H1N1 Strain Research Articles

Severe forms of influenza infections admitted in intensive care units: Analysis of mortality factors.

The severe forms of influenza infection requiring intensive care unit (ICU) admission remain a medical challenge due to its high mortality. New H1N1 strains were hypothesized to increase mortality. The studies below represent a large series focusing on ICU-admitted influenza patients over the last decade with an emphasis on factors related to death. A retrospective study of patients admitted in ICU for influenza infection over the 2010-2019 period in Réunion Island (a French overseas territory) was conducted. Demographic data, underlying conditions, and therapeutic management were recorded. A univariate analysis was performed to assess factors related to ICU mortality. Three hundred and fifty adult patients were analyzed. Overall mortality was 25.1%. Factors related to higher mortality were found to be patient age >65, cancer history, need for intubation, early intubation within 48 h after admission, invasive mechanical ventilation (MV), acute respiratory distress syndrome (ARDS), vaso-support drugs, extracorporal oxygenation by membrane (ECMO), dialysis, bacterial coinfection, leucopenia, anemia, and thrombopenia. History of asthma and oseltamivir therapy were correlated with a lower mortality. H1N1 did not impact mortality. Patient's underlying conditions influence hospital admission and secondary ICU admission but were not found to impact ICU mortality except in patients age >65, history of cancer, and bacterial coinfections. Pulmonary involvement was often present, required MV, and often evolved toward ARDS. ICU mortality was strongly related to ARDS severity. We recommend rapid ICU admission of patients with influenza-related pneumonia, management of bacterial coinfection, and early administration of oseltamivir.

A fully human monoclonal antibody possesses antibody-dependent cellular cytotoxicity (ADCC) activity against the H1 subtype of influenza A virus by targeting a conserved epitope at the HA1 protomer interface.

The DiversitabTM system produces target specific high titer fully human polyclonal IgG immunoglobulins from transchromosomic (Tc) bovines shown to be safe and effective against multiple virulent pathogens in animal studies and Phase 1, 2 and 3 human clinical trials. We describe the functional properties of a human monoclonal antibody (mAb), 38C2, identified from this platform, which recognizes recombinant H1 hemagglutinins (HAs) and induces appreciable antibody-dependent cellular cytotoxicity (ADCC) activity in vitro. Interestingly, 38C2 monoclonal antibody demonstrated no detectable neutralizing activity against H1N1 virus in both hemagglutination inhibition and virus neutralization assays. Nevertheless, this human monoclonal antibody induced appreciable ADCC against cells infected with multiple H1N1 strains. The HA-binding activity of 38C2 was also demonstrated in flow cytometry using Madin-Darby canine kidney cells infected with multiple influenza A H1N1 viruses. Through further investigation with the enzyme-linked immunosorbent assay involving the HA peptide array and 3-dimensional structural modeling, we demonstrated that 38C2 appears to target a conserved epitope located at the HA1 protomer interface of H1N1 influenza viruses. A novel mode of HA-binding and in vitro ADCCactivity pave the way for further evaluation of 38C2 as a potential therapeutic agent to treat influenza virus infections in humans.

Humoral immune response following the inactivated quadrivalent influenza vaccination among HIV-infected and HIV-uninfected adults

