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.

Recombinant parainfluenza virus 5 expressing clade 2.3.4.4b H5 hemagglutinin protein confers broad protection against H5Ny influenza viruses.

The global circulation of clade 2.3.4.4b H5Ny highly pathogenic avian influenza viruses (HPAIVs) in poultry and wild birds, increasing mammal infections, continues to pose a public health threat and may even form a pandemic. An efficacious vaccine against H5Ny HPAIVs is crucial for emergency use and pandemic preparedness. In this study, we developed a parainfluenza virus 5 (PIV5)-based vaccine candidate expressing hemagglutinin (HA) protein of clade 2.3.4.4b H5 HPAIV, termed rPIV5-H5, and evaluated its safety and efficacy in mice and ferrets. Our results demonstrated that intranasal immunization with a single dose of rPIV5-H5 could stimulate H5-specific antibody responses, moreover, a prime-boost regimen using rPIV5-H5 stimulated robust humoral, cellular, and mucosal immune responses in mice. Challenge study showed that rPIV5-H5 prime-boost regimen provided sterile immunity against lethal clade 2.3.4.4b H5N1 virus infection in mice and ferrets. Notably, rPIV5-H5 prime-boost regimen provided protection in mice against challenge with lethal doses of heterologous clades 2.2, 2.3.2, and 2.3.4 H5N1, and clade 2.3.4.4h H5N6 viruses. These results revealed that rPIV5-H5 can elicit protective immunity against a diverse clade of highly pathogenic H5Ny virus infection in mammals, highlighting the potential of rPIV5-H5 as a pan-H5 influenza vaccine candidate for emergency use.IMPORTANCEClade 2.3.4.4b H5Ny highly pathogenic avian influenza viruses (HPAIVs) have been widely circulating in wild birds and domestic poultry all over the world, leading to infections in mammals, including humans. Here, we developed a recombinant PIV5-vectored vaccine candidate expressing the HA protein of clade 2.3.4.4b H5 virus. Intranasal immunization with rPIV5-H5 in mice induced airway mucosal IgA responses, high levels of antibodies, and robust T-cell responses. Importantly, rPIV5-H5 conferred complete protection in mice and ferrets against clade 2.3.4.4b H5N1 virus challenge, the protective immunity was extended against heterologous H5Ny viruses. Taken together, our data demonstrate that rPIV5-H5 is a promising vaccine candidate against diverse H5Ny influenza viruses in mammals.

Extraction of the CDRH3 sequence of the mouse antibody repertoire selected upon influenza virus infection by subtraction of the background antibody repertoire.

Historically, antibody reactivity to pathogens and vaccine antigens has been evaluated using serological measurements of antigen-specific antibodies. However, it is difficult to evaluate all antibodies that contribute to various functions in a single assay, such as the measurement of the neutralizing antibody titer. Bulk antibody repertoire analysis using next-generation sequencing is a comprehensive method for analyzing the overall antibody response; however, it is unreliable for estimating antigen-specific antibodies due to individual variation. To address this issue, we propose a method to subtract the background signal from the repertoire of data of interest. In this study, we analyzed changes in antibody diversity and inferred the heavy-chain complementarity-determining region 3 (CDRH3) sequences of antibody clones that were selected upon influenza virus infection in a mouse model using bulk repertoire analysis. A decrease in the diversity of the antibody repertoire was observed upon viral infection, along with an increase in neutralizing antibody titers. Using kernel density estimation of sequences in a high-dimensional sequence space with background signal subtraction, we identified several clusters of CDRH3 sequences induced upon influenza virus infection. Most of these repertoires were detected more frequently in infected mice than in uninfected control mice, suggesting that infection-specific antibody sequences can be extracted using this method. Such an accurate extraction of antigen- or infection-specific repertoire information will be a useful tool for vaccine evaluation in the future. IMPORTANCE: As specific interactions between antigens and cell-surface antibodies trigger the proliferation of B-cell clones, the frequency of each antibody sequence in the samples reflects the size of each clonal population. Nevertheless, it is extremely difficult to extract antigen-specific antibody sequences from the comprehensive bulk antibody sequences obtained from blood samples due to repertoire bias influenced by exposure to dietary antigens and other infectious agents. This issue can be addressed by subtracting the background noise from the post-immunization or post-infection repertoire data. In the present study, we propose a method to quantify repertoire data from comprehensive repertoire data. This method allowed subtraction of the background repertoire, resulting in more accurate extraction of expanded antibody repertoires upon influenza virus infection. This accurate extraction of antigen- or infection-specific repertoire information is a useful tool for vaccine evaluation.

Harnessing preexisting influenza virus-specific immunity increases antibody responses against SARS-CoV-2.

In pandemic scenarios involving novel human pathogenic viruses, it is highly desirable that vaccines induce strong neutralizing antibodies as quickly as possible. However, current vaccine strategies require multiple immunization doses to produce high titers of neutralizing antibodies and are poorly protective after a single vaccination. We therefore wished to design a vaccine candidate that would induce increased protective immune responses following the first vaccine dose. We hypothesized that antibodies against the receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein could be increased by drawing upon immunity to a previous infection. We generated a fusion protein containing the influenza H1N1 PR8 virus nucleoprotein (NP) and the SARS-CoV-2 spike RBD. Mice with or without preexisting immunity to PR8 were then vaccinated with NP/RBD. We observed significantly increased SARS-CoV-2 neutralizing antibodies in mice with PR8 immunity compared to mice without preexisting PR8 immunity. Vaccination with NP/RBD protected mice from SARS-CoV-2-induced morbidity and mortality after a single dose. Additionally, we compared SARS-CoV-2 virus titers in the lungs and nasal turbinates 4 days post-challenge of mice vaccinated with NP/RBD. SARS-CoV-2 virus was detectable in the lungs and nasal turbinate of mice without preexisting PR8 immunity, while SARS-CoV-2 virus was completely undetectable in mice with preexisting PR8 immunity. We also found that CD4-positive T cells in mice with preexisting immunity to PR8 play an essential role in producing the increased antibody response against RBD. This vaccine strategy potentially can be modified to target other pathogens of concern and offers extra value in future pandemic scenarios.IMPORTANCEIncreased globalization and changes in human interactions with wild animals has increased the likelihood of the emergence of novel viruses with pandemic potential. Vaccines can be effective in preventing severe disease caused by pandemic viruses. However, it takes time to develop protective immunity via prime-boost vaccination. More effective vaccine designs should quickly induce protective immunity. We propose leveraging preexisting immunity to a different pathogen to boost protection against emerging viruses. We targeted SARS-CoV-2 as a representative pandemic virus and generated a fusion protein vaccine that combines the nucleoprotein from influenza A virus and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Our vaccine design significantly increased the production of RBD-specific antibodies in mice that had previously been exposed to influenza virus, compared to those without previous exposure. This enhanced immunity reduced SARS-CoV-2 replication in mice. Our results offer a vaccine design that could be valuable in a future pandemic setting.

Immunity induced by vaccination with recombinant influenza B virus neuraminidase protein breaks viral transmission chains in guinea pigs in an exposure intensity-dependent manner.

IMPORTANCE: Vaccines that can slow respiratory virus transmission in the population are urgently needed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza virus. Here, we describe how a recombinant neuraminidase-based influenza virus vaccine reduces transmission in vaccinated guinea pigs in an exposure intensity-based manner.

Adenoviral-vectored universal influenza vaccines administered intranasally reduce lung inflammatory responses upon viral challenge 15 months post-vaccination.

IMPORTANCE: Vaccines targeting highly conserved proteins can protect broadly against diverse viral strains. When a vaccine is administered to the respiratory tract, protection against disease is especially powerful. However, it is important to establish that this approach is safe. When vaccinated animals later encounter viruses, does reactivation of powerful local immunity, including T cell responses, damage the lungs? This study investigates the safety of mucosal vaccination of the respiratory tract. Non-replicating adenoviral vaccine vectors expressing conserved influenza virus proteins were given intranasally. This vaccine-induced protection persists for at least 15 months. Vaccination did not exacerbate inflammatory responses or tissue damage upon influenza virus infection. Instead, vaccination with nucleoprotein reduced cytokine responses and histopathology, while neutrophil and T cell responses resolved earlier. The results are promising for safe vaccination at the site of infection and thus have implications for the control of influenza and other respiratory viruses.

Report on influenza viruses received and tested by the Melbourne WHO Collaborating Centre for Reference and Research on Influenza during 2022.

As part of its role in the World Health Organization's (WHO) Global Influenza Surveillance and Response System (GISRS), the WHO Collaborating Centre for Reference and Research on Influenza in Melbourne received a record total of 12,073 human influenza positive samples during 2022. Viruses were analysed for their antigenic, genetic and antiviral susceptibility properties. Selected viruses were propagated in qualified cells or embryonated hen's eggs for potential use in seasonal influenza virus vaccines. In 2022, influenza A(H3N2) viruses predominated over influenza A(H1N1)pdm09 and B viruses, accounting for 77% of all viruses analysed. The majority of A(H1N1)pdm09, A(H3N2) and influenza B viruses analysed at the Centre were found to be antigenically and genetically similar to the respective WHO recommended vaccine strains for the southern hemisphere in 2022. Of 3,372 samples tested for susceptibility to the neuraminidase inhibitors oseltamivir and zanamivir, two A(H1N1)pdm09 viruses showed highly reduced inhibition against oseltamivir.

Immunogenicity and safety of the quadrivalent inactivated split-virion influenza vaccine in populations aged ≥ 3 years: A phase 3, randomized, double-blind, non-inferiority clinical trial.

This study aimed to evaluate the immunogenicity non-inferiority and safety of the quadrivalent inactivated split-virion influenza vaccine in participants ≥ 3 years old. A total of 3,328 participants were enrolled. Participants 3-8 years old were administered one or two doses of the investigational vaccine or one dose of the control vaccine, whereas the other participants were administered only one dose of the investigational or control vaccine. The immunogenicity and occurrence of adverse events (AEs) after 30 days of full-course vaccination and serious adverse events (SAEs) within 6 months after full-course vaccination were assessed. The sero-conversion rates (SCRs) of anti-H1N1, H3N2, B(Y), and B(V) antibodies in the test group were 74.64%, 87.40%, 82.66%, and 78.89%, respectively, and their geometric mean titers were 1:250.13, 1:394.54, 1:200.84, and 1:94.91, respectively, which were non-inferior to those in the control group. The SCRs and sero-protection rates in the two-dose group of participants 3-8 years old were greater than those in the one-dose group. The incidences of total AEs and adverse reactions in the test group were 31.6% and 21.7%, respectively, which were close to those in the control group. In the two-dose group, the incidence of adverse reactions was considerably lower in the second dose (5.5%) than in the first dose (14.7%). Most AEs were grade 1 in severity, and no SAEs were recorded. The investigational vaccine had immunogenicity non-inferior to the control vaccine, and two doses were more effective than one dose in participants 3-8 years old, with a good overall safety.Trial registration: CTR20200715.

People in China are frequently infected by influenza viruses in specific seasons, causing a large burden of disease. Influenza viruses have distinct phenotypes depending on the season. Therefore, vaccines that can effectively prevent the infection of various influenza virus phenotypes need to be developed. The quadrivalent inactivