Virus Strains Research Articles

Targeted Immunization Strategies and Designing Vaccine against Indian Nipah Virus Strain (NiV B) and Malaysian Variant (NiV M)

Targeted Immunization Strategies and Designing Vaccine against Indian Nipah Virus Strain (NiV B) and Malaysian Variant (NiV M)

Structure-guided in vitro evolution of nanobodies targeting new viral variants.

A major challenge in antiviral antibody therapy is keeping up with the rapid evolution of viruses. Our research shows that nanobodies - single-domain antibodies derived from camelids - can be rapidly re-engineered to combat new viral strains through structure-guided in vitro evolution. Specifically, for viral mutations occurring at nanobody-binding sites, we introduce randomized amino acid sequences into nanobody residues near these mutations. We then select nanobody variants that effectively bind to the mutated viral target from a phage display library. As a proof of concept, we used this approach to adapt Nanosota-3, a nanobody originally identified to target the receptor-binding domain (RBD) of early Omicron subvariants, making it highly effective against recent Omicron subvariants. Remarkably, this adaptation process can be completed in less than two weeks, allowing drug development to keep pace with viral evolution and provide timely protection to humans.

The Ebola Data Platform: A prospective, standardised, clinical dataset collected during the 2013-2016 West African Ebola outbreak.

The Ebola Data Platform (EDP) was developed to strengthen knowledge and capacity across health, research, and humanitarian communities to reduce the impact of Ebola through responsible data use. This collaborative initiative was established by West African governments, NGOs, academic organisations, and intra-governmental health organisations directly involved in the 2013-2016 West African Ebola outbreak. The platform was established to provide a centralised, standardised dataset of individual patient data collected during the outbreak for the purpose of research to improve Ebola treatment and control, and includes over 13,600 patient records of individuals infected and treated from 22 different Ebola treatment centres across Guinea, Sierra Leone, Liberia, and Nigeria. Patient data are available from treatment centre triage and admission, inpatient clinical observations, and outcomes, with outpatient follow-up available for some datasets. Data include signs and symptoms, pre-existing comorbidities, vital signs, laboratory testing, treatments, complications, dates of admission and discharge, mortality, viral strains, and other data. This publication describes characteristics of the EDP dataset, its architecture, methods for data access and tools for utilising the dataset.

The I7L protein of African swine fever virus is involved in viral pathogenicity by antagonizing the IFN-γ-triggered JAK-STAT signaling pathway through inhibiting the phosphorylation of STAT1.

Cell-passage-adapted strains of African swine fever virus (ASFV) typically exhibit substantial genomic alterations and attenuated virulence in pigs. We have indicated that the human embryonic kidney (HEK293T) cells-adapted ASFV strain underwent genetic alterations and the I7L gene in the right variable region was deleted compared with the ASFV HLJ/2018 strain (ASFV-WT). A recent study has revealed that the deletion of the I7L-I11L genes results in attenuation of virulent ASFV in vivo, but the underlying mechanism remains largely unknown. Therefore, we hypothesized that the deletion of the I7L gene may be related to the pathogenicity of ASFV in pigs. We generated the I7L gene-deleted ASFV mutant (ASFV-ΔI7L) and found that the I7L gene deletion does not influence the replication of ASFV in primary porcine alveolar macrophages (PAMs). Using transcriptome sequencing analysis, we identified that the differentially expressed genes in the PAMs infected with ASFV-ΔI7L were mainly involved in antiviral immune responses induced by interferon gamma (IFN-γ) compared with those in the ASFV-WT-infected PAMs. Meanwhile, we further confirmed that the I7L protein (pI7L) suppressed the IFN-γ-triggered JAK-STAT signaling pathway. Mechanistically, pI7L interacts with STAT1 and inhibits its phosphorylation and homodimerization, which depends on the tyrosine at position 98 (Y98) of pI7L, thereby preventing the nuclear translocation of STAT1 and leading to the decreased production of IFN-γ-stimulated genes. Importantly, ASFV-ΔI7L exhibited reduced replication and virulence compared with ASFV-WT in pigs, likely due to the increased production of IFN-γ-stimulated genes, indicating that pI7L is involved in the virulence of ASFV. Taken together, our findings demonstrate that pI7L is associated with pathogenicity and antagonizes the IFN-γ-triggered JAK-STAT signaling pathway via inhibiting the phosphorylation and homodimerization of STAT1 depending on the Y98 residue of pI7L and the Src homology 2 domain of STAT1, which provides more information for understanding the immunoevasion strategies and designing the live attenuated vaccines against ASFV infection.