BackgroundA limited amount of information is available about the immunogenicity of the quadrivalent inactivated influenza vaccine among human immunodeficiency virus (HIV)-infected individuals, especially in low and middle-income countries (LMICs). MethodsHIV-infected adults and HIV-uninfected adults received a dose of quadrivalent inactivated influenza vaccine including strains of H1N1, H3N2, BV and BY. Enzyme-linked immunosorbent assay (ELISA) and hemagglutination-inhibition assay (HAI) were used to determine IgA, IgG antibody concentration and geometric mean titers (GMT) at day 0 and day 28, respectively. Associated factors contributing to seroconversion or GMT changes were analyzed using simple logistic regression model. ResultsA total of 131 HIV-infected and 55 HIV-uninfected subjects were included in the study. In both HIV-infected and uninfected arms, IgG and IgA against influenza A and B all increased significantly at day 28 after receiving QIV (P < 0.001). GMTs of post-vaccination at day 28 showed that HIV-infected persons with CD4 + T cell counts ≤ 350 cells/mm3 were statistically less immunogenic to all strains of QIV than HIV-uninfected ones (P < 0.05). HIV-infected participants with CD4 + T cell counts ≤ 350 cells/mm3 were less likely to achieve seroconversion to QIV (H1N1, BY and BV) than HIV-uninfected individuals at day 28 after vaccination (P < 0.05). Compared with HIV-infected patients with baseline CD4 + T cell counts ≤ 350 cells/mm3, individuals with baseline CD4 + T cell counts > 350 cell/mm3 seemed more likely to generate antibody responses to H1N1 (OR:2.65, 95 %CI: 1.07–6.56) and BY (OR: 3.43, 95 %CI: 1.37–8.63), and showed a higher probability of seroconversion to BY (OR: 3.59, 95 %CI: 1.03–12.48). Compared with nadir CD4 + T cell count ≤ 350 cell/mm3, individuals with nadir CD4 + T cell count > 350 cell/mm3 showed a higher probability of seroconversion to H1N1(OR: 3.15, 95 %CI: 1.14–8.73). ConclusionInfluenza vaccination of HIV-infected adults might be effective despite variable antibody responses. HIV-positive populations with CD4 + T cell counts ≤ 350 are less likely to achieve seroconversion. Further vaccination strategies could be developed for those with low CD4 T cell counts.

Department of Microbiology, Collage of Veterinary Medicine, University of Diyal, Iraq

This study was done at the college of Veterinary Medicine, University of Diyala, from September 2021 to April 2022 to assess the seroprevalence and molecular detection of avian influenza virus (AIV) H5N8 in layer chickens. All samples came from 8 commercial flocks of layer chickens. Clinical sings included, severe respiratory infection with enlargement of the head, neck, and prominent edema of the face, followed by excessive lacrimation, sinusitis, severe rales, and cyanosis in all parts of unfeather skin predominantly the wattles comb, and the legs. In this regard, ELISA was used as a serological test and RT-PCR as a molecular detection technique. Accordingly, (364) serum samples from 8 probable AIV (H5N8) infected layer farms were collected and subjected to ELISA test using commercial kit that specific for IgG antibodies to AIV H5N8. The results showed a significant increase of IgG antibodies in such serum samples at days 70 and 200 of age. According to the instruction manual of ELISA kit manufacturer and to mean titers of layers of present study, all farms are infected with AIVH5N8 strain. However, at the age of 200 days, 48 postmortem tissue samples including trachea, lung and liver of affected birds were collected from clinically and sub-clinically infected flocks from all farms. These samples were processed, RNA extracted and submitted to RT-PCR using specific primers for H5N8 strain. The results showed that 32 out of the 48 tissue samples (66.6%) tested positive for H5N8.The resulted Amplicon (320bp) was commercially sequenced and analyzed. The sequencing of the local AIV H5N8 revealed a 99% sequence identity with the reference sequences. The detected strain was registered in GenBank data (NCBI) under acc.number ON247929.1. Phylogenetic tree for locally detected virus in comparison to data from of NCBI was created and showed that the investigated S1(ON247929.1, AIV (A/laying hens/Iraq/(H5N8) segment 4 hemagglutinin (HA) gene, partial cds, local sample is closely related to reference isolates from the NCBI acc. no. of MW961428.1, MW961444.1, MW961476.1, MW961436.,1 and MW961484.1. These strains of the Influenza H5 virus have been deposited from Nigeria.

A Novel Triple Reassortment H3N8 Avian Influenza Virus: Characteristics, Pathogenicity, and Transmissibility

An increasing number of new subtypes of avian influenza viruses (AIVs) are reported to be infecting humans, including H3N2, H5N1, H7N9, H10N8, and the recently emerged H3N8 virus in China in 2022. However, the genetic and biological properties of the currently prevalent H3N8 AIVs are not yet fully understood. This study reports the isolation of a novel triple reassortment H3N8 virus (GD-H3N8) from chicken flocks in Guangdong province, China, in 2022. The GD-H3N8 virus contains the Eurasian avian duck-origin H3 gene, the North American avian N8 gene, and dynamic internal genes of the H9N2 virus, and shows high homology with human H3N8 strains. The GD-H3N8 isolate has multiple mammalian adaptive mutations associated with receptor binding and virulence. Growth kinetics assays demonstrate that the GD-H3N8 isolate is capable of efficient replication in avian, mammalian, and human cells in vitro. In vivo, the GD-H3N8 isolate can replicate efficiently in mice without preadaptation, in addition to establishing systemic infection and transmission by direct contact in chickens. These findings underscore the need for continued surveillance of H3N8 viruses to identify circulating strains that may potentially threaten human health.

In Silico Design of an Oseltamivir Derivative with Increased Affinity against Wild-Type and Mutant Variants of Neuraminidase and Hemagglutinin of Influenza A H1N1 Virus.

Antiviral resistance has turned into a world concern nowadays. Influenza A H1N1 emerged as a problem at the world level due to the neuraminidase (NA) mutations. The NA mutants conferred resistance to oseltamivir and zanamivir. Several efforts were conducted to develop better anti-influenza A H1N1 drugs. Our research group combined in silico methods to create a compound derived from oseltamivir to be tested in vitro against influenza A H1N1. Here we show the results of a new compound derived from oseltamivir but with specific chemical modifications, with significant affinity either on NA (in silico and in vitro assays) or HA (in silico) from influenza A H1N1 strain. We include docking and molecular dynamics (MD) simulations of the oseltamivir derivative at the binding site onto NA and HA of influenza A H1N1. Additionally, the biological experimental results show that oseltamivir derivative decreases the lytic-plaque formation on viral susceptibility assays, and it does not show cytotoxicity. Finally, oseltamivir derivative assayed on viral NA showed a concentration-dependent inhibition behavior at nM, depicting a high affinity of the compound for the enzyme, corroborated with the MD simulations results, placing our designed oseltamivir derivative as a potential antiviral against influenza A H1N1.

The Interferon-Induced Protein with Tetratricopeptide Repeats Repress Influenza Virus Infection by Inhibiting Viral RNA Synthesis.

Influenza A virus (IAV) is an eight-segment negative-sense RNA virus and is subjected to gene recombination between strains to form novel strains, which may lead to influenza pandemics. Seasonal influenza occurs annually and causes great losses in public healthcare. In this study, we examined the role of interferon-induced protein with tetratricopeptide repeats 1 and 2 (IFIT1 and IFIT2) in influenza virus infection. Knockdown of IFIT1 or IFIT2 using a lentiviral shRNA increased viral nucleoprotein (NP) and nonstructural protein 1 (NS1) protein levels, as well as progeny virus production in A/Puerto Rico/8/34 H1N1 (PR/8)-infected lung epithelial A549 cells. Overexpression of IFIT1 or IFIT2 reduced viral NP and NS1 RNA and protein levels in PR/8-infected HEK293 cells. Overexpression of IFIT1 or IFIT2 also inhibited influenza virus infection of various H1N1 strains, including PR/8, A/WSN/1933, A/California/07/2009 and A/Oklahoma/3052/2009, as determined by a viral reporter luciferase assay. Furthermore, knockdown of IFIT1 or IFIT2 increased while overexpression of IFIT1 or IFIT2 decreased viral RNA, complementary RNA, and mRNA levels of NP and NS1, as well as viral polymerase activities. Taken together, our results support that both IFIT1 and -2 have anti-influenza virus activities by inhibiting viral RNA synthesis.

Evolutionary dynamics of influenza A/H1N1 virus circulating in India from 2011 to 2021

Influenza A viruses (IAV) are fast-evolving pathogens with a very high mutation rate (2.0×10-6 to 2.0×10-4) compared to the influenza B (IBV) and influenza C (ICV) viruses. Generally, the tropical regions are considered as the reservoir for the IAV's genetic and antigenic evolutionary modification to be reintroduced into the temperate region. Therefore, in connection to the above facts, the present study emphasized on the evolutionary dynamic of the pandemic-2009 H1N1 (pdmH1N1) influenza virus in India. A total of Ninety-two whole genome sequences of pdmH1N1 viruses circulating in India during the 2009 post-pandemic era were analysed. The temporal signal of the study, indicating strict molecular clock evolutionary process and the overall substitution rate is 2.21×10-3/site/year. We are using the nonparametric Bayesian Skygrid coalescent model to estimates the effective past population dynamic or size over time. The study exhibits a strong relation between the genetic distances and collection dates of the Indian pdmH1N1 strain. The skygrid plot represents the highest exponential growth of IAV in rainy and winter seasons. All the genes of Indian pdmH1N1 were under purifying selective pressure. The Bayesian time-imprinted phylogenetic tree represents the following clade distributions in the country within the last 10years; I) clade 6, 6C, and 7 were co-circulating between the 2011 to 2012 flu season; II) the clade 6B was introduced into circulation in the late seasons of 2012; III) lastly, the clade 6B remain existing in the circulation and segregated into subclade 6B.1 with five different subgroup (6B.1A, 6B.1A.1, 6B.1A.5a, 6B.1A.5a.2, 6B.1A.7). The recent circulating strain of Indian H1N1 strain represent the insertion of basic-amino acid arginine (R) in the cleavage site (325/K-R) of HA protein and amino acid mutation (314/I-M) on the lateral head surface domain of NA protein. Moreover, the study indicates the sporadic presence of the oseltamivir-resistant (275/H-Y) H1N1 variant in circulation. The present study suggests the purifying selective pressure and stochastic ecological factors for the existence and adaptation of a certain clade 6B in the host populations and additional information on the emergence of mutated strains in the circulation.

A pan-influenza antibody inhibiting neuraminidase via receptor mimicry

Rapidly evolving influenza A viruses (IAVs) and influenza B viruses (IBVs) are major causes of recurrent lower respiratory tract infections. Current influenza vaccines elicit antibodies predominantly to the highly variable head region of haemagglutinin and their effectiveness is limited by viral drift1 and suboptimal immune responses2. Here we describe a neuraminidase-targeting monoclonal antibody, FNI9, that potently inhibits the enzymatic activity of all group 1 and group 2 IAVs, as well as Victoria/2/87-like, Yamagata/16/88-like and ancestral IBVs. FNI9 broadly neutralizes seasonal IAVs and IBVs, including the immune-evading H3N2 strains bearing an N-glycan at position 245, and shows synergistic activity when combined with anti-haemagglutinin stem-directed antibodies. Structural analysis reveals that D107 in the FNI9 heavy chain complementarity-determinant region 3 mimics the interaction of the sialic acid carboxyl group with the three highly conserved arginine residues (R118, R292 and R371) of the neuraminidase catalytic site. FNI9 demonstrates potent prophylactic activity against lethal IAV and IBV infections in mice. The unprecedented breadth and potency of the FNI9 monoclonal antibody supports its development for the prevention of influenza illness by seasonal and pandemic viruses.

Development of PREDAC-H1pdm to model the antigenic evolution of influenza A/(H1N1) pdm09 viruses

The Influenza A (H1N1) pdm09 virus caused a global pandemic in 2009 and has circulated seasonally ever since. As the continual genetic evolution of hemagglutinin in this virus leads to antigenic drift, rapid identification of antigenic variants and characterization of the antigenic evolution are needed. In this study, we developed PREDAC-H1pdm, a model to predict antigenic relationships between H1N1pdm viruses and identify antigenic clusters for post-2009 pandemic H1N1 strains. Our model performed well in predicting antigenic variants, which was helpful in influenza surveillance. By mapping the antigenic clusters for H1N1pdm, we found that substitutions on the Sa epitope were common for H1N1pdm, whereas for the former seasonal H1N1, substitutions on the Sb epitope were more common in antigenic evolution. Additionally, the localized epidemic pattern of H1N1pdm was more obvious than that of the former seasonal H1N1, which could make vaccine recommendation more sophisticated. Overall, the antigenic relationship prediction model we developed provides a rapid determination method for identifying antigenic variants, and the further analysis of evolutionary and epidemic characteristics can facilitate vaccine recommendations and influenza surveillance for H1N1pdm.

Immunoinformatic based designing of potential immunogenic novel mRNA and peptide-based prophylactic vaccines against H5N1 and H7N9 avian influenza viruses

