadic outbreaks of hemorrhagic fever with high mortality rates. No countermeasures are currently available for the prevention or treatment of MARV infection. Monoclonal antibodies (mAbs) are promising candidates to display high neutralizing activity against MARV infection in vitro and in vivo. Recently, growing evidence has shown that immune effector function including antibody-dependent cell-mediated cytotoxicity (ADCC) is also required for in vivo efficacy of a panel of antibodies. Glyco-engineered methods are widely utilized to augment ADCC function of mAbs. In this study, we generated a fucose-knockout MARV GP-specific mAb named AF-04 and showed that afucosylation dramatically increased its binding affinity to polymorphic FcγRIIIa (F176/V176) compared with the parental AF-03. Accordingly, AF-04-mediated NK cell activation and NFAT expression downstream of FcγRIIIa in effector cells were also augmented. In conclusion, this work demonstrates that AF-04 represents a novel avenue for the treatment of MARV-caused disease.