Surface display of the COE antigen of porcine epidemic diarrhoea virus on Bacillus subtilis spores.

Porcine epidemic diarrhoea virus (PEDV) infects pigs of all ages by invading small intestine, causing acute diarrhoea, vomiting, and dehydration with high morbidity and mortality among newborn piglets. However, current PEDV vaccines are not effective to protect the pigs from field epidemic strains because of poor mucosal immune response and strain variation. Therefore, it is indispensable to develop a novel oral vaccine based on epidemic strains. Bacillus subtilis spores are attractive delivery vehicles for oral vaccination on account of the safety, high stability, and low cost. In this study, a chimeric gene CotC-Linker-COE (CLE), comprising of the B. subtilis spore coat gene cotC fused to the core neutralizing epitope CO-26 K equivalent (COE) of the epidemic strain PEDV-AJ1102 spike protein gene, was constructed. Then recombinant B. subtilis displaying the CLE on the spore surface was developed by homologous recombination. Mice were immunized by oral route with B. subtilis 168-CLE, B. subtilis 168, or phosphate-buffered saline (PBS) as control. Results showed that the IgG antibodies and cytokine (IL-4, IFN-γ) levels in the B. subtilis 168-CLE group were significantly higher than the control groups. This study demonstrates that B. subtilis 168-CLE can generate specific systemic immune and mucosal immune responses and is a potential vaccine candidate against PEDV infection.

Multi-modal generative modeling for joint analysis of single-cell T cell receptor and gene expression data.

Recent advances in single-cell immune profiling have enabled the simultaneous measurement of transcriptome and T cell receptor (TCR) sequences, offering great potential for studying immune responses at the cellular level. However, integrating these diverse modalities across datasets is challenging due to their unique data characteristics and technical variations. Here, to address this, we develop the multimodal generative model mvTCR to fuse modality-specific information across transcriptome and TCR into a shared representation. Our analysis demonstrates the added value of multimodal over unimodal approaches to capture antigen specificity. Notably, we use mvTCR to distinguish T cell subpopulations binding to SARS-CoV-2 antigens from bystander cells. Furthermore, when combined with reference mapping approaches, mvTCR can map newly generated datasets to extensive T cell references, facilitating knowledge transfer. In summary, we envision mvTCR to enable a scalable analysis of multimodal immune profiling data and advance our understanding of immune responses.

Production of Recombinant Viral Antigens Using the Baculovirus-Insect Cell Expression System.

Insect cell expression has been successfully used for the production of viral antigens as part of commercial vaccine development. As expression host, insect cells offer advantage over bacterial system by presenting the ability of performing post-translational modifications (PTMs) such as glycosylation and phosphorylation thus preserving the native functionality of the proteins especially for viral antigens. Insect cells have limitation in exactly mimicking some proteins which require complex glycosylation pattern. The recent advancement in insect cell engineering strategies could overcome this limitation to some extent. Moreover, cost efficiency, timelines, safety, and process adoptability make insect cells a preferred platform for production of subunit antigens for human and animal vaccines. In this chapter, we describe the method for producing the SARS-CoV2 spike ectodomain subunit antigen for human vaccine development and the virus like particle (VLP), based on capsid protein of porcine circovirus virus 2 (PCV2d) antigen for animal vaccine development using two different insect cell lines, SF9 & Hi5, respectively. This methodology demonstrates the flexibility and broad applicability of insect cell as expression host.

Identification of two novel B cell epitopes on E184L protein of African swine fever virus using monoclonal antibodies.

African swine fever virus (ASFV) is a large double-stranded DNA virus with a complex structural architecture and encodes more than 150 proteins, where many are with unknown functions. E184L has been reported as one of the immunogenic ASFV proteins that may contribute to ASFV pathogenesis and immune evasion. However, the antigenic epitopes of E184L are not yet characterized. In this study, recombinant E184L protein was expressed in prokaryotic expression system and four monoclonal antibodies (mAbs), designated as 1A10, 2D2, 3H6, and 4C10 were generated. All four mAbs reacted specifically with ASFV infected cells. To identify the epitopes of the mAbs, a series of overlapped peptides of E184L were designed and expressed as maltose binding fusion proteins. Accordingly, the expressed fusion proteins were probed with each E184L mAb separately by using Western blot. Following a fine mapping, the minimal linear epitope recognized by mAb 1A10 was identified as 119IQRQGFL125, and mAbs 2D2, 3H6, and 4C10 recognized a region located between 153DPTEFF158. Alignment of amino acids of E184L revealed that the two linear epitopes are highly conserved among different ASFV isolates. Furthermore, the potential application of the two epitopes in ASFV diagnosis was assessed through epitope-based ELISA using 24 ASFV positive and 18 negative pig serum and the method were able to distinguish positive and negative samples, indicating the two epitopes are dominant antigenic sites. To our knowledge, this is the first study to characterize the B cell epitopes of the antigenic E184L protein of ASFV, offering valuable tools for future research, as well as laying a foundation for serological diagnosis and epitope-based marker vaccine development.

