Isohemagglutinin titration in pooled and apheresis platelets.

BACKGROUND: Platelet inventory constraints necessitate ABO-incompatible platelet transfusion. Many minimize the hemolytic impact by confirming low titre (LT) donor isohemagglutinins. This process is costly. Pathogen-reduced platelets (PRP) in platelet additive solutions (PAS) will dilute plasma and decrease high-titre isohemagglutinins (HT). We determined the proportion of HT platelets and incompatible transfusions for units suspended in plasma to reassess the need for titres following introduction of PRP/PAS. STUDY DESIGN AND METHODS: Our titre method is manual tube (1:50) dilution of platelet supernatant from apheresis or whole blood derived buffy coat pools suspended in plasma, tested with A1/B red cells. Testing included 49,058 pooled and 11,738 apheresis platelets over 4 years. The HT proportion, rate of out-of-group transfusions, and hemolytic reactions were determined. The impact of PAS dilution was estimated. RESULTS: Totally 60,796 platelet units were tested. Group O pooled and group B apheresis platelets had HT in 6.6% and 5.7%, respectively. Group A pooled and apheresis platelets included 2% with HT. Approximately 25% of platelets transfused were ABO-incompatible and no hemolytic reactions were reported. Based on the proportions of PAS-E and plasma for PRP platelets, plasma from each donor comprises 11 mL (6% of total volume) vs 20-257 mL in untreated pools. PAS-E will replace and dilute residual plasma by at least 50%. DISCUSSION: Rare platelet pools may demonstrate HT. PRP platelets with PAS will reduce titres and may abrogate the need for titration. A strategy of group specific transfusion or transfusion of group A PRP platelet transfusions may be a safe alternative.

Expression of recombinant Florida clade 2 hemagglutinin in baculovirus expression system: A step for subunit vaccine development against H3N8 equine influenza virus.

Background: Equine influenza (EI) is a transmissible viral respiratory sickness of the Equidae family. Two viruses, H7N7 and H3N8 caused EI; however, H7N7 has not been detected for decades. H3N8 has circulated and bifurcated into Eurasian and American lineages. The latter subsequently diversified into Kentucky, South America, and Florida sub-lineages. Florida clade 1 (FC1) and Florida clade 2 (FC2) strains are the only circulating EI viruses (EIVs) in the meantime. Immunization is considered the major means for the prevention and control of EI infection. Using disparate technologies and platforms, several vaccines have been developed and commercialized. According to the recommendations of the World Organization for Animal Health (WOAH), all commercial vaccines shall comprise representatives of both FC1 and FC2 strains. Unfortunately, most of the commercially available vaccines were not updated to incorporate a representative of FC2 strains. Aim: The purpose of this research was to develop a new EI vaccine candidate that incorporates the hemagglutinin (HA) antigen from the currently circulating FC2. Methods: In this study, we report the expression of the full-length recombinant HA gene of FC2 in the baculovirus expression system. Results: The HA recombinant protein has been proven to maintain its biological characteristics by hemadsorption (HAD) and hemagglutination tests. Moreover, using a reference-specific serum, the specificity of the HA has been confirmed through the implementation of immunoperoxidase and western immunoblotting assays. Conclusion: In conclusion, we report the expression of specific biologically active recombinant HA of FC2, which would act as a foundation for the generation of an updated EI subunit or virus vector vaccine candidates.

mRNA vaccines encoding influenza virus hemagglutinin (HA) elicits immunity in mice from influenza A virus challenge.