Influenza viruses are the most common cause of serious respiratory illnesses worldwide and are responsible for a significant number of annual fatalities. Therefore, it is crucial to look for new immunogenic sites that might trigger an effective immune response. In the present study, bioinformatics tools were used to design mRNA and multiepitope-based vaccines against H5N1 and H7N9 subtypes of avian influenza viruses. Several Immunoinformatic tools were employed to extrapolate T and B lymphocyte epitopes of HA and NA proteins of both subtypes. The molecular docking approach was used to dock the selected HTL and CTL epitopes with the corresponding MHC molecules. Eight (8) CTL, four (4) HTL, and Six (6) linear B cell epitopes were chosen for the structural arrangement of mRNA and of peptide-based prophylactic vaccine designs. Different physicochemical characteristics of the selected epitopes fitted with suitable linkers were analyzed. High antigenic, non-toxic, and non-allergenic features of the designed vaccines were noted at a neutral physiological pH. Codon optimization tool was used to check the GC content and CAI value of constructed MEVC-Flu vaccine, which were recorded to be 50.42% and 0.97 respectively. the GC content and CAI value verify the stable expression of vaccine in pET28a + vector. In-silico immunological simulation the MEVC-Flu vaccine construct revealed a high level of immune responses. The molecular dynamics simulation and docking results confirmed the stable interaction of TLR-8 and MEVC-Flu vaccine. Based on these parameters, vaccine constructs can be regarded as an optimistic choice against H5N1 and H7N9 strains of the influenza virus. Further experimental testing of these prophylactic vaccine designs against pathogenic avian influenza strains may clarify their safety and efficacy. Communicated by Ramaswamy H. Sarma

Emergence of a novel reassortant H3N6 canine influenza virus.

Although the natural hosts of avian influenza viruses (AIVs) are wild birds, multiple subtypes of AIVs have established epidemics in numerous mammals due to their cross-species spillover. Replication and evolution in intermedia mammalian hosts may facilitate AIV adaptation in humans. Because of their large population and intimacy with humans, dogs could act as such an intermedia host. To monitor the epidemiology of canine influenza viruses (CIVs) in Liaoning, China, we performed three surveillances in November 2018, March 2019, and April 2019. Five H3N2 and seven novel H3N6 CIVs had been isolated. Since the N6 neuraminidase (NA) genes were clustered with the H5N6 AIV, there is a high possibility that these H3N6 CIVs were generated from a H3N2 CIVs and H5N6 AIVs reassortment case. In addition, the H3N6 CIV showed increased mammalian adaptation ability compared to all the H3N2 strains in both in vitro and in vivo studies. Even though isolated 3 months later, the March 2019 isolated H3N2 viruses replicated more efficiently than the November 2018 isolated viruses. Our study indicated that H3 CIVs were undergoing an evolution process, through both genetic mutations and gene reassortment, at an incredible speed.

Detection and Genomic Characterization of an Avian Influenza Virus Subtype H5N1 (Clade 2.3.4.4b) Strain Isolated from a Pelican in Peru.

Surveillance helps us identify and monitor strains with zoonotic potential. A tracheal swab from a pelican on a Peruvian beach was H5N1 positive (clade 2.3.4.4b) using Oxford Nanopore's MinION platform. The near-complete genome sequence of strain VFAR-140 will aid us in understanding avian influenza epidemiology and spread.

Combination adjuvants and antigens provide heterosubtypic protection against lethal influenza infection.

Abstract Influenza A virus (IAV) is a negative strand RNA virus that undergoes antigenic drift and shift, leading to serologically distinct strains that can cause tens of thousands of deaths annually in the US. The wide variety of hemagglutinin (HA) and neuraminidase (NA) proteins necessitates yearly reformulation of the vaccine to protect against novel seasonal and pandemic strains. Current IAV vaccines do not contain adjuvants, however, immune modulators such as pattern recognition receptor (PRR) agonists may enhance antigen presentation to T cells and provide broader protection against IAV strains that differ in HA and NA outer coat proteins. We set out to investigate whether TLR4 (lipopolysaccharide) and TLR3 (poly IC) agonists in combination with HA (H3N2) and nucleoprotein (NP) or NP and matrix (M1) proteins could provide protection against lethal challenge with mismatched IAV strain H1N1. Mice given H3 and NP with LPS + pIC in a prime/boost strategy demonstrated 80% survival after heterosubtypic H1N1 challenge while H3 + NP in PBS showed 20% survival. Similar results were seen using LPS + pIC in combination with NP and M1 protein with 90% survival. High endpoint titers of anti-NP antibodies (that also bound H1N1 virus) correlated with heterosubtypic protection suggesting a role for non-neutralizing antibodies in promoting survival. In addition, LPS and pIC enhanced dendritic cell (DC) function in vitro and increased migratory monocyte DC in the lymph node in vivo, coupled with early upregulation of T cell marker CD69. This strategy may provide increased efficacy over current IAV vaccines by inducing non-neutralizing antibodies and enhanced T cell responses to internal proteins. Supported by Trudeau Institute and Clarkson University.