N-glycosylation on hemagglutinin head reveals inter-branch antigenic variability of avian influenza virus H5-subtypes.

H5-subtype avian influenza virus (AIV) is globally prevalent and undergoes frequent antigenic drift, necessitating regular updates to vaccines. One of the many influencing elements that cause incompatibility between vaccinations and epidemic strains is the dynamic alteration of glycosylation sites. However, the biological significance of N-glycosylation in the viral evolution and antigenic changes is unclear. Here, we performed a systematic analysis of glycosylation sites on the HA1 subunit of H5N1, providing insights into the changes of primary glycosylation sites, including 140 N, 156 N, and 170 N within the antigenic epitopes of HA1 protein. Multiple recombinant viruses were then generated based on HA genes of historical vaccine strains and deactivated for immunizing SPF chickens. Inactivated recombinant strains showed relatively closer antigenicity compared to which has identical N-glycosylation patterns. The N-glycosylation modification discrepancy highlights the inter-branch antigenic diversity of H5-subtype viruses in avian influenza and serves as a vital foundation for improving vaccination tactics.

Circulating rotavirus strains in children with acute gastroenteritis in Iran, 1986 to 2023 and their genetic/antigenic divergence compared to approved vaccines strains (Rotarix, RotaTeq, ROTAVAC, ROTASIIL) before mass vaccination: Clues for vaccination policy makers.

In the present study, first, rotaviruses that caused acute gastroenteritis in children under five years of age during the time before the vaccine was introduced in Iran (1986 to 2023) are reviewed. Subsequently, the antigenic epitopes of the VP7 and VP4/VP8 proteins in circulating rotavirus strains in Iran and that of the vaccine strains were compared and their genetic differences in histo-blood group antigens (HBGAs) and the potential impact on rotavirus infection susceptibility and vaccine efficacy were discussed. Overall data indicate that rotavirus was estimated in about 38.1 % of samples tested. The most common genotypes or combinations were G1 and P[8], or G1P[8]. From 2015 to 2023, there was a decline in the prevalence of G1P[8], with intermittent peaks of genotypes G3P[8] and G9P[8]. The analyses suggested that the monovalent Rotarix vaccine or monovalent vaccines containing the G1P[8] component might be proper in areas with a similar rotavirus genotype pattern and genetic background as the Iranian population where the G1P[8] strain is the most predominant and has the ability to bind to HBGA secretors. While the same concept can be applied to RotaTeq and RotasIIL vaccines, their complex vaccine technology, which involves reassortment, makes them less of a priority. The ROTASIIL vaccine, despite not having the VP4 arm (P[5]) as a suitable protection option, has previously shown the ability to neutralize not only G9-lineage I strains but also other G9-lineages at high titers. Thus, vaccination with the ROTASIIL vaccine may be more effective in Iran compared to RotaTeq. However, considering the rotavirus genotypic pattern, ROTAVAC might not be a good choice for Iran. Overall, the findings of this study provide valuable insights into the prevalence of rotavirus strains and the potential effectiveness of different vaccines in the Iranian and similar populations.

Protein Nanoparticles as Vaccine Platforms for Human and Zoonotic Viruses.

