Hemagglutination Inhibition Research Articles

Identification of key antigenic sites in hemagglutinin of H10N3 avian influenza virus

The H10 avian influenza viruses (AIV) have been detected in both birds and mammals. Recently, the cases of human infection with H10N8 and H10N3 in China pose high risk to public health. However, the antigenic sites in hemagglutinin (HA) of H10 are poorly understood. In this study, 3 monoclonal antibodies (MAb), designated as 1F4, 6B3 and 6G12, against the HA protein of the H10N3 strain A/chicken/Taizhou/498/2021(H10N3) (TZ498), were first generated. All of these MAb could effectively inhibit TZ498 in haemagglutination inhibition assay and microneutralization assay. Four novel antigenic sites at positions 135, 208, 227, and 266 (H10 numbering) were identified in the HA of TZ498 through escape mutants selected by these 3 MAb. Moreover, natural mutations at positions 135 and 227 were found in the H10 field strains. All these not only provide novel insights into the molecular markers for monitoring the antigenic variation of H10 but also be helpful for developing efficient control strategies against H10.

Rapid Onset of Innate Response, Cytokine Signaling and Humoral Immunity in Inactivated LPAI-H9N2-Vaccinated Broilers

The development of effective and innovative vaccination strategies is urgently needed to better control the spread and transmission of the low-pathogenic avian influenza H9N2 subtype (LPAI-H9N2) in poultry. In addition, the enhancement of innate immunity by some of these innovative inactivated vaccines has not yet been investigated. Here, an experiment was conducted in commercial broiler chickens to compare the immune response to two different inactivated H9N2 vaccines. For this, Group 1 (G1) broilers were vaccinated with vaccine 1 [Nobilis® H9N2-P (pathogen-associated molecular patterns—PAMP) technology], broilers in G2 were vaccinated with vaccine 2 [an inactivated whole H9N2 virus (IWV) autogenous oil emulsion vaccine], while birds in G3 were not vaccinated. The study lasted 34 days. Innate immune parameters (phagocytic activity, nitric oxide, and lysozyme), cytokine signaling (IL-1β, IL-6, IL-8), humoral immunity using the hemagglutination inhibition (HI) test, and the gene expressions of IFN-γ and TLR-21 were assessed. The results showed a significant increase in innate immunity and modulatory cytokines at 24–48 h after the vaccination of G1 broilers, with a continuous increase until the end of the experiment. In addition, a significant increase in geometric mean HI titers was observed in G1 at 11 days post-vaccination (dpv), and a significant (p ˂ 0.05) upregulation of IFN-γ and TLR-21 was observed in the same group, G1, at 31 dpv compared to G2 and G3. Nobilis® H9N2-P may induce faster and stronger innate and active humoral immunity compared to another IWV, which may contribute to the protection of broilers against early H9N2 infections. However, challenge protection studies for several IWV vaccines, including PAMP-H9N2 against LPAI-H9N2, should be further evaluated in both specific pathogen-free (SPF) and commercial broilers.

A Modified Novel Validated High-Throughput Hemagglutinin Inhibition Assay Using Recombinant Virus-like Particles and Human Red Blood Cells for the Objective Evaluation of Recombinant Hemagglutinin Nanoparticle Seasonal Influenza Vaccine.

Currently available seasonal influenza vaccines confer variable protection due to antigenic changes resulting from the accumulation of diverse mutations. The analysis of new seasonal influenza vaccines is challenging in part due to the limitations of the traditional hemagglutination inhibition (HAI) assay with A/H3N2 strains. An improved and objective novel HAI assay was developed with recombinant virus-like particles (VLPs) and an egg-derived virus as agglutinins, the oseltamivir treatment of VLPs, human red blood cells, and using an automated image reader-based analysis of hemagglutination. HAI validation was demonstrated using four VLPs and egg-derived strains, with 46-56 serum samples tested 12 times in duplicate per strain. The validated HAI assay was precise as indicated by the percent geometric coefficient of variation for intra-, inter-, and total assay precision, as well as accurate as evidenced by percent bias measurements. The assay exhibited linearity, specificity for homologous type/subtype strains, and sensitivity with a starting dilution of 1:10. Assay robustness and sample stability were demonstrated as a percentage difference compared to reference condition. Validated HAI results were equivalent for the single and duplicate sample testing and correlated well with a qualified live wild-type influenza microneutralization assay. These findings demonstrate the suitability of this high-throughput novel modified validated HAI assay for evaluating vaccine immunogenicity and efficacy.

Highly Pathogenic H5N1 Influenza A Virus (IAV) in Blue-Winged Teal in the Mississippi Flyway Is Following the Historic Seasonal Pattern of Low-Pathogenicity IAV in Ducks.

Highly pathogenic H5N1 (HP H5N1) influenza A virus (IAV) has been detected annually in North American ducks since its introduction during 2021, but it is unknown if this virus will follow the same seasonal and geographic patterns that have been observed with low-pathogenicity (LP) IAV in this reservoir. We monitored blue-winged teal in the Mississippi flyway prior to the detection of HP H5N1 and during two post-introduction migration cycles from spring 2022 to spring 2024, testing birds for infection and antibodies to IAV nucleoprotein (NP), hemagglutinin subtype H5, and neuraminidase subtype N1. Antigens representing clade 2.3.4.4b HP H5 and LP North American H5 were used for hemagglutination inhibition (HI) and virus neutralization (VN) tests for H5 antibodies. Virologic results were consistent with historic seasonal and geographic patterns reported for LP IAV with peak infections occurring in pre-migration staging areas in Minnesota during fall 2022. However, the high prevalence of the H5 subtype was exceptional compared to historic prevalence estimates at this same site and for the Mississippi flyway. HP H5N1 was detected on wintering areas in Louisiana and Texas during the fall of that same year and this was followed by an increase in estimated antibody prevalence to NP, H5, and N1 with no HP H5N1 detections during the wintering or spring migration periods of 2022/2023. HP H5N1 was not detected in Minnesota during fall 2023 but was detected from a single bird in Louisiana. However, a similar increase in antibody prevalence was observed during the winter and spring period of 2023 and 2024. Over the two migration cycles, there was a temporal shift in observed prevalence and relative titers against the H5 antigens with a higher proportion of ducks testing positive to the 2.3.4.4b H5 antigen and higher relative titer to that antigen compared to the representative LP North American H5 antigen. The seasonal and geographic patterns observed appear to be driven by population immunity during the migration cycle. Results support an initial high infection rate of HP H5N1 in blue-winged teal in the Mississippi flyway followed by a high prevalence of antibodies to NP, H5, and N1. Although prevalence was much reduced in the second migration cycle following introduction, it is not known if this pattern will persist in the longer term or affect historic patterns of subtype diversity in this reservoir.

Antiviral Effect of Amentoflavone Against Influenza Viruses.

Amentoflavone (AF) is a biflavonoid compound found in many plants. In this study, we first demonstrate that AF has a potent antiviral effect against the influenza virus via the inhibition of viral attachment and virucidal effects. The anti-influenza-viral effect of AF was evaluated using green fluorescent protein-tagged Influenza A virus (IAV) with fluorescent microscopy and flow cytometry analysis. AF decreased the GFP expression by viral infection, dose-dependently. Fifty micromoles of AF suppressed the GFP expression by virus infection of up to 70% of untreated infected control cells. Consistently, immunofluorescence results showed the inhibitory effect of AF on viral protein expression. Time-of-addition and hemagglutination assays revealed that AF inhibits viral binding to cells by interfering with the hemagglutinin (HA) of IAV. Furthermore, AF has a virucidal effect and blocks cytopathic effects caused by the Influenza B virus and H3N2 IAV. Additionally, AF represses the neuraminidase (NA) activity of IAV. In silico analysis confirmed the potential interaction of AF with both HA and NA. Our findings indicate that AF has antiviral effects by modulating HA and NA during the attachment and release stages of influenza viral infection.