Influenza viruses cause epidemics and can cause pandemics with substantial morbidity with some mortality every year. Seasonal influenza vaccines have incomplete effectiveness and elicit a narrow antibody response that often does not protect against mutations occurring in influenza viruses. Thus, various vaccine approaches have been investigated to improve safety and efficacy. Here, we evaluate an mRNA influenza vaccine encoding hemagglutinin (HA) proteins in a BALB/c mouse model. The results show that mRNA vaccination elicits neutralizing and serum antibodies to each influenza virus strain contained in the current quadrivalent vaccine that is designed to protect against four different influenza viruses including two influenza A viruses (IAV) and two influenza B (IBV), as well as several antigenically distinct influenza virus strains in both hemagglutination inhibition assay (HAI) and virus neutralization assays. The quadrivalent mRNA vaccines had antibody titers comparable to the antibodies elicited by the monovalent vaccines to each tested virus regardless of dosage following an mRNA booster vaccine. Mice vaccinated with mRNA encoding an H1 HA had decreased weight loss and decreased lung viral titers compared to mice not vaccinated with an mRNA encoding an H1 HA. Overall, this study demonstrates the efficacy of mRNA-based seasonal influenza vaccines are their potential to replace both the currently available split-inactivated, and live-attenuated seasonal influenza vaccines.

HA2-FimA DNA Vaccine Treats Experimental Periodontitis.

PURPOSE: To study the therapeutic effect of hemagglutinin-2 and fimbrial (HA2-FimA) vaccine on experimental periodontitis in rats. MATERIALS AND METHODS: The first batch of rats was divided into two groups and immunised with pure water or pVAX1-HA2-FimA at the age of 6, 7, and 9 weeks. After sacrificing the animals, total RNA was extracted from the spleens for RNA high-throughput sequencing (RNA-Seq) analysis. The second batch of rats was divided into four groups (A, B, C, D), and an experimental periodontitis rat model was established by suturing silk thread around the maxillary second molars of rats in groups B, C, and D for 4 weeks. The rats were immunised with pure water, pVAX1-HA2-FimA vaccine, empty pVAX1 vector, and pure water at 10, 11, and 13 weeks of age, respectively. Secretory immunoglobulin A (SIgA) antibodies and cathelicidin antimicrobial peptide (CAMP) levels in saliva were measured by enzyme-linked immunosorbent assay (ELISA). All rats were euthanised at 17 weeks of age, and alveolar bone loss was examined using micro-computed tomography (Micro-CT). RESULTS: Through sequencing analysis, six key genes, including Camp, were identified. Compared with the other three groups, the rats in the periodontitis+pVAX1-HA2-FimA vaccine group showed higher levels of SIgA and CAMP (p < 0.05). Micro-CT results showed significantly less alveolar bone loss in the periodontitis+pVAX1-HA2-FimA vaccine group compared to the periodontitis+pVAX1 group and periodontitis+pure water group (p < 0.05). CONCLUSION: HA2-FimA DNA vaccine can increase the levels of SIgA and CAMP in the saliva of experimental periodontitis model rats and reduce alveolar bone loss.

Expression of the SARS-CoV-2 receptor-binding domain by live attenuated influenza vaccine virus as a strategy for designing a bivalent vaccine against COVID-19 and influenza.

Influenza and SARS-CoV-2 are two major respiratory pathogens that cocirculate in humans and cause serious illness with the potential to exacerbate disease in the event of co-infection. To develop a bivalent vaccine, capable of protecting against both infections, we inserted the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein into hemagglutinin (HA) molecule or into the open reading frame of the truncated nonstructural protein 1 (NS1) of live attenuated influenza vaccine (LAIV) virus and assessed phenotypic characteristics of the rescued LAIV-RBD viruses, as well as their immunogenicity in mouse and Syrian hamster animal models. A panel of 9 recombinant LAIV-RBD viruses was rescued using the A/Leningrad/17 backbone. Notably, only two variants with RBD insertions into the HA molecule could express sufficient quantities of RBD protein in infected MDCK cells. Intranasal immunization of mice induced high levels of anti-influenza antibody responses in all chimeric LAIV-RBD viruses, which was comparable to the LAIV virus vector. The RBD-specific antibody responses were most pronounced in the variant expressing RBD194 fragment as a chimeric HA protein. This candidate was further tested in Syrian hamsters and was shown to be immunogenic and capable of protecting animals against both infections.

mRNA vaccines encoding computationally optimized hemagglutinin elicit protective antibodies against future antigenically drifted H1N1 and H3N2 influenza viruses isolated between 2018-2020.