Vaccines are one of the most effective medical interventions, playing a pivotal role in treating infectious diseases. Although traditional vaccines comprise killed, inactivated, or live-attenuated pathogens that have resulted in protective immune responses, the negative consequences of their administration have been well appreciated. Modern vaccines have evolved to contain purified antigenic subunits, epitopes, or antigen-encoding mRNAs, rendering them relatively safe. However, reduced humoral and cellular responses pose major challenges to these subunit vaccines. Protein nanoparticle (PNP)-based vaccines have garnered substantial interest in recent years for their ability to present a repetitive array of antigens for improving immunogenicity and enhancing protective responses. Discovery and characterisation of naturally occurring PNPs from various living organisms such as bacteria, archaea, viruses, insects, and eukaryotes, as well as computationally designed structures and approaches to link antigens to the PNPs, have paved the way for unprecedented advances in the field of vaccine technology. In this review, we focus on some of the widely used naturally occurring and optimally designed PNPs for their suitability as promising vaccine platforms for displaying native-like antigens from human viral pathogens for protective immune responses. Such platforms hold great promise in combating emerging and re-emerging infectious viral diseases and enhancing vaccine efficacy and safety.

Complete coding region sequence analyses and antigenic characterization of emerging lineage G-IX of foot- and-mouth disease virus serotype Asia1.

Foot-and-mouth disease Virus (FMDV) serotype Asia1 is prevalent in the Indian subcontinent, with only G-III and G-VIII reported in India until 2020. However, in 2019, a novel genetic group within serotype Asia1, designated as G-IX, emerged in Bangladesh, followed by its detection in India in 2020. This report presents analyses of the complete coding region sequences of the G-IX lineage isolates. The length of the open reading frame (ORF) of the two G-IX isolates was 6990 nucleotides without any deletion or insertion. The G-IX isolates showed the highest sequence similarity with an isolate of G-III at the ORF, L, P2, and P3 regions, and with an isolate of G-VIII at the P1 region. Phylogenetic analysis based on the capsid region (P1) supports the hypothesis that G-VIII and G-IX originated from a common ancestor, as speculated earlier. Further, VP1 region-based phylogenetic analyses revealed the re-emergence of G-VIII after a gap of 3 years. One isolate of G-VIII collected during 2023 revealed a codon insertion in the G-H loop of VP1. The vaccine matching studies support the suitability of the currently used Indian vaccine strain IND63/1972 to contain outbreaks due to viruses belonging to G-IX.

Proscan: a structure-based proline design web server.

The ability to control protein conformations and dynamics through structure-based design has been useful in various scenarios, including engineering of viral antigens for vaccines. One effective design strategy is the substitution of residues to proline amino acids, which due to its unique cyclic side chain can favor and rigidify key backbone conformations. To provide the community with a means to readily identify and explore proline designs for target proteins of interest, we developed the Proscan web server. Proscan provides assessment of backbone angles, energetic and deep learning-based favorability scores, and other parameters for proline substitutions at each position of an input structure, along with interactive visualization of backbone angles and candidate substitution sites on structures. It identifies known favorable proline substitutions for viral antigens, and was benchmarked against datasets of proline substitution stability effects from deep mutational scanning and thermodynamic measurements. This tool can enable researchers to identify and prioritize designs for prospective vaccine antigen targets, or other designs to favor stability of key protein conformations. Proscan is available at: https://proscan.ibbr.umd.edu.

Identification of a norovirus GII-specific antigenic epitope.

Noroviruses (NoVs) are the chief cause of acute viral gastroenteritis worldwide. By employing the major capsid protein VP1 of a GII.6 NoV strain as an immunogen, we generated two monoclonal antibodies (mAbs) with wide-spectrum binding activities against NoV genogroup II (GII) VP1 proteins. One mAb (10G7) could bind to native and denatured GII-specific VP1 proteins. The other mAb (10F2) could bind to all tested native GII VP1 proteins, but not to denatured GII.3, GII.4, GII.7, or GII.17 VP1 proteins. Using GII.6/GII.4 fusion proteins, the mAb 10F2 binding region was confirmed to be located in the C-terminal P1 domain. An enzyme-linked immunosorbent assay based on peptides covering the P domain did not detect any binding. Using a panel of VP1 proteins with swapped regions, deletions, and mutations, the mAb 10F2 binding region was determined to be located between residues 496 and 513. However, the residue(s) responsible for its varied binding affinity for different denatured GII VP1 proteins remain to be identified. In summary, two NoV GII-specific cross-reactive mAbs were generated, and their binding regions were determined. Our results might facilitate the detection and immunogenic study of NoVs.

An indirect ELISA for detecting anti-SARS-CoV-2 antibodies in human sera using a baculovirus-expressed recombinant nucleocapsid antigen.

This study focuses on the development and initial assessment of an indirect IgG enzyme-linked immunosorbent assay (ELISA) specifically designed to detect of anti-SARS-CoV-2 antibodies. The unique aspect of this ELISA method lies in its utilization of a recombinant nucleocapsid (N) antigen, produced through baculovirus expression in insect cells. Our analysis involved 292 RT-qPCR confirmed positive serum samples and 54 pre-pandemic healthy controls. The process encompassed cloning, expression, and purification of the SARS-CoV-2 N gene in insect cells, with the resulted purified protein employed in our ELISA tests. Statistical analysis yielded an Area Under the Curve of 0.979, and the optimized cut-off exhibited 92 % sensitivity and 94 % specificity. These results highlight the ELISA's potential for robust and reliable serological detection of SARS-CoV-2 antibodies. Further assessments, including a larger panel size, reproducibility tests, and application in diverse populations, could enhance its utility as a valuable biotechnological solution for diseases surveillance.

Antigenic epitope analysis of Pakistani G3 and G9 rotavirus strains compared to vaccine strains revealed multiple amino acid differences.

Rotaviruses belong to genotype VP4-P[8] are a significant cause of severe loose diarrhea in infants and young children. In the present study, we characterised the complete genome of three of the Pakistani P[8]b RVA strains by Illumina HiSeq sequencing technology to determine the complete genotype constellation providing insight into the evolutionary dynamics of their genes using maximum likelihood analysis. The maximum genomic sequences of our study strains were similar to more recent human Wa-Like G1P[8]a, G3P[8]a, G4P[6], G4P[8], G9P[4], G9P[8]a, G11P[25],G12P[8]a and G12P[6] strains circulating around the world. Therefore, strains PAK274, PAK439 and PAK624 carry natively distinctive VP4 gene with universally common human Wa-Like genetic backbone. Comparing our study P[8]b strains with vaccines strains RotarixTM and RotaTeqTM, multiple amino acid differences were examined between vaccine virus antigenic epitopes and Pakistani isolates. Over time, these differences may result in the selection for strains that will escape the vaccine-induced RVA-neutralizing-antibody effect.

KSHV genome harbors both constitutive and lytically induced enhancers.

Kaposi's sarcoma-associated herpesvirus (KSHV) belongs to the gamma-herpesvirus family and is a well-known human oncogenic virus. In infected cells, the viral genome of 165 kbp is circular DNA wrapped in chromatin. The tight control of gene expression is critical for latency, the transition into the lytic phase, and the development of viral-associated malignancies. Distal cis-regulatory elements, such as enhancers and silencers, can regulate gene expression in a position- and orientation-independent manner. Open chromatin is another characteristic feature of enhancers. To systematically search for enhancers, we cloned all the open chromatin regions in the KSHV genome downstream of the luciferase gene and tested their enhancer activity in infected and uninfected cells. A silencer was detected upstream of the latency-associated nuclear antigen promoter. Two constitutive enhancers were identified in the K12p-OriLyt-R and ORF29 Intron regions, where ORF29 Intron is a tissue-specific enhancer. The following promoters: OriLyt-L, PANp, ALTp, and the terminal repeats (TRs) acted as lytically induced enhancers. The expression of the replication and transcription activator (RTA), the master regulator of the lytic cycle, was sufficient to induce the activity of lytic enhancers in uninfected cells. We propose that the TRs that span about 24 kbp region serve as a "viral super-enhancer" that integrates the repressive effect of the latency-associated nuclear antigen (LANA) with the activating effect of RTA. Utilizing CRISPR activation and interference techniques, we determined the connections between these enhancers and their regulated genes. The silencer and enhancers described here provide an additional layer to the complex gene regulation of herpesviruses.IMPORTANCEIn this study, we performed a systematic functional assay to identify cis-regulatory elements within the genome of the oncogenic herpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV). Similar to other herpesviruses, KSHV presents both latent and lytic phases. Therefore, our assays were performed in uninfected cells, during latent infection, and under lytic conditions. We identified two constitutive enhancers, one of which seems to be a tissue-specific enhancer. In addition, four lytically induced enhancers, which are all responsive to the replication and transcription activator (RTA), were identified. Furthermore, a silencer was identified between the major latency promoter and the lytic gene locus. Utilizing CRISPR activation and interference techniques, we determined the connections between these enhancers and their regulated genes. The terminal repeats, spanning a region of about 24 kbp, seem like a "viral super-enhancer" that integrates the repressive effect of the latency-associated nuclear antigen (LANA) with the activating effect of RTA to regulate latency to lytic transition.