The introduction of a highly virulent PRRSV strain in pig farms is associated with a change in the pattern of influenza A virus infection in nurseries

The present study aimed to determine the dynamics of influenza A virus (IAV) infection in two endemically infected farms (F1 and F2), where a longitudinal follow-up of piglets was performed from birth to 8–12 weeks of age. During the study, a highly virulent isolate of porcine reproductive and respiratory syndrome virus (PRRSV) was introduced on both farms. This allowed us to examine the impact of such introduction on the patterns of infection, disease, and the antibody response of pigs to IAV infection. The introduction of the new PRRSV strain coincided with a change in the dynamics of IAV infection on both farms. In F1, the cumulative incidence of IAV increased from 20% before the outbreak to 67.5%, together with the existence of animals that tested positive for IAV (RT‒qPCR) in nasal swabs for two or more consecutive samples. In F2, the cumulative incidence of IAV increased from 50% before the PRRSV outbreak to 70%, and the proportion of prolonged IAV shedders increased sharply. Additionally, some animals were infected with the same IAV twice during the observation period. In contrast to previous reports, our study revealed that prolonged shedding was not related to the titres of maternally derived antibodies at the time of infection but was significantly (p < 0.05) related to PRRSV infection status. Notably, both before and after the PRRSV outbreak, a high proportion of IAV-infected piglets did not seroconvert, which was significantly (p < 0.05) related to the hemagglutination inhibition titres against IAV when infected.

Exploring potential of graphene oxide as an alternative antiviral approach for influenza A H1N1.

Aim: Graphene oxide (GO), known for its distinctive physicochemical properties, shows promise as a nanomaterial capable of combating infectious agents. This study investigates the efficacy of GO nanoparticles in restricting influenza A H1N1 replication in MDCK cells.Methods: GO nanoparticles were synthesized. After evaluating the toxicity of GO nanoparticles, the antiviral activity of the highest nontoxic concentration of GO against influenza A H1N1 in MDCK cells was studied.Results: GO treatments resulted in substantial decreases in virus titers, as shown via hemagglutination assay, TCID50 assay and real-time PCR analysis.Conclusion: This study emphasizes that GO nanoparticles have a high level of effectiveness against influenza A H1N1 viruses, making them an intriguing option for various antiviral uses.

Protective efficacy of a recombinant adenovirus expressing novel dual F and HN proteins of bovine parainfluenza virus type 3

Bovine parainfluenza virus type 3 (BPIV3) is a viral respiratory pathogen that infects cattle and causes significant economic losses. We generated a recombinant adenovirus called rHAd5-F + HN by expressing the fusion (F) and hemagglutinin-neuraminidase (HN) glycoprotein of BPIV3 using the human adenovirus serotype 5 (rHAd5). We evaluated its effects on humoral and cellular immune responses in mice (n = 45) and calves (n = 9). Serum antibody responses were assessed by enzyme-linked immunosorbent assay (ELISA), hemagglutination inhibition (HI), and neutralising antibodies (NAb). After boosting immunity with rHAd5-F + HN, mice produced significantly higher levels of antibodies against the BPIV3 genotype A and genotype C strains. The production of antibodies exceeded those produced by adenoviruses rHAd5-F and rHAd5-HN, which express the F and HN glycoprotein, respectively. The percentages of splenic CD3+/CD8+T lymphocytes and IL-4+ cytokines in rHAd5-F + HN mice were considerably higher than those in the control group. Mice immunised with rHAd5-F + HN exhibited much lower viral loads in the lungs and tracheas compared to the control group. Additionally, the lungs of mice vaccinated with rHAd5-F + HN showed no notable histopathological changes. On the other hand, rHAd5-F + HN produced a humoral immune response in calves. Following the booster intramuscular injection with the rHAd5-F + HN, the serum antibody levels against BPIV3 genotype C strain were 1:20 452, 1:1024, and 1:426 in calves, as detected by ELISA, HI, and NAb, respectively. The HI and NAb levels against the BPIV3 genotype A strain were 1:213 and 1:85 in calves, respectively. These results indicate that rHAd5-F + HN effectively induced immunity against BPIV3 infection.

Hemagglutination-Inhibition Antibodies and Protection against Influenza Elicited by Inactivated and Live Attenuated Vaccines in Children.