Background: The implementation of mRNA vaccines against COVID-19 has successfully validated the safety and efficacy of the platform, while at the same time revealing the potential for their applications against other infectious diseases. Traditional seasonal influenza vaccines often induce strain specific antibody responses that offer limited protection against antigenically drifted viruses, leading to reduced vaccine efficacy. Modern advances in viral surveillance and sequencing have led to the development of in-silico methodologies for generating computationally optimized broadly reactive antigens (COBRAs) to improve seasonal influenza vaccines. Methods: In this study, immunologically naïve mice were intramuscularly vaccinated with mRNA encoding H1 and H3 COBRA hemagglutinins (HA) or wild-type (WT) influenza HAs encapsulated in lipid nanoparticles (LNPs). Results: Mice vaccinated with H1 and H3 COBRA HA-encoding mRNA vaccines generated robust neutralizing serum antibody responses against more antigenically distinct contemporary and future drifted H1N1 and H3N2 influenza strains than those vaccinated with WT H1 and H3 HA-encoding mRNA vaccines. The H1 and H3 COBRA HA-encoding mRNA vaccines also prevented influenza illness, including severe disease in the mouse model against H1N1 and H3N2 viruses. Conclusions: This study highlights the potential benefits of combining universal influenza antigen design technology with modern vaccine delivery platforms and exhibits how these vaccines can be advantageous over traditional WT vaccine antigens at eliciting superior protective antibody responses against a broader number of influenza virus isolates.

Immunogenicity of chimeric hemagglutinins delivered by an orf virus vector platform against swine influenza virus.

Orf virus (ORFV) is a large DNA virus that can harbor and efficiently deliver viral antigens in swine. Here we used ORFV as a vector platform to deliver chimeric hemagglutinins (HA) of Influenza A virus of swine (IAV-S). Vaccine development against IAV-S faces limitations posed by strain-specific immunity and the antigenic diversity of the IAV-S strains circulating in the field. A promising alternative aiming at re-directing immune responses on conserved epitopes of the stalk segment of the hemagglutinin (HA2) has recently emerged. Sequential immunization with chimeric HAs comprising the same stalk but distinct exotic head domains can potentially induce cross-reactive immune responses against conserved epitopes of the HA2 while breaking the immunodominance of the head domain (HA1). Here, we generated two recombinant ORFVs expressing chimeric HAs encoding the stalk region of a contemporary H1N1 IAV-S strain and exotic heads derived from either H6 or H8 subtypes, ORFVΔ121cH6/1 and ORFVΔ121cH8/1, respectively. The resulting recombinant viruses were able to express the heterologous protein in vitro. Further, the immunogenicity and cross-protection of these vaccine candidates were assessed in swine after sequential intramuscular immunization with OV-cH6/1 and OV-cH8/1, and subsequent challenge with divergent IAV-S strains. Humoral responses showed that vaccinated piglets presented increasing IgG responses in sera. Additionally, cross-reactive IgG and IgA antibody responses elicited by immunization were detected in sera and bronchoalveolar lavage (BAL), respectively, by ELISA against different viral clades and a diverse range of contemporary H1N1 IAV-S strains, indicating induction of humoral and mucosal immunity in vaccinated animals. Importantly, viral shedding was reduced in nasal swabs from vaccinated piglets after intranasal challenge with either Oh07 (gamma clade) or Ca09 (npdm clade) IAV-S strains. These results demonstrated the efficiency of ORFV-based vectors in delivering chimeric IAV-S HA-based vaccine candidates and underline the potential use of chimeric-HAs for prevention and control of influenza in swine.

C1q enables influenza hemagglutinin stem binding antibodies to block viral attachment and broadens the antibody escape repertoire.

Antigenic drift, the gradual accumulation of amino acid substitutions in the influenza virus hemagglutinin (HA) receptor protein, enables viral immune evasion. Antibodies (Abs) specific for the drift-resistant HA stem region are a promising universal influenza vaccine target. Although anti-stem Abs are not believed to block viral attachment, here we show that complement component 1q (C1q), a 460-kilodalton protein with six Ab Fc-binding domains, confers attachment inhibition to anti-stem Abs and enhances their fusion and neuraminidase inhibition. As a result, virus neutralization activity in vitro is boosted up to 30-fold, and in vivo protection from influenza PR8 infection in mice is enhanced. These effects reflect increased steric hindrance and not increased Ab avidity. C1q greatly expands the anti-stem Ab viral escape repertoire to include residues throughout the HA, some of which cause antigenic alterations in the globular region or modulate HA receptor avidity. We also show that C1q enhances the neutralization activity of non-receptor binding domain anti-SARS-CoV-2 spike Abs, an effect dependent on spike density on the virion surface. These findings demonstrate that C1q can greatly expand Ab function and thereby contribute to viral evolution and immune escape.

Recent Advances, Approaches and Challenges in the Development of Universal Influenza Vaccines.

Every year, influenza virus infections cause significant morbidity and mortality worldwide. They pose a substantial burden of disease, in terms of not only health but also the economy. Owing to the ability of influenza viruses to continuously evolve, annual seasonal influenza vaccines are necessary as a prophylaxis. However, current influenza vaccines against seasonal strains have limited effectiveness and require yearly reformulation due to the virus undergoing antigenic drift or shift. Vaccine mismatches are common, conferring suboptimal protection against seasonal outbreaks, and the threat of the next pandemic continues to loom. Therefore, there is a great need to develop a universal influenza vaccine (UIV) capable of providing broad and durable protection against all influenza virus strains. In the quest to develop a UIV that would obviate the need for annual vaccination and formulation, a multitude of strategies is currently underway. Promising approaches include targeting the highly conserved epitopes of haemagglutinin (HA), neuraminidase (NA), M2 extracellular domain (M2e) and internal proteins of the influenza virus. The identification and characterization of broadly neutralizing antibodies (bnAbs) targeting conserved regions of the viral HA protein, in particular, have provided important insight into novel vaccine designs and platforms. This review discusses universal vaccine approaches presently under development, with an emphasis on those targeting the highly conserved stalk of the HA protein, recent technological advancements used and the future prospects of a UIV in terms of its advantages, developmental obstacles and potential shortcomings.

The seroepidemiology of immunoglobulin G antibodies against pertussis toxin and filamentous hemagglutinin in the east of China during the COVID-19 pandemic.

This study employed sero-epidemiological methods to estimate the incidence of pertussis within a healthy population located in eastern China. The aim was to gain deeper insights into the epidemiological characteristics and burden of pertussis within the country. Blood samples were collected from healthy individuals in Jiangsu Province between June 2019 and December 2022. The levels of IgG antibodies against pertussis toxin (anti-PT) and filamentous hemagglutinin (anti-FHA) in the serum were quantitatively measured using enzyme-linked immunosorbent assay (ELISA). Additionally, pertussis case data reported in Jiangsu Province were collected from the China Information System for Disease Control and Prevention and compared with the results of this study. In 2022, the reported incidence of pertussis stood at 1.0 per 100,000 individuals, marking the highest rate observed in the past two decades. Among 1,909 patients examined, the geometric mean concentration (GMC) of anti-PT IgG antibody was 20.2 (18.5-21.9) IU/ml, while that of anti-FHA IgG antibody was 27.0 (25.4-28.7) IU/ml. The IgG-PT and IgG-FHA seropositivity rate (>20.0 IU/ml) was highest in the 1 ~ 2 y old group and decreased rapidly to the lowest in the 3 ~ 4 y old group and then increased gradually with age. The estimated rate of pertussis infection based on seroprevalence was approximately 25,625-fold higher than the reported notification rate in the ≥15 year age group. Our findings highlight decreased immunity post-vaccination, stressing the importance of additional booster shots for adolescents and adults to maintain immunity and reduce severe illness. Additionally, they offer vital guidance for policymakers to enhance immunization strategies.

A novel data augmentation approach for influenza A subtype prediction based on HA proteins.