Rational design and production of a chimeric antigen targeting Zika virus that induces neutralizing antibodies in mice.

The Zika virus (ZIKV) is considered a public health problem worldwide due to its association with the development of microcephaly and the Guillain-Barré syndrome. Currently, there is no specific treatment or vaccine approved to combat this disease, and thus, developing safe and effective vaccines is a relevant goal. In this study, a multi-epitope protein called rpZDIII was designed based on a series of ZIKV antigenic sequences, a bacterial carrier, and linkers. The analysis of the predicted 3D structure of the rpZDIII chimeric antigen was performed on the AlphaFold 2 server, and it was produced in E. coli and purified from inclusion bodies, followed by solubilization and refolding processes. The yield achieved for rpZDIII was 11 mg/L in terms of pure soluble recombinant protein per liter of fermentation. rpZDIII was deemed immunogenic since it induced serum IgG and IgM responses in mice upon subcutaneous immunization in a three-dose scheme. Moreover, sera from mice immunized with rpZDIII showed neutralizing activity against ZIKV. Therefore, this study reveals rpZDIII as a promising immunogen for the development of a rationally designed multi-epitope vaccine against ZIKV, and completion of its preclinical evaluation is guaranteed.

Characterizing the antigenic evolution of pandemic influenza A (H1N1) pdm09 from 2009 to 2023.

The H1N1pdm09 virus has been a persistent threat to public health since the 2009 pandemic. Particularly, since the relaxation of COVID-19 pandemic mitigation measures, the influenza virus and SARS-CoV-2 have been concurrently prevalent worldwide. To determine the antigenic evolution pattern of H1N1pdm09 and develop preventive countermeasures, we collected influenza sequence data and immunological data to establish a new antigenic evolution analysis framework. A machine learning model (XGBoost, accuracy = 0.86, area under the receiver operating characteristic curve = 0.89) was constructed using epitopes, physicochemical properties, receptor binding sites, and glycosylation sites as features to predict the antigenic similarity relationships between influenza strains. An antigenic correlation network was constructed, and the Markov clustering algorithm was used to identify antigenic clusters. Subsequently, the antigenic evolution pattern of H1N1pdm09 was analyzed at the global and regional scales across three continents. We found that H1N1pdm09 evolved into around five antigenic clusters between 2009 and 2023 and that their antigenic evolution trajectories were characterized by cocirculation of multiple clusters, low-level persistence of former dominant clusters, and local heterogeneity of cluster circulations. Furthermore, compared with the seasonal H1N1 virus, the potential cluster-transition determining sites of H1N1pdm09 were restricted to epitopes Sa and Sb. This study demonstrated the effectiveness of machine learning methods for characterizing antigenic evolution of viruses, developed a specific model to rapidly identify H1N1pdm09 antigenic variants, and elucidated their evolutionary patterns. Our findings may provide valuable support for the implementation of effective surveillance strategies and targeted prevention efforts to mitigate the impact of H1N1pdm09.

Seasonal antigenic prediction of influenza A H3N2 using machine learning.

Antigenic characterization of circulating influenza A virus (IAV) isolates is routinely assessed by using the hemagglutination inhibition (HI) assays for surveillance purposes. It is also used to determine the need for annual influenza vaccine updates as well as for pandemic preparedness. Performing antigenic characterization of IAV on a global scale is confronted with high costs, animal availability, and other practical challenges. Here we present a machine learning model that accurately predicts (normalized) outputs of HI assays involving circulating human IAV H3N2 viruses, using their hemagglutinin subunit 1 (HA1) sequences and associated metadata. Each season, the model learns an updated nonlinear mapping of genetic to antigenic changes using data from past seasons only. The model accurately distinguishes antigenic variants from non-variants and adaptively characterizes seasonal dynamics of HA1 sites having the strongest influence on antigenic change. Antigenic predictions produced by the model can aid influenza surveillance, public health management, and vaccine strain selection activities.