Hemagglutinin (HA)-inhibiting antibodies contribute to the immune defense against influenza infection. However, there are insufficient data on the extent of correlation between vaccine-elicited HA antibodies and protection in children against different influenza strains, particularly when comparing live attenuated influenza vaccines (LAIV) versus inactivated influenza vaccines (IIV). We measured postvaccination hemagglutination-inhibition (HAI) titers in 3-15-year-old participants of a cluster-randomized controlled trial of trivalent LAIV(3) versus IIV(3) in Canadian Hutterite colonies. We assessed HAI titers as predictors of symptomatic, reverse transcription polymerase chain reaction (RT-PCR)-confirmed influenza over 3 influenza seasons using Cox proportional hazards regression models with vaccine type as a covariate. For each log2 unit increase in postvaccination HAI against A/H1N1 in 2013-2014, A/H3N2 2014-2015, and B/Yamagata in 2013-2014 (each the predominant circulating strain for the respective influenza season), the reduction in the risk of confirmed influenza was equal to 29.6% (95% confidence interval [CI], 17.1%-39.5%), 34.8% (95% CI, 17.2%-47.9%), and 31.8% (95% CI, 23.8%-38.5%), respectively. No reduction in the risk of influenza was observed with B/Yamagata-specific HAI titers in 2012-2013, which was dominated by a mixture of Yamagata and Victoria strains. Despite the overall lower HAI titers in the LAIV3 group, both H1N1 and H3N2 HAI titers were associated with protection against subtype matched influenza. Both LAIV3- and IIV3-elicited HA antibodies are associated with protection against influenza infection in seasons when the vaccine strains match the circulating influenza strain subtypes, supporting the use of HAI as a correlate of protection for both vaccine types in children.

Reconstructed influenza A/H3N2 infection histories reveal variation in incidence and antibody dynamics over the life course.

Humans experience many influenza infections over their lives, resulting in complex and varied immunological histories. Although experimental and quantitative analyses have improved our understanding of the immunological processes defining an individual's antibody repertoire, how these within-host processes are linked to population-level influenza epidemiology in humans remains unclear. Here, we used a multilevel mathematical model to jointly infer antibody dynamics and individual-level lifetime influenza A/H3N2 infection histories for 1,130 individuals in Guangzhou, China, using 67,683 haemagglutination inhibition (HI) assay measurements against 20 A/H3N2 strains from repeat serum samples collected between 2009 and 2015. These estimated infection histories allowed us to reconstruct historical seasonal influenza patterns in humans and to investigate how influenza incidence varies over time, space, and age in this population. We estimated median annual influenza infection rates to be approximately 19% from 1968 to 2015, but with substantial variation between years; 88% of individuals were estimated to have been infected at least once during the study period (2009 to 2015), and 20% were estimated to have 3 or more infections in that time. We inferred decreasing infection rates with increasing age, and found that annual attack rates were highly correlated across all locations, regardless of their distance, suggesting that age has a stronger impact than fine-scale spatial effects in determining an individual's antibody profile. Finally, we reconstructed each individual's expected antibody profile over their lifetime and inferred an age-stratified relationship between probability of infection and HI titre. Our analyses show how multi-strain serological panels provide rich information on long-term epidemiological trends, within-host processes, and immunity when analysed using appropriate inference methods, and adds to our understanding of the life course epidemiology of influenza A/H3N2.

Development and Evaluation of a Novel Chimeric Genotype VII Newcastle Disease Vaccine: Overcoming Maternal Antibody Interference and Spray Administration.

Newcastle disease virus (NDV) poses a significant threat to the poultry industry, with the emergence of genotype VII NDV leading to extensive outbreaks and economic losses. Vaccination is the primary means of controlling NDV, but the presence of maternal antibodies (MDAs) can interfere with the immunological effect of live virus vaccines. Thus, we constructed a chimeric NDV live virus vaccine, LX-OAI4S, by replacing the extracellular regions of the F and HN genes of the NDV LX strain with the corresponding regions of the A-VII vaccine strain. The chimeric vaccine LX-OAI4S demonstrated high genetic stability, good safety, and strong reproductive capacity in chicken embryos. The LX-OAI4S vaccine induced rapid antibody production in specific pathogen-free (SPF) and commercial chickens via the intranasal and intraocular (IN/IO) routes, with hemagglutination inhibition (HI) antibody titers reaching 4.71 ± 1.03 log2 at 7 days post-vaccination (dpv), significantly higher than those of the two classical vaccine strains La Sota and VG/GA. The LX-OAI4S vaccine group provided effective protection against the challenge of genotype VII NDV virulent strain JS2/06 and inhibited viral shedding. When administered via spray, the LX-OAI4S vaccine elicited high systemic immunity against NDV in both SPF and commercial chickens, effectively protecting against clinical disease and reducing viral shedding. The chickens were exposed to high-dose vaccine for spray vaccination, and no adverse reactions were observed after vaccination. Despite the presence of anti-NDV MDAs in chickens, the NDV-specific antibody titers were significantly greater in the vaccinated groups than in the unvaccinated group. The vaccine exhibited high immunogenicity and the potential to overcome maternal antibody interference. The LX-OAI4S vaccine is a promising candidate for an ND vaccine. Its administration via spray can effectively prevent the occurrence of ND, making it a valuable tool for the poultry industry.

Highly pathogenic avian influenza (HPAI) H5 virus exposure in domestic cats and rural stray cats, the Netherlands, October 2020 to June 2023.

BackgroundHighly pathogenic avian influenza (HPAI) H5Nx and human H1N1pdm2009 influenza viruses can infect cats. Infections in cats may result in viral adaptations or recombinant viruses, which may facilitate zoonotic transfer.AimWe aimed to investigate the presence of HPAI H5 clade 2.3.4.4 and H1 influenza viruses and antibodies to these viruses in domestic and rural stray cats in the Netherlands and factors associated with exposure.MethodsSera from stray and domestic cats, sampled 2020-2023, were analysed by ELISA and confirmed by hemagglutination inhibition assay (HAI) and pharyngeal swabs and lung tissue for influenza A virus by RT-qPCR.ResultsIn 701 stray cats, 83 (11.8%; 95% confidence interval (CI): 9.5-14.5) sera were positive for HPAI H5 and 65 findings were confirmed. In HAI, two sera were positive for both HPAI H5 and H1. In 871 domestic cats, four (0.46%; 95% CI: 0.13-1.2) sera were HPAI H5 positive and none were confirmed but 40 (4.6%; 95% CI: 3.3-6.2) sera were seropositive for H1 and 26 were confirmed. Stray cats living in nature reserves (odds ratio (OR) = 5.4; 95% CI: 1.5-20.1) and older cats (OR = 3.8; 95% CI: 2.7-7.1) were more likely to be HPAI H5 seropositive. No influenza A virus was detected in 230 cats.ConclusionThe higher HPAI H5 seroprevalence in stray cats compared with domestic cats suggests more frequent viral exposure, most likely due to foraging on wild birds. In contrast, exposure to H1 was more common in domestic cats compared with stray cats.

Protective Impact of Influenza Vaccination on Healthcare Workers.

Influenza vaccine uptake among healthcare workers is crucial for preventing influenza infections, yet its effectiveness needs further investigation. This prospective observational study aimed to assess the protective effect of influenza vaccination among healthcare workers in Shenzhen. We enrolled 100 participants, with 50 receiving the 2023-2024 quadrivalent influenza vaccine (QIV) and 50 serving as unvaccinated controls. Epidemiological data were collected when the participants presented influenza-like illness. Serum samples were collected at three time points (pre-vaccination and 28 and 180 days after vaccination). Hemagglutination inhibition (HI) assay was performed against the strains included in the 2023-2024 QIV (H1N1, H3N2, BV and BY strains) to assess antibody protection levels. Demographics comparisons revealed no significant differences between the vaccinated and control groups (p > 0.05), ensuring group comparability. The incidence of influenza-like illness was significantly lower in the vaccinated (18%) compared to the control group (36%; p = 0.046; OR = 0.39; 95% CI: 0.15 to 0.98). The vaccinated group also exhibited a higher rate of consecutive two-year vaccinations (48% vs. 24% in the control group, p < 0.05). Additionally, the vaccinated healthcare workers were more inclined to recommend vaccination to their families (80% vs. 48%, p < 0.05). HI titers against H1N1 (p < 0.01), H3N2 (p < 0.01), BV (p < 0.001) and BY (p < 0.01) significantly increased in the vaccinated group at 28 days post-vaccination. Moreover, a marked and sustained increase in HI titers against the H3N2 strain (p < 0.001) was observed at 180 days post-vaccination, highlighting the vaccine's enduring impact on the immune response. The fold change in the HI titers, indicative of the magnitude of the immune response, was significantly higher for H1N1 (p < 0.01), H3N2 (p < 0.001), BV (p < 0.01) and BY (p < 0.05) among the vaccinated individuals compared to the control group, underscoring the vaccine's efficacy in eliciting a robust and sustained antibody response. Influenza vaccination significantly reduces the incidence of influenza-like illness among healthcare workers and promotes a sustained immune response. The study supports the importance of annual vaccination for this group to enhance personal and public health.

Purification, Structural Characterization, and Bioactivity of Amaranthus hypochondriacus Lectin.

Lectin extracted from Amaranthus hypochondriacus was purified using an affinity column with an agarose-fetuin matrix specific to the lectin of interest. Purification was confirmed by SDS-PAGE, revealing a single protein band with a molecular mass of 34.4 kDa. A hemagglutination assay showed that the lectin had a higher affinity for human type A erythrocytes, and its hemagglutinating activity was inhibited only by fetuin, not by mono-, di-, or trisaccharides. This demonstrated the lectin's selectivity for the N-acetylgalactosamine present on the surface of type A erythrocytes and fetuin. Amaranth lectin exhibited antioxidant activity, which was attributed to the phenolic compounds, amino acids, and specific peptides within the protein structure that are known for their antioxidant properties. Infrared (IR) spectroscopy provided a structural analysis and confirmed lectin glycosylation, a crucial factor in its stability and its ability to bind specific glycans on cell surfaces. Cu2+, Mn2+, and Zn2+ ions were found in the lectin, and these ions were strongly bound to the protein, as dialysis against ethylenediaminetetraacetic acid (EDTA) did not remove them. pH and temperature influenced lectin stability, with higher hemagglutinating activity observed at pH 7, and it remained thermostable at 25 °C.

Immunogenicity and Antibody Persistence of the Inactivated Quadrivalent Influenza Vaccine in Pediatric Patients Post-Chemotherapy or Allogeneic Hematopoietic Stem Cell Transplantation Versus Healthy Controls.

Pediatric patients who have undergone hematopoietic stem cell transplantation (HSCT) or chemotherapy are at increased risk for severe influenza complications, necessitating annual vaccination. This study evaluated the immunogenicity and antibody persistence of the 2021-2022 seasonal quadrivalent influenza vaccine in pediatric patients post-HSCT or chemotherapy, compared to healthy controls. A prospective cohort study included 80 pediatric participants divided into three groups: chemotherapy (n = 33), HSCT (n = 27), and healthy controls (n = 20). All participants were vaccinated with the 2021-2022 GC FLU Quadrivalent vaccine. Hemagglutination inhibition (HI) assays measured seroprotection rates (SPR), geometric mean titers (GMT), and seroconversion rates (SCR) for the four vaccine antigens (A/H1N1, A/H3N2, B/Victoria, B/Yamagata) at one, three, and six months post-vaccination. At one month post-vaccination, all groups met the 70% SPR threshold for A/H1N1 and A/H3N2, but not for B/Victoria. For B/Yamagata, the SPR was low in the chemotherapy and HSCT groups (18.18% and 33.33%, respectively), compared to 80.00% in controls (p < 0.0001 and p = 0.0015). While A/H1N1 and A/H3N2 GMTs were protective in all groups, only controls achieved protective levels for B/Yamagata. Over time, the control group maintained >70% SPR for A/H1N1 up to six months, but the chemotherapy and HSCT groups declined by three and six months, respectively. For A/H3N2, the SPR in controls dropped below 70% at three months, while it remained above 70% in the chemotherapy and HSCT groups until three months. None of the groups achieved protective GMTs for B strains at three or six months. Pediatric patients post-HSCT or chemotherapy demonstrated a comparable immune response to healthy controls for A/H1N1 and A/H3N2, but the rapid decline in A/H1N1 antibody levels suggests the need for ongoing monitoring and adjusted vaccination schedules. The poor response to B antigens, particularly B/Yamagata, underscores the need for improved vaccination strategies in these vulnerable populations.