Influenza, a pervasive viral respiratory illness, remains a significant global health concern. The influenza A virus, capable of causing pandemics, necessitates timely identification of specific subtypes for effective prevention and control, as highlighted by the World Health Organization. The genetic diversity of influenza A virus, especially in the hemagglutinin protein, presents challenges for accurate subtype prediction. This study introduces PreIS as a novel pipeline utilizing advanced protein language models and supervised data augmentation to discern subtle differences in hemagglutinin protein sequences. PreIS demonstrates two key contributions: leveraging pre-trained protein language models for influenza subtype classification and utilizing supervised data augmentation to generate additional training data without extensive annotations. The effectiveness of the pipeline has been rigorously assessed through extensive experiments, demonstrating a superior performance with an impressive accuracy of 94.54% compared to the current state-of-the-art model, the MC-NN model, which achieves an accuracy of 89.6%. PreIS also exhibits proficiency in handling unknown subtypes, emphasizing the importance of early detection. Pioneering the classification of HxNy subtypes solely based on the hemagglutinin protein chain, this research sets a benchmark for future studies. These findings promise more precise and timely influenza subtype prediction, enhancing public health preparedness against influenza outbreaks and pandemics. The data and code underlying this article are available in https://github.com/CBRC-lab/PreIS.

Adjuvanted nanoliposomes displaying six hemagglutinins and neuraminidases as an influenza virus vaccine.

Inclusion of defined quantities of the two major surface proteins of influenza virus, hemagglutinin (HA) and neuraminidase (NA), could benefit seasonal influenza vaccines. Recombinant HA and NA multimeric proteins derived from three influenza serotypes, H1N1, H3N2, and type B, are surface displayed on nanoliposomes co-loaded with immunostimulatory adjuvants, generating "hexaplex" particles that are used to immunize mice. Protective immune responses to hexaplex liposomes involve functional antibody elicitation against each included antigen, comparable to vaccination with monovalent antigen particles. When compared to contemporary recombinant or adjuvanted influenza virus vaccines, hexaplex liposomes perform favorably in many areas, including antibody production, T cell activation, protection from lethal virus challenge, and protection following passive sera transfer. Based on these results, hexaplex liposomes warrant further investigation as an adjuvanted recombinant influenza vaccine formulation.

Multiplexed Capillary-Flow Driven Immunoassay for Respiratory Illnesses.

Multiplexed analysis in medical diagnostics is widely accepted as a more thorough and complete method compared to single-analyte detection. While analytical methods like polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA) exist for multiplexed detection of biomarkers, they remain time-consuming and expensive. Lateral flow assays (LFAs) are an attractive option for point-of-care testing, and examples of multiplexed LFAs exist. However, these devices are limited by spatial resolution of test lines, large sample volume requirements, cross-reactivity, and poor sensitivity. Recent work has developed capillary-flow microfluidic ELISA platforms as a more sensitive alternative to LFAs; however, multiplexed detection on these types of devices has yet to be demonstrated. In the aftermath of the initial SARS-CoV-2 pandemic, the need for rapid, sensitive point-of-care devices has become ever clearer. Moving forward, devices that can distinguish between diseases with similar presenting symptoms would be the ideal home diagnostic. Here, the first example of a multiplexed capillary-flow immunoassay device for the simultaneous detection of multiple biomarkers is reported. From a single sample addition step, the reagents and washing steps required for two simultaneous ELISAs are delivered to spatially separated test strips. Visual results can be obtained in <15 min, and images captured with a smartphone can be analyzed for quantitative data. This device was used to distinguish between and quantify H1N1 hemagglutinin (HA) and SARS-CoV-2 nucleocapsid protein (N-protein). Using this device, analytical detection limits of 840 and 133 pg/mL were obtained for hemagglutinin and nucleocapsid protein, respectively. The presence of one target in the device did not increase the signal on the other test line, indicating no cross-reactivity between the assays. Additionally, simultaneous detection of both N-protein and HA was performed as well as simultaneous detection of N-protein and human C-reactive protein (CRP). Elevated levels of CRP in a patient infected with SARS-CoV-2 have been shown to correlate with more severe outcomes and a greater risk of death as well. To further expand on the simultaneous detection of two biomarkers, CRP and N-protein were detected simultaneously, and the presence of SARS-CoV-2 N-protein did not interfere with the detection of CRP when both targets were present in the sample.

Nucleoprotein reassortment enhanced transmissibility of H3 1990.4.a clade influenza A virus in swine.

The increased detection of H3 C-IVA (1990.4.a) clade influenza A viruses (IAVs) in US swine in 2019 was associated with a reassortment event to acquire an H1N1pdm09 lineage nucleoprotein (pdmNP) gene, replacing a TRIG lineage NP (trigNP). We hypothesized that acquiring the pdmNP conferred a selective advantage over prior circulating H3 viruses with a trigNP. To investigate the role of NP reassortment in transmission, we identified two contemporary 1990.4.a representative strains (NC/19 and MN/18) with different evolutionary origins of the NP gene. A reverse genetics system was used to generate wild-type (wt) strains and swap the pdm and TRIG lineage NP genes, generating four viruses: wtNC/19-pdmNP, NC/19-trigNP, wtMN/18-trigNP, and MN/18-pdmNP. The pathogenicity and transmission of the four viruses were compared in pigs. All four viruses infected 10 primary pigs and transmitted to five indirect contact pigs per group. Pigs infected via contact with MN/18-pdmNP shed virus 2 days earlier than pigs infected with wtMN/18-trigNP. The inverse did not occur for wtNC/19-pdmNP and NC/19-trigNP. This suggests that pdmNP reassortment resulted in a combination of genes that improved transmission efficiency when paired with the 1990.4.a hemagglutinin (HA). This is likely a multigenic trait, as replacing the trigNP gene did not diminish the transmission of a wild-type IAV in swine. This study demonstrates how reassortment and evolutionary change of internal genes can result in more transmissible viruses that influence HA clade detection frequency. Thus, rapidly identifying novel reassortants paired with dominant hemagglutinin/neuraminidase may improve the prediction of strains to include in vaccines.IMPORTANCEInfluenza A viruses (IAVs) are composed of eight non-continuous gene segments that can reassort during coinfection of a host, creating new combinations. Some gene combinations may convey a selective advantage and be paired together preferentially. A reassortment event was detected in swine in the United States that involved the exchange of two lineages of nucleoprotein (NP) genes (trigNP to pdmNP) that became a predominant genotype detected in surveillance. Using a transmission study, we demonstrated that exchanging the trigNP for a pdmNP caused the virus to shed from the nose at higher levels and transmit to other pigs more rapidly. Replacing a pdmNP with a trigNP did not hinder transmission, suggesting that transmission efficiency depends on interactions between multiple genes. This demonstrates how reassortment alters IAV transmission and that reassortment events can provide an explanation for why genetically related viruses with different internal gene combinations experience rapid fluxes in detection frequency.

Crossprotection induced by virus-like particles containing influenza dual-hemagglutinin and M2 ectodomain.

Aims: To develop an effective universal vaccine against antigenically different influenza viruses. Materials & methods: We generated influenza virus-like particles (VLPs) expressing the H1 and H3 antigens with or without M2e5x. VLP-induced immune responses and crossprotection against H1N1, H3N2 or H5N1 viruses were assessed to evaluate their protective efficacy. Results: H1H3M2e5x immunization elicited higher crossreactive IgG antibodies than H1H3 VLPs. Upon challenge, both VLPs enhanced lung IgG, IgA and germinal center B-cell responses compared with control. While these VLPs conferred protection, H1H3M2e5x showed greater lung viral load reduction than H1H3 VLPs with minimal body weight loss. Conclusion: Utilizing VLPs containing dual-hemagglutinin, along with M2e5x, can be a vaccination strategy for inducing crossprotection against influenza A viruses.

Mosaic quadrivalent influenza vaccine single nanoparticle characterization.