Seroconversion of a Swine Herd in a Free-Range Rural Multi-Species Farm against HPAI H5N1 2.3.4.4b Clade Virus.

Starting from October 2021, several outbreaks of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 were reported in wild and domestic birds in Italy. Following the detection of an HPAIV in a free-ranging poultry farm in Ostia, province of Rome, despite the lack of clinical signs, additional virological and serological analyses were conducted on samples collected from free-ranging pigs, reared in the same holding, due to their direct contact with the infected poultry. While the swine nasal swabs were all RT-PCR negative for the influenza type A matrix (M) gene, the majority (%) of the tested pigs resulted serologically positive for the hemagglutination inhibition test and microneutralization assay, using an H5N1 strain considered to be homologous to the virus detected in the farm. These results provide further evidence of the worrisome replicative fitness that HPAI H5Nx viruses of the 2.3.4.4b clade have in mammalian species. Moreover, our report calls for additional active surveillance, to promptly intercept occasional spillover transmissions to domestic mammals in close contact with HPAI affected birds. Strengthened biosecurity measures and efficient separation should be prioritized in mixed-species farms in areas at risk of HPAI introduction.

Structural characterisation of hemagglutinin from seven Influenza A H1N1 strains reveal diversity in the C05 antibody recognition site

Influenza virus (IV) causes several outbreaks of the flu each year resulting in an economic burden to the healthcare system in the billions of dollars. Several influenza pandemics have occurred during the last century and estimated to have caused 100 million deaths. There are four genera of IV, A (IVA), B (IVB), C (IVC), and D (IVD), with IVA being the most virulent to the human population. Hemagglutinin (HA) is an IVA surface protein that allows the virus to attach to host cell receptors and enter the cell. Here we have characterised the high-resolution structures of seven IVA HAs, with one in complex with the anti-influenza head-binding antibody C05. Our analysis revealed conserved receptor binding residues in all structures, as seen in previously characterised IV HAs. Amino acid conservation is more prevalent on the stalk than the receptor binding domain (RBD; also called the head domain), allowing the virus to escape from antibodies targeting the RBD. The equivalent site of C05 antibody binding to A/Denver/57 HA appears hypervariable in the other H1N1 IV HAs. Modifications within this region appear to disrupt binding of the C05 antibody, as these HAs no longer bind the C05 antibody by analytical SEC. Our study brings new insights into the structural and functional recognition of IV HA proteins and can contribute to further development of anti-influenza vaccines.

An Orf-Virus (ORFV)-Based Vector Expressing a Consensus H1 Hemagglutinin Provides Protection against Diverse Swine Influenza Viruses.

Influenza A viruses (IAV-S) belonging to the H1 subtype are endemic in swine worldwide. Antigenic drift and antigenic shift lead to a substantial antigenic diversity in circulating IAV-S strains. As a result, the most commonly used vaccines based on whole inactivated viruses (WIVs) provide low protection against divergent H1 strains due to the mismatch between the vaccine virus strain and the circulating one. Here, a consensus coding sequence of the full-length of HA from H1 subtype was generated in silico after alignment of the sequences from IAV-S isolates obtained from public databases and was delivered to pigs using the Orf virus (ORFV) vector platform. The immunogenicity and protective efficacy of the resulting ORFVΔ121conH1 recombinant virus were evaluated against divergent IAV-S strains in piglets. Virus shedding after intranasal/intratracheal challenge with two IAV-S strains was assessed by real-time RT-PCR and virus titration. Viral genome copies and infectious virus load were reduced in nasal secretions of immunized animals. Flow cytometry analysis showed that the frequency of T helper/memory cells, as well as cytotoxic T lymphocytes (CTLs), were significantly higher in the peripheral blood mononuclear cells (PBMCs) of the vaccinated groups compared to unvaccinated animals when they were challenged with a pandemic strain of IAV H1N1 (CA/09). Interestingly, the percentage of T cells was higher in the bronchoalveolar lavage of vaccinated animals in relation to unvaccinated animals in the groups challenged with a H1N1 from the gamma clade (OH/07). In summary, delivery of the consensus HA from the H1 IAV-S subtype by the parapoxvirus ORFV vector decreased shedding of infectious virus and viral load of IAV-S in nasal secretions and induced cellular protective immunity against divergent influenza viruses in swine.