Efficient signal sequence of mRNA vaccines enhances the antigen expression to expand the immune protection against viral infection.

The signal sequence played a crucial role in the efficacy of mRNA vaccines against virus pandemic by influencing antigen translation. However, limited research had been conducted to compare and analyze the specific mechanisms involved. In this study, a novel approach was introduced by substituting the signal sequence of the mRNA antigen to enhance its immune response. Computational simulations demonstrated that various signal peptides differed in their binding capacities with the signal recognition particle (SRP) 54 M subunit, which positively correlated with antigen translation efficiency. Our data revealed that the signal sequences of tPA and IL-6-modified receptor binding domain (RBD) mRNA vaccines sequentially led to higher antigen expression and elicited more robust humoral and cellular immune protection against the SARS-CoV-2 compared to the original signal sequence. By highlighting the importance of the signal sequence, this research provided a foundational and safe approach for ongoing modifications in signal sequence-antigen design, aiming to optimize the efficacy of mRNA vaccines.

Design and Development of an Antigen Test for SARS-CoV-2 Nucleocapsid Protein to Validate the Viral Quality Assurance Panels.

The continuing mutability of the SARS-CoV-2 virus can result in failures of diagnostic assays. To address this, we describe a generalizable bioinformatics-to-biology pipeline developed for the calibration and quality assurance of inactivated SARS-CoV-2 variant panels provided to Radical Acceleration of Diagnostics programs (RADx)-radical program awardees. A heuristic genetic analysis based on variant-defining mutations demonstrated the lowest genetic variance in the Nucleocapsid protein (Np)-C-terminal domain (CTD) across all SARS-CoV-2 variants. We then employed the Shannon entropy method on (Np) sequences collected from the major variants, verifying the CTD with lower entropy (less prone to mutations) than other Np regions. Polyclonal and monoclonal antibodies were raised against this target CTD antigen and used to develop an Enzyme-linked immunoassay (ELISA) test for SARS-CoV-2. Blinded Viral Quality Assurance (VQA) panels comprised of UV-inactivated SARS-CoV-2 variants (XBB.1.5, BF.7, BA.1, B.1.617.2, and WA1) and distractor respiratory viruses (CoV 229E, CoV OC43, RSV A2, RSV B, IAV H1N1, and IBV) were assembled by the RADx-rad Diagnostics core and tested using the ELISA described here. The assay tested positive for all variants with high sensitivity (limit of detection: 1.72-8.78 ng/mL) and negative for the distractor virus panel. Epitope mapping for the monoclonal antibodies identified a 20 amino acid antigenic peptide on the Np-CTD that an in-silico program also predicted for the highest antigenicity. This work provides a template for a bioinformatics pipeline to select genetic regions with a low propensity for mutation (low Shannon entropy) to develop robust 'pan-variant' antigen-based assays for viruses prone to high mutational rates.

Rotavirus trends and distribution of genotypes before and during COVID-19 pandemic era: Bangladesh, 2017-2021.

Rotavirus gastroenteritis is accountable for an estimated 128 500 deaths among children younger than 5 years worldwide, and the majority occur in low-income countries. Although the clinical trials of rotavirus vaccines in Bangladesh revealed a significant reduction of severe rotavirus disease by around 50%, the vaccines are not yet included in the routine immunization program. The present study was designed to provide data on rotavirus diarrhea with clinical profiles and genotypes before (2017-2019) and during the COVID-19 pandemic period (2020-2021). Fecal samples were collected from 2% of the diarrheal patients at icddr,b Dhaka hospital of all ages between January 2017 and December 2021 and were tested for VP6 rotavirus antigen using ELISA. The clinical manifestations such as fever, duration of diarrhea and hospitalization, number of stools, and dehydration and so on were collected from the surveillance database (n = 3127). Of the positive samples, 10% were randomly selected for genotyping using Sanger sequencing method. A total of 12 705 fecal samples were screened for rotavirus A antigen by enzyme immunoassay. Overall, 3369 (27%) were rotavirus antigen-positive, of whom children <2 years had the highest prevalence (88.6%). The risk of rotavirus A infection was 4.2 times higher in winter than in summer. Overall, G3P[8] was the most prominent genotype (45.3%), followed by G1P[8] (32.1%), G9P[8] (6.8%), and G2P[4] (6.1%). The other unusual combinations, such as G1P[4], G1P[6], G2P[6], G3P[4], G3P[6], and G9P[6], were also present. Genetic analysis on Bangladeshi strains revealed that the selection pressure (dN/dS) was estimated as <1. The number of hospital visits showed a 37% drop during the COVID-19 pandemic relative to the years before the pandemic. Conversely, there was a notable increase in the rate of rotavirus positivity during the pandemic (34%, p < 0.00) compared to the period before COVID-19 (23%). Among the various clinical symptoms, only the occurrence of watery stool significantly increased during the pandemic. The G2P[4] strain showed a sudden rise (19%) in 2020, which then declined in 2021. In the same year, G1P[8] was more prevalent than G3P[8] (40% vs. 38%, respectively). The remaining genotypes were negligible and did not exhibit much fluctuation. This study reveals that the rotavirus burden remained high during the COVID-19 prepandemic and pandemic in Bangladesh. Considering the lack of antigenic variations between the circulating and vaccine-targeted strains, integrating the vaccine into the national immunization program could reduce the prevalence of the disease, the number of hospitalizations, and the severity of cases.

The antigenic landscape of human influenza N2 neuraminidases from 2009 until 2017.

Human H3N2 influenza viruses are subject to rapid antigenic evolution which translates into frequent updates of the composition of seasonal influenza vaccines. Despite these updates, the effectiveness of influenza vaccines against H3N2-associated disease is suboptimal. Seasonal influenza vaccines primarily induce hemagglutinin-specific antibody responses. However, antibodies directed against influenza neuraminidase (NA) also contribute to protection. Here, we analysed the antigenic diversity of a panel of N2 NAs derived from human H3N2 viruses that circulated between 2009 and 2017. The antigenic breadth of these NAs was determined based on the NA inhibition (NAI) of a broad panel of ferret and mouse immune sera that were raised by infection and recombinant N2 NA immunisation. This assessment allowed us to distinguish at least four antigenic groups in the N2 NAs derived from human H3N2 viruses that circulated between 2009 and 2017. Computational analysis further revealed that the amino acid residues in N2 NA that have a major impact on susceptibility to NAI by immune sera are in proximity of the catalytic site. Finally, a machine learning method was developed that allowed to accurately predict the impact of mutations that are present in our N2 NA panel on NAI. These findings have important implications for the renewed interest to develop improved influenza vaccines based on the inclusion of a protective NA antigen formulation.

Two proteins, the hemagglutinin and the neuraminidase, protrude from the surface of the influenza virus. Their detection by the immune system allows the host organism to mount defences against the viral threat. The virus evolves in response to this pressure, which manifests as changes in the appearance of its hemagglutinin and neuraminidase. This process, known as antigenic drift, leads to the proteins evading detection. It is also why flu vaccines require frequent updates, as they rely on 'training' the immune system to recognise the most important strains in circulation ­ primarily by exposing it to appropriate versions of hemagglutinin. While the antigenic drift of hemagglutinin has been extensively studied, much less is known about how the neuraminidase accumulates mutations, and how these affect the immune response. To investigate this question, Catani et al. selected 43 genetically distant neuraminidases from human viral samples isolated between 2009 and 2017. Statistical analyses were applied to define their relatedness, revealing that a group of closely related neuraminidases predominated from 2009 to 2015, before they were being taken over by a second group. A third group, which was identified in viruses isolated in 2013, was remarkably close to the neuraminidase of strains that circulated in the late 1990s. The fourth and final group of neuraminidases was derived from influenza viruses that normally circulate in pigs but can also occasionally infect humans. Next, Catani et al. examined the immune response that these 43 neuraminidases could elicit in mice, as well as in ferrets ­ the animal most traditionally used in influenza research. This allowed them to pinpoint which changes in the neuraminidase sequences were important to escape recognition by the host. Data obtained from the two model species were comparable, suggesting that these experiments could be conducted on mice going forward, which are easier to work with than ferrets. Finally, Catani et al. used machine learning to build a computational model that could predict how strongly the immune system would respond to a specific neuraminidase variant. These findings could help guide the development of new vaccines that include neuraminidases tailored to best prime and train the immune system against a larger variety of strains. This may aid the development of 'supra-seasonal' vaccines that protect against a broad range of influenza viruses, reducing the need for yearly updates.