Immunogenicity and Efficacy of Combined mRNA Vaccine Against Influenza and SARS-CoV-2 in Mice Animal Models.

Background. The combined or multivalent vaccines are actively used in pediatric practice and offer a series of advantages, including a reduced number of injections and visits to the doctor, simplicity of the vaccination schedule and minimization of side effects, easier vaccine monitoring and storage, and lower vaccination costs. The practice of widespread use of the combined vaccines has shown the potential to increase vaccination coverage against single infections. The mRNA platform has been shown to be effective against the COVID-19 pandemic and enables the development of combined vaccines. There are currently no mRNA-based combined vaccines approved for use in humans. Some studies have shown that different mRNA components in a vaccine can interact to increase or decrease the immunogenicity and efficacy of the combined vaccine. Objectives. In the present study, we investigated the possibility of combining the mRNA vaccines, encoding seasonal influenza and SARS-CoV-2 antigens. In our previous works, both vaccine candidates have shown excellent immunogenicity and efficacy profiles in mice. Methods. The mRNA-LNPs were prepared by microfluidic mixing, immunogenicity in mice was assessed by hemagglutination inhibition assay, enzyme-linked immunoassay and virus neutralization assay. Immunological efficacy was assessed in a mouse viral challenge model. Results. In this work, we demonstrated that the individual mRNA components of the combined vaccine did not affect the immunogenicity level of each other. The combined vaccine demonstrated excellent protective efficacy, providing a 100% survival rate when mice were infected with the H1N1 influenza virus and reducing the viral load in the lungs. Four days after the challenge with SARS-CoV-2 EG.5.1.1., no viable virus and low levels of detectable viral RNA were observed in the lungs of vaccinated mice. Conclusions. The combination does not lead to mutual interference between the individual vaccines. We believe that such a combined mRNA-based vaccine could be a good alternative to separated human vaccinations for the prevention of COVID-19 and influenza.

Voltammetric-based immunosensing of Newcastle disease virus on polyethylene glycol-containing self-assembled monolayer modified gold electrode

A voltammetric immunosensor for the detection of Newcastle disease virus (NDV) has been developed by employing polyclonal antibody targeting NDV (anti-NDV) as a bioreceptor. Anti-NDV was immobilized on polyethylene glycol (PEG)-containing self-assembled monolayer (SAM) which was activated with N-(3-dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (EDC) and N-hydroxy succinimide (NHS) coupling on screen-printed gold electrode (SPGE). The introduction of PEG-containing SAM on the SPGE allowed the bioreceptor to covalently bound to the electrode surface whilst still providing a hydrophilic layer on the electrode which is important to greatly reduce non-specific bindings. The bioreceptor functionalized electrode was then allowed to be incubated with NDV-spiked samples. The electrode surface modification with PEG-containing SAM, immobilization of anti-NDV as bioreceptor, up to the detection of NDV were characterized electrochemically through differential pulse voltammetry (DPV) analysis in [Fe(CN)6]3- as the redox probe. Decrement of anodic current peak (Ipa) of [Fe(CN)6]3- was seen as the concentration of NDV increased from 0.156 to 20 HA μL−1 with the limit of detection (LoD) of 1.50 HA μL−1 at 3σ m−1. The detection of NDV in HA μL−1 unit in this study would ease interlaboratory interpretation as it was the same unit used in hemagglutination (HA) assay of conventional NDV diagnosis. The specificity of anti-NDV used as bioreceptor towards NDV was confirmed through western blot analysis, whilst the selectivity of the bioreceptor-functionalized electrode has been tested with allantoic fluid as the negative control in which no apparent changes of anodic peak (Ipa) has been seen. This simple, fast, and less laborious electrochemical detection method could become an alternative to the conventional method for NDV detection.

Efficacy of the H7N9 vaccine as a candidate for the Korean avian influenza antigen bank

The avian influenza A virus (H7N9), first detected in China in 2013, is a zoonotic virus that remains persistent in bird populations despite a decline in human cases owing to control measures. Therefore, this study aimed to develop a vaccine as one preventive strategy in anticipation of the potential entry of H7N9 into Korea. Using the hemagglutinin and neuraminidase consensus sequences of H7N9 from 2018–2019, a recombinant H7N9 vaccine, rgAPQAH7N9, was developed, and its protective efficacy in specific pathogen-free chickens was evaluated. The rgAPQAH7N9 vaccine exhibited proliferation in eggs and demonstrated high immunogenicity, with a hemagglutination inhibition titer of 9.3 log2. Furthermore, the vaccine provided complete protection, as vaccinated chickens did not exhibit clinical signs or viral shedding. Moreover, when the rgAPQAH7N9 vaccine was boosted, the resulting immunity was long-lasting, with hemagglutination inhibition titers > 7 log2 persisting after 6 months. Therefore, the rgAPQAH7N9 vaccine virus may be considered a potential candidate for inclusion in the avian influenza antigen bank to ensure preparedness for emergency vaccination in poultry.

Immune History Modifies Disease Severity to HPAI H5N1 Clade 2.3.4.4b Viral Challenge.

The most recent outbreak of highly pathogenic avian H5 influenza (HPAI) virus in cattle is now widespread across the U.S. with spillover events happening to other mammals, including humans. Several human cases have been reported with clinical signs ranging from conjunctivitis to respiratory illness. However, most of those infected report mild to moderate symptoms, while previously reported HPAI H5Nx infections in humans have had mortality rates upwards of 50%. We recently reported that mice with pre-existing immunity to A/Puerto Rico/08/1934 H1N1 virus were protected from lethal challenge from highly pathogenic clade 2.3.4.4b H5N1 influenza virus. Here, we demonstrate that mice infected with the 2009 pandemic H1N1 virus strain A/California/04/2009 (Cal09) or vaccinated with a live-attenuated influenza vaccine (LAIV) were moderately-to-highly protected against a lethal A/bovine/Ohio/B24OSU-439/2024 H5N1 virus challenge. We also observed that ferrets with mixed pre-existing immunity-either from LAIV vaccination and/or from Cal09 infection-showed protection against a HPAI H5N1 clade 2.3.4.4b virus isolated from a cat. Notably, this protection occurred independently of any detectable hemagglutination inhibition titers (HAIs) against the H5N1 virus. To explore factors that may contribute to protection, we conducted detailed T cell epitope mapping using previously published sequences from H1N1 strains. This analysis revealed a high conservation of amino acid sequences within the internal proteins of our bovine HPAI H5N1 virus strain. These data highlight the necessity to explore additional factors that contribute to protection against HPAI H5N1 viruses, such as memory T cell responses, in addition to HA-inhibition or neutralizing antibodies.

Recombinant avian-derived antiviral proteins cIFITM1, cIFITM3, and cViperin as effective adjuvants in inactivated H9N2 subtype avian influenza vaccines

Vaccine adjuvants, serving as non-specific immune enhancers, play a pivotal role in the immunoprevention and control of animal diseases. This study utilized prokaryotic expression systems to express and purify chicken-derived cIFITM1, cIFITM3, and cViperin, which were then formulated as adjuvants with H9N2 avian influenza virus antigens to create inactivated vaccines. These vaccines were administered to SPF chickens to investigate their immunopotentiating functions. Additionally, the proteins were assessed for their ability to act as standalone immune enhancers. The results demonstrated that cIFITM1, cIFITM3, and cViperin significantly elevated serum hemagglutination inhibition (HI) antibody titers. Notably, when used individually, these proteins markedly enhanced the antiviral capabilities of challenged chickens, leading to alleviated clinical symptoms, reduced tracheal virus replication, diminished virus shedding, and lessened histopathological damage, with cIFITM1 exhibiting the most pronounced effect. Furthermore, the protective efficacy of two H9N2 recombinant virus inactivated vaccines supplemented with cIFITM1 adjuvant was validated, achieving a 100 % vaccine protection efficiency. In conclusion, cIFITM1, cIFITM3, and cViperin as adjuvants for influenza vaccines effectively inhibit virus replication and shedding, highlighting their significant potential in influenza prevention and control.