H3N2 influenza hemagglutination inhibition method qualification with data driven statistical methods for human clinical trials.

Hemagglutination inhibition (HAI) antibody titers to seasonal influenza strains are important surrogates for vaccine-elicited protection. However, HAI assays can be variable across labs, with low sensitivity across diverse viruses due to lack of standardization. Performing qualification of these assays on a strain specific level enables the precise and accurate quantification of HAI titers. Influenza A (H3N2) continues to be a predominant circulating subtype in most countries in Europe and North America since 1968 and is thus a focus of influenza vaccine research. As a part of the National Institutes of Health (NIH)-funded Collaborative Influenza Vaccine Innovation Centers (CIVICs) program, we report on the identification of a robust assay design, rigorous statistical analysis, and complete qualification of an HAI assay using A/Texas/71/2017 as a representative H3N2 strain and guinea pig red blood cells and neuraminidase (NA) inhibitor oseltamivir to prevent NA-mediated agglutination. This qualified HAI assay is precise (calculated by the geometric coefficient of variation (GCV)) for intermediate precision and intra-operator variability, accurate calculated by relative error, perfectly linear (slope of -1, R-Square 1), robust (<25% GCV) and depicts high specificity and sensitivity. This HAI method was successfully qualified for another H3N2 influenza strain A/Singapore/INFIMH-16-0019/2016, meeting all pre-specified acceptance criteria. These results demonstrate that HAI qualification and data generation for new influenza strains can be achieved efficiently with minimal extra testing and development. We report on a qualified and adaptable influenza serology method and analysis strategy to measure quantifiable HAI titers to define correlates of vaccine mediated protection in human clinical trials.

Genetic Diversity and Evolutionary Kinetics of Influenza A Virus H3N2 Subtypes Circulating in Riyadh, Saudi Arabia.

Presence of a large foreign workforce and the annual gathering of people for pilgrimage from around the globe have significantly contributed to the emergence and diversity of respiratory viruses in Saudi Arabia. Here, we report the sequence and phylogenetic analysis of the H3N2 subtype of influenza A virus (IAV) in clinical samples collected from Riyadh, Saudi Arabia. Based on RT-PCR, IAV was found in 88 (28.3%) of the 311 samples screened. Of the 88-IAV positive samples, 43 (48.8%) were H1N1 subtype while the remaining 45 (51.2%) were found to be of the H3N2 subtype. Complete sequencing of HA and NA genes of H3N2 revealed, twelve and nine amino acid (AA) substitutions respectively, and importantly, these variations are absent in the current vaccine strains. Based on the phylogenetic analysis, the majority of H3N2 strains were grouped in the same clades as the vaccine strains. Importantly, the N-glycosylation sites at AA 135(NSS) were found to be unique to 6 strains in the investigated HA1 protein and were absent in the current vaccine strains. These data may have significant clinical implications in designing novel and population-based vaccines for IAV and underscore the need for regular monitoring of efficacy of vaccines due to emerging variants.

Recombinant hemagglutinin protein and DNA-RNA-combined nucleic acid vaccines harbored by yeast elicit protective immunity against H9N2 avian influenza infection

A safe, convenience, and effective vaccine for controlling avian influenza virus infection is crucial in scale poultry production. Yeasts are considered useful vaccine vehicles for the delivery of antigens, which has been used to protect human and animal health. We report here the development of H9N2 strain hemagglutinin (HA)-based recombinant protein vaccines (rH9HA) and DNA-RNA-combined vaccine (rH9-DNA-RNA) in Saccharomyces cerevisiae for the first time. The immunogenicity assay indicated that both rH9HA and rH9-DNA-RNA could induce robust production of serum IgG, mucosal sIgA, and cellular immune responses. The reshape and diversification of gut microbiota and an enriched Lactobacillus, Debaryomyces were observed after oral immunization with rH9HA or rH9-DNA-RNA yeast vaccine, which might contribute to modulate the intestinal mucosal immunity and antiviral process. Oral immunized birds with either rH9HA or rH9-DNA-RNA were effectively protected from H9N2 virus challenge. Our findings suggested that yeast-derived H9N2 HA-based recombinant protein vaccines and DNA-RNA-combined nucleic acid vaccines are feasible and efficacious, opening up a new avenue for rapid and cost-effective production of avian influenza vaccines to achieve good protection effect.