Rabies in free-ranging capybaras (Hydrochoerus hydrochaeris) on Anchieta Island, Ubatuba, Brazil.

Between December 2019 and January 2020, three cases of rabies were reported in free-ranging capybaras on Anchieta Island, Ubatuba-SP, Brazil. This 8.28km² island is located 540m offshore from the mainland. Two of the capybaras exhibited signs of hindlimb paralysis, and one was found dead. Rabies was diagnosed using the direct fluorescent antibody test (dFAT), while RT-qPCR and phylogenetic analysis of nucleotide sequences confirmed the presence of the vampire bat rabies virus (RABV) strain. Although no visible bat bite marks were found on the capybaras, vampire bats are known to inhabit the island. Other wildlife tested negative for rabies during this period, and no further rabies outbreaks have been observed since. Environmental changes and human activities, such as the disturbance of bat roosting sites, may have contributed to the incident. The detection of rabies in capybaras suggests a potential spillover from a vampire bat reservoir. Further investigation is needed to determine whether capybaras act as dead-end hosts or play a role in maintaining the rabies transmission cycle.

Analysis of neuraminidase activity of human parainfluenza viruses using enzyme-linked lectin assay and BTP3-Neu5Ac assay.

Human parainfluenza viruses (hPIVs) are causative agents of upper and lower respiratory tract infections and they have four serotypes. The virion surface displays hemagglutinin-neuraminidase (HN), having hemagglutinating (HA) and neuraminidase (NA) activities in a single molecule. The HA activity binds the virion to sialic acid on the viral receptor on host cells and the NA releases the progeny viruses from the cell surface. There are several methods for assaying viral NA activity, such as the thiobarbituric acid assay, 4-methylumbelliferyl-N-acetyl-α-d-neuraminic acid assay, NA-Star assay, and enzyme-linked lectin assay (ELLA). However, these are mainly used for influenza viruses and not for hPIVs. A fluorescent-based cytochemical NA assay using BTP3-Neu5Ac as the substrate was recently developed and used for orthomyxo- and paramyxoviruses, including types 1 and 3 hPIVs. In this study, we used the ELLA, and BTP-Neu5Ac assay for 14 field isolate strains of hPIVs including all four serotypes. The reaction in ELLA at pH 6.5 using peanut agglutinin (PNA) as a lectin was very low for all tested viruses except a type 3 virus strain with the maximum reaction at pH 6.5 and the acidic conditions did not enhance the reaction. ELLA with another lectin, Erythrina cristagalli agglutinin exhibited significant and stronger reactions than with PNA in some strains of types 1 and 3 viruses. The BTP3-Neu5Ac assay showed a fluorescent signal on cells infected with all the viruses except the hPIV1/Sendai/713/2018 strain in LLC-MK2 and/or MNT-1. The signal was detected in cell-free virus, as well, in all the viruses except the hPIV4a/Sendai/3935/2003 strain. The strength of the signal varied among viral strains but it was stronger in the reaction at pH 4.0 than pH 7.0 and strongest in type 2 hPIVs.

A transgenic mouse with a humanised B cell repertoire mounts an antibody response to influenza infection and vaccination.

The development of a universal influenza vaccine likely requires an understanding of previous exposure to influenza virus (through vaccination or infection) and how that shapes the antibody repertoire to vaccination, sometimes called Original Antigenic Sin or antigenic imprinting. Whilst animal models can have a much more defined exposure history, they lack a human B cell repertoire. Transgenic mice with the complete human immunoglobulin locus enable studies of controlled infection history leading to human-like antibody evolution. Here we evaluated responses to influenza in the Intelliselect Transgenic mouse (the Kymouse). We show the Kymouse is susceptible to disease following infection with either H1N1, H3N2 or B/Yam influenza viruses and that it induces a robust binding and neutralising antibody response to all three strains of influenza virus. This study demonstrates that human B cell repertoire mice can be used for influenza virus studies, providing a tool for further interrogation of the antibody response.

Genetic characteristics of influenza A and B viruses circulating in Russia in 2019-2023.

Relevance. Influenza viruses have a high potential for genetic change. These viruses are monitored annually around the world, including Russia, to determine the dominant genetic groups and select the strains to be included in influenza vaccines. Aims of the study include: analysis of the circulation of influenza viruses in Russia in 2019-2023, phylogenetic and molecular analysis of hemagglutinin (HA) sequences of influenza virus strains, detection of drug resistance mutations to neuraminidase (NA) inhibitors and M2-protein (M2) ion channel inhibitors. Materials and methods. Biological samples containing RNA of influenza viruses were studied: 417 A(H1N1)pdm09, 147 A(H3N2) and 167 B(Victoria). Sequencing of the PCR fragments was performed on the 3500xL Genetic Analyzer (Applied Biosytems). Data processing and analysis were carried out using DNASTAR, Nextclade, FluSurver and BioNumerics v.6.6 software. Results. Influenza A(H1N1)pdm09, A(H3N2), B(Victoria) viruses circulating in 2019-2023 were investigated. The highest variability of HA was observed in A(H3N2) viruses. 3% of A(H1N1)pdm09 viruses had the D222N mutation in the receptor-binding site of HA, which is associated with more severe disease. The oseltamivir and zanamivir resistance mutation H275Y in NA was detected in 2% of influenza A(H1N1)pdm09 viruses. No oseltamivir and zanamivir resistance mutations in NA were detected in all influenza A(H3N2) and B viruses tested. In all investigated influenza A(H1N1)pdm09 and A(H3N2) viruses the adamantane resistance mutation S31N in M2 was identified. Conclusions. The detection of amino acid substitutions in HA antigenic sites and resistance mutations in NA and M2 confirms the evolution of influenza viruses and the need for continuous genetic surveillance.

MECHANISMS OF TECOVIRIMAT ANTIVIRAL ACTIVITY AND POXVIRUS RESISTANCE.

Mpox is a zoonotic disease endemic in central and west Africa. However, since 2022, human-adapted mpox virus (MPXV) strains are causing large outbreaks spreading outside these regions, leading the World Health Organization to declare public health emergency twice. Tecovirimat, the most widely used drug to treat these infections, blocks viral egress through a poorly understood mechanism. Tecovirimat-resistant strains, all with mutations in the viral phospholipase F13, pose public health concerns. Herein, we report the structure of an F13 homodimer, both alone and in complex with tecovirimat. We demonstrate that tecovirimat acts as a molecular glue, inducing the dimerization of the phospholipase. F13 escape mutations in MPXV clinical isolates are at the dimer interface and prevent drug-induced dimerization in solution and cells. These findings, which decipher tecovirimat's mode of action, will allow better monitoring of poxvirus outbreaks and pave the way for developing more potent and resilient therapeutics.

Mycoplasma: Properties, Detection, and Decontamination Methods of Cell Cultures and Viral Strains (Review)

Mycoplasma: Properties, Detection, and Decontamination Methods of Cell Cultures and Viral Strains (Review)

COBRA N2 NA vaccines induce protective immune responses against influenza viral infection

ABSTRACT Influenza neuraminidase (NA) is a promising target for a broadly protective vaccine. In this study, the Computationally Optimized Broadly Reactive Antigen (COBRA) methodology was used to develop N2 NA vaccine candidates. The unique wild type (WT) N2 sequences of human and swine influenza strains isolated between 1957 and 2019 were used to design the COBRA N2-A NA vaccine, while the unique WT N2 sequences of human influenza strains isolated between 2000 and 2019 were used to design the COBRA N2-B NA vaccine. Sera collected from COBRA N2 NA vaccinated mice showed more broadly reactive antibody responses against a broad panel of H×N2 influenza virus strains than sera collected from mice vaccinated with WT N2 NA vaccines. Antibodies elicited by COBRA or WT N2 NA antigens cross react with recent human H3N2 influenza viruses from different clades, while the antibodies elicited by A/Switzerland/9715293/2013 hemagglutinin (HA) reacted with viruses from the same clade. Furthermore, mice vaccinated with COBRA N2-B NA vaccine had lower viral lung titers compared to mock vaccinated mice when challenged with human H3N2 influenza viruses. Thus, the COBRA N2 NA vaccines elicit broadly protective murine anti-NA antibodies against multiple strains across subtypes and the viral loads were significantly decreased in the lungs of the mice in the COBRA N2 NA vaccine groups, compared to the mice in the mock vaccinated group, indicating that the COBRA-based N2 subtype NA vaccines have a potential to be a component in a universal influenza vaccine.