Recent work by our laboratory and others indicates that co-display of multiple antigens on protein-based nanoparticles may be key to induce cross-reactive antibodies that provide broad protection against disease. To reach the ultimate goal of a universal vaccine for seasonal influenza, a mosaic influenza nanoparticle vaccine (FluMos-v1) was developed for clinical trial (NCT04896086). FluMos-v1 is unique in that it is designed to co-display four recently circulating haemagglutinin (HA) strains; however, current vaccine analysis techniques are limited to nanoparticle population analysis, thus, are unable to determine the valency of an individual nanoparticle. For the first time, we demonstrate by total internal reflection fluorescence microscopy and supportive physical-chemical methods that the co-display of four antigens is indeed achieved in single nanoparticles. Additionally, we have determined percentages of multivalent (mosaic) nanoparticles with four, three, or two HA proteins. The integrated imaging and physicochemical methods we have developed for single nanoparticle multivalency will serve to further understand immunogenicity data from our current FluMos-v1 clinical trial.

Amino acid 138 in the HA of a H3N2 subtype influenza A virus increases affinity for the lower respiratory tract and alveolar macrophages in pigs.

Influenza A virus (FLUAV) infects a wide range of hosts and human-to-swine spillover events are frequently reported. However, only a few of these human viruses have become established in pigs and the host barriers and molecular mechanisms driving adaptation to the swine host remain poorly understood. We previously found that infection of pigs with a 2:6 reassortant virus (hVIC/11) containing the hemagglutinin (HA) and neuraminidase (NA) gene segments from the human strain A/Victoria/361/2011 (H3N2) and internal gene segments of an endemic swine strain (sOH/04) resulted in a fixed amino acid substitution in the HA (A138S, mature H3 HA numbering). In silico analysis revealed that S138 became predominant among swine H3N2 virus sequences deposited in public databases, while 138A predominates in human isolates. To understand the role of the HA A138S substitution in the adaptation of a human-origin FLUAV HA to swine, we infected pigs with the hVIC/11A138S mutant and analyzed pathogenesis and transmission compared to hVIC/11 and sOH/04. Our results showed that the hVIC/11A138S virus had an intermediary pathogenesis between hVIC/11 and sOH/04. The hVIC/11A138S infected the upper respiratory tract, right caudal, and both cranial lobes while hVIC/11 was only detected in nose and trachea samples. Viruses induced a distinct expression pattern of various pro-inflammatory cytokines such as IL-8, TNF-α, and IFN-ß. Flow cytometric analysis of lung samples revealed a significant reduction of porcine alveolar macrophages (PAMs) in hVIC/11A138S-infected pigs compared to hVIC/11 while a MHCIIlowCD163neg population was increased. The hVIC/11A138S showed a higher affinity for PAMs than hVIC/11, noted as an increase of infected PAMs in bronchoalveolar lavage fluid (BALF), and showed no differences in the percentage of HA-positive PAMs compared to sOH/04. This increased infection of PAMs led to an increase of granulocyte-monocyte colony-stimulating factor (GM-CSF) stimulation but a reduced expression of peroxisome proliferator-activated receptor gamma (PPARγ) in the sOH/04-infected group. Analysis using the PAM cell line 3D4/21 revealed that the A138S substitution improved replication and apoptosis induction in this cell type compared to hVIC/11 but at lower levels than sOH/04. Overall, our study indicates that adaptation of human viruses to the swine host involves an increased affinity for the lower respiratory tract and alveolar macrophages.

Bivalent Hemagglutinin Cleavage-Site Peptide Vaccines Protect Chickens from Lethal Infections with Highly Pathogenic H5N1 and H5N6 Avian Influenza Viruses.

BACKGROUND: Outbreaks of highly pathogenic avian influenza viruses cause huge economic losses to the poultry industry worldwide. Vaccines that can protect chickens from infections caused by various variants of highly pathogenic H5Nx avian influenza viruses are needed owing to the continuous emergence of new variants. We previously showed that vaccines containing the H5 cleavage-site peptide from clade 2.3.4.4. H5N6 avian influenza virus protects chickens from infection with homologous clade 2.3.4.4. H5N6 avian influenza virus, but not from infection with the heterologous clade 1 H5N1 avian influenza virus. Therefore, we developed bivalent peptide vaccines containing H5 cleavage sites of viruses from both clades to protect chickens from both H5N1 and H5N6 avian influenza viruses. METHODS: Chickens were vaccinated with two doses of a combined peptide vaccine containing cleavage-site peptides from clade 1 and clade 2.3.4.4. highly pathogenic H5N1 and H5N6 avian influenza viruses and then challenged with both viruses. The infected chickens were monitored for survival and their tracheae and cloacae were sampled to check for viral shedding based on the median tissue culture infectious dose of 50 (log10TCID50/mL) in Madin-Darby canine kidney cells. RESULTS: Antibody production was induced at similar levels in the sera of chickens immunized with two doses of the combined peptide vaccines containing cleavage-site peptides from highly pathogenic H5N1 and H5N6 avian influenza viruses. The immunized chickens were protected from infection with both H5N1 and H5N6 avian influenza viruses without viral shedding in the tracheae and cloacae. CONCLUSIONS: Dual-peptide vaccines containing cleavage-site peptides of both clades can protect chickens from highly pathogenic avian influenza virus infections.