Research Note: The infectious bursal disease (Gumboro disease) vaccination scheme affects the quantitative and qualitative carcass characteristics and the immune response of Ross 308 broiler chickens

After different vaccinations, the study aimed to assess carcass composition, meat quality, and the level of the infectious bursal disease antibody titers in broiler chickens. In the experiment, 4 (30 chicks per group, 120 total) groups of Ross 308 broiler chickens (males and females) were included. Then, they were vaccinated at the hatchery. Group I was immunized against infectious avian bronchitis (IB) and Newcastle disease (ND) using vaccine A (20 mL/100 birds) and vaccine B (20 mL/100 birds), respectively, with injectable vaccine C (0.2 mL/each) targeting Infectious bursal' and Marek's diseases (MD). Group II received the same vaccinations for IB and ND, with separate injectable D vaccines for MD (0.2 mL/unit) and E for infectious bursal' disease (0.2 mL/each). Group III was similarly vaccinated for IB and ND; however, they received only vaccine D (0.2 mL/each) for MD, and on d 23, vaccine F was administered via drinking water against Gumboro disease (0.05 mL/each). Group IV served as the control and was not vaccinated. Ten carcasses from each group on d 42 were taken for carcass composition analysis and meat quality, including pH, color, and water-holding capacity (WHC). During rearing, on d 14, 28, and 42, blood was collected to determine the level of antibodies against infectious bursal' disease. Additionally, hematocrit (HCT), hemoglobin (HGB), and red blood cells were analyzed. In group I, a higher bursa of Fabricius weight than in the other groups was found (P < 0.001), and the liver weight was lower compared to group II (P = 0.020). In breast muscle, the pH24 was higher in the control group than in group I (P = 0.036). Lightness (L*) in breast and leg muscles from group I was higher than in group II (P < 0.05). WHC was higher in group I than in group II (P = 0.025). The antibody titers of infectious bursal' disease was higher in group I on d 28 than in other groups (P < 0.05). Vaccine C (1 dose 0.2 mL/1 bird) of the reconstituted vaccine with the live recombinant virus strain vHVT013-69, not less than 3.6 - 5.0 log10 PFU) had a beneficial effect.

Assessing different next-generation sequencing technologies for wastewater-based epidemiology

Wastewater-based epidemiology has proven to be an important public health asset during the COVID-19 pandemic. It can provide less biassed and more cost-effective population-level monitoring of the disease burden as compared to clinical testing. An essential component of SARS-CoV-2 wastewater monitoring is next-generation sequencing, providing genomic data to identify and quantify circulating viral strains rapidly. However, the specific choice of sequencing method influences the quality and timeliness of generated data and hence its usefulness for wastewater-based pathogen surveillance. Here, we systematically benchmarked Illumina Novaseq 6000, Element Aviti, ONT R9.4.1 MinION flow cell, and ONT R9.4.1 Flongle flow cell sequencing data to facilitate the selection of sequencing technology. Using a time series of wastewater samples from influent of six wastewater treatment plants throughout Switzerland, along with spike-in experiments, we show that higher sequencing error rates of ONT Nanopore sequencing reduce the accuracy of estimates of the relative abundance of viral variants, but the overall trend is in good concordance among all technologies. We find that the sequencing runtime for ONT Nanopore flow cells can be reduced to as little as five hours without significant impact on the quality of variant estimates. Our findings suggest that SARS-CoV-2 variant tracking is readily achievable with all tested technologies, albeit with different tradeoffs in terms of cost, timeliness and accuracy.

Antiviral effect of oversulfated kappa-carrageenan derivatives against COVID-19 for spray coating application on facemasks

The COVID-19 pandemic caused by SARS-CoV-2 has spurred the urgent need for effective antiviral strategies. In this work, we explored the potential of oversulfated kappa-carrageenan (OSKC) in spray-coated facemasks for SARS-CoV-2 inhibition pathway. The sulfated derivative was synthesized with sulfur trioxide pyridine complex in dimethylformamide solution. The antiviral efficacy of OSKC at different concentrations and spray-coated facemasks was evaluated using betacoronavirus Murine Hepatitis Virus strain 3, revealing a significant reduction in viral load compared to commercial kappa-carrageenan. Furthermore, the characterization techniques assessed the effect of the position of the introduced sulfate groups on the antiviral activity and on the physicochemical characteristics. OSKC is able to bind specific proteins of enveloped viruses, preventing viral attachment into target cells. Overall, this study demonstrates the feasibility and effectiveness of OSKC spray coating for breathable facemasks with antimicrobial properties, offering a promising approach to enhancing personal protective equipment against viral transmission in healthcare and community settings.

Influenza virus antibodies inhibit antigen-specific de novo B cell responses in mice.

Antibody responses to influenza vaccines tend to be focused on epitopes encountered during prior influenza exposures, with little production of de novo responses to novel epitopes. To examine the contribution of circulating antibodies to this phenomenon, we passively transferred a hemagglutinin (HA)-specific monoclonal antibody (mAb) into mice before immunizing with whole inactivated virions. The HA mAb inhibited de novo HA-specific antibodies, plasmablasts, germinal center B cells, and memory B cells, while responses to a second antigen in the vaccine, neuraminidase (NA), were uninhibited. The HA mAb potently inhibited de novo antibody responses against epitopes near the HA mAb binding site. The HA mAb also promoted IgG1 class switching, an effect that, unlike the inhibition of HA responses, relied on signaling through Fc-gamma receptors. These studies suggest that circulating antibodies inhibit de novo B cell responses in an antigen-specific manner, which likely contributes to differences in antibody specificities elicited during primary and secondary influenza virus exposures.IMPORTANCEMost humans are exposed to influenza viruses in childhood and then subsequently exposed to antigenically drifted influenza variants later in life. It is unclear if antibodies elicited by earlier influenza virus exposures impact immunity against new influenza virus strains. Here, we used a mouse model to investigate how an anti-hemagglutinin (HA) monoclonal antibody (mAb) affects de novo B cell and antibody responses to the protein targeted by the monoclonal antibody (HA) and a second protein not targeted by the monoclonal antibody [neuraminidase (NA)]. Collectively, our studies suggest that circulating anti-influenza virus antibodies can potently modulate the magnitude and specificity of antibody responses elicited by secondary influenza virus exposures.

The 5'UTR of porcine reproductive and respiratory syndrome virus strain JXwn06 harbors a uORF that regulates cellular inflammation.

PRRSV has remained a major challenge to the world swine industry, but we still do not know much about its biology and pathogenesis. Here, we provide evidence to show that the 5'UTR of PRRSV strain JXwn06 harbors a functional uORF that has the coding capacity and regulates induction of inflammation as demonstrated by in vitro assays and animal experiment. The findings reveal a novel viral factor that regulates cellular inflammation and provide insight into the understanding of PRRSV pathogenesis.

Establishment of in-house assay for screening of anti-SARS-CoV-2 protein inhibitors

Developing a potent antiviral agent to combat Coronavirus Disease-19 (COVID-19) is of critical importance as we may be at risk of the emergence of new virus strains or another pandemic recurrence. The interaction between the SARS-CoV-2 spike protein and Angiotensin Converting Enzyme 2 (ACE2) is the main protein-protein interaction (PPI) implicated in the virus entry into the host cells. Spike-ACE2 PPI represents a major target for drug intervention. We have repurposed a previously described protein-protein interaction detection method to be utilized as a drug screening assay. The assay was standardized using Chitosan nanoparticles (CNPs) as the drug and SARS-CoV-2 spike-ACE2 interaction as the PPI model. The assay was then used to screen four natural bioactive compounds: Curcumin (Cur), Gallic acid (GA), Quercetin (Q), and Silymarin (Sil), and their cytotoxicity was evaluated in vitro. Production of the spike protein and the evaluation of its activity in comparison to a standard commercial protein was part of our work as well. Here we describe a novel simple immunofluorescent screening assay to identify potential SARS-CoV-2 inhibitors that could assess the inhibitory effect of any ligand against any PPI.Graphical

Varicella-zoster virus recapitulates its immune evasive behaviour in matured hiPSC-derived neurospheroids.

Varicella-zoster virus (VZV) encephalitis and meningitis are potential central nervous system (CNS) complications following primary VZV infection or reactivation. With Type-I interferon (IFN) signalling being an important first line cellular defence mechanism against VZV infection by the peripheral tissues, we here investigated the triggering of innate immune responses in a human neural-like environment. For this, we established and characterised 5-month matured hiPSC-derived neurospheroids (NSPHs) containing neurons and astrocytes. Subsequently, NSPHs were infected with reporter strains of VZV (VZVeGFP-ORF23) or Sendai virus (SeVeGFP), with the latter serving as an immune-activating positive control. Live cell and immunocytochemical analyses demonstrated VZVeGFP-ORF23 infection throughout the NSPHs, while SeVeGFP infection was limited to the outer NSPH border. Next, NanoString digital transcriptomics revealed that SeVeGFP-infected NSPHs activated a clear Type-I IFN response, while this was not the case in VZVeGFP-ORF23-infected NSPHs. Moreover, the latter displayed a strong suppression of genes related to IFN signalling and antigen presentation, as further demonstrated by suppression of IL-6 and CXCL10 production, failure to upregulate Type-I IFN activated anti-viral proteins (Mx1, IFIT2 and ISG15), as well as reduced expression of CD74, a key-protein in the MHC class II antigen presentation pathway. Finally, even though VZVeGFP-ORF23-infection seems to be immunologically ignored in NSPHs, its presence does result in the formation of stress granules upon long-term infection, as well as disruption of cellular integrity within the infected NSPHs. Concluding, in this study we demonstrate that 5-month matured hiPSC-derived NSPHs display functional innate immune reactivity towards SeV infection, and have the capacity to recapitulate the strong immune evasive behaviour towards VZV.

Development and validation of highly sensitive multiplex real-time RT-PCR assay for detection of classical swine fever virus genome

Classical swine fever (CSF) remains a challenge for pig farming industrial lover the world despite the measures taken. The last CS case in the Russian Federation was reported in 2020, however, the threat of the disease emerging still persists. A set of anti-epidemic measures including mainly preventive vaccination and annual diagnostic monitoring using molecular-genetic and serological methods is required for CSF virus introduction prevention and rapid eradication of potential disease out breaks. Therefore, areal-time reverse transcription-polymerase chain reaction using an internal control sample has been developed. Therefore, areal time reverse transcription-polymerase chain reaction using an internal control sample has been developed. Modified primers (locked nucleic acids containing conformationally blocked nucleosides) providing a higher affinity to the DNA matrix and physico-chemical stability and a FAM-labeled TaqMan probe were selected for 5’-untranslatedregion of the genome. The following validation parameters were defined: accuracy, repeatability, reproducibility, specificity and sensitivity. For comparative analysis of the developed as say sensitivity, swabs, samples of organs and tissues collected from pigs experimentally infected with an epizootic strain of the classical swine fever virus (spleen, kidney, liver, blood, lymph nodes, rectal and oral smears), animal-contaminated feed and virus-containing material with known virus titres were also tested in parallel with coded test systems No. x1 andx2. The developed assay was shown to have 100% diagnostic sensitivity and detection limit of 0,23 lgCCID50/cm3. Therewith, there sults of analysis of test systems No. x1, x2 based on above parameters were lower that could give rise to false positive real-time RT-PCR results and incorrect diagnosis. Thus, described assay can be used for extensive monitoring of classical swine fever in the Russian Federation

CircRNA based multivalent neuraminidase vaccine induces broad protection against influenza viruses in mice

Developing broad-spectrum influenza vaccines is crucial for influenza control and potential pandemic preparedness. Here, we reported a novel vaccine design utilizing circular RNA (circRNA) as a delivery platform for multi-subtype neuraminidases (NA) (influenza A N1, N2, and influenza B Victoria lineage NA) immunogens. Individual NA circRNA lipid nanoparticles (LNP) elicited robust NA-specific antibody responses with neuraminidase inhibition activity (NAI), preventing the virus from egressing and infecting neighboring cells. Additionally, the administration of circRNA LNP induced cellular immunity in mice. To achieve a universal influenza vaccine, we combined all three subtypes of NA circRNA-LNPs to generate a trivalent circRNA vaccine. The trivalent vaccine elicited a balanced antibody response against all three NA subtypes and a Th1-biased immune response in mice. Moreover, it protected mice against the lethal challenge of matched and mismatched H1N1, H3N2, and influenza B viruses, encompassing circulating and ancestral influenza virus strains. This study highlights the potential of delivering multiple NA antigens through circRNA-LNPs as a promising strategy for effectively developing a universal influenza vaccine against diverse influenza viruses.