Antigenic commonality and divergence of hemagglutinin-esterase-fusion protein among influenza D virus lineages revealed using epitope mapping.

Influenza D virus (IDV) is one of the causative agents of bovine respiratory disease complex, which is the most common and economically burdensome disease affecting the cattle industry, and the need for an IDV vaccine has been proposed to enhance disease control. IDVs are classified into five genetic lineages based on the coding sequences of the hemagglutinin-esterase-fusion (HEF) protein, an envelope glycoprotein, which is the main target of protective antibodies against IDV infection. Herein, we prepared a panel of monoclonal antibodies (mAbs) against the HEF protein of viruses of various lineages to investigate the antigenic characteristics of IDVs and found that the mAbs could be largely separated into three groups. The first, second, and third groups demonstrated lineage-specific reactivity, cross-reactivity to viruses of multiple but not all lineages, and cross-reactivity to viruses of all lineages, respectively. Analyzing the escape mutant viruses from virus-neutralizing mAbs revealed that the receptor-binding region of the HEF molecule harbors virus-neutralizing epitopes that are conserved across multiple lineage viruses. In contrast, the apex region of the molecule possessed epitopes unique to each lineage virus. Furthermore, reverse genetics-generated recombinant viruses with point mutations revealed that amino acids within positions 210-214 of the HEF protein determined the antigenic specificity of each lineage virus. Taken together, this study reveals considerable antigenic variation among IDV lineages, although they are presumed to form a single serotype in terms of HEF antigenicity. Characterization of the antigenic epitope structure of HEF may contribute to selecting and creating effective vaccine viruses against IDV.IMPORTANCEInfluenza D viruses (IDVs) are suggested to create cross-reactive single serotypes in hemagglutinin-esterase-fusion (HEF) antigenicity, as indicated by serological analyses among distinct HEF lineage viruses. This is supported by the high identities of HEF gene sequences among strains, unlike the hemagglutinin (HA) genes of the influenza A virus that exhibit HA subtypes. Herein, we analyzed HEF antigenicity using a monoclonal antibody panel prepared from several virus lineages and found the existence of lineage-conserved and lineage-specific epitopes in HEF molecules. These findings confirm the HEF commonality and divergence among IDVs and provide useful information for constructing a vaccine containing a recombinant IDV virus with an engineered HEF gene, thereby leading to broad immunogenicity.

A neonatal piglet model reveals interactions between nasal microbiota and influenza A virus pathogenesis.

While vaccination and therapeutics for prevention/treatment of influenza are available, new strategies are needed to combat influenza disease in susceptible populations, particularly young children and newborns. Host associated microbiota play an important role in modulating the virulence of numerous pathogens, including the influenza A virus. In this study, we examined microbiome-influenza interactions in a neonatal piglet model system. The nasal microbiome of newborn piglets was longitudinally sampled before and after intranasal infection with recombinant viruses expressing hemagglutinins (HAs) derived from distinct zoonotic H1 subtypes. We found that viruses expressing different parental HAs manifested unique patterns of pathogenicity, and varied impacts on microbial community diversity. Despite these virus specific differences, a consistent microbial signature of viral infection was detected. Our results indicate that influenza A virus infection associates with the restructuring of nasal microbiome and such shifts in microbial diversity may contribute to outcomes of viral infection in neonatal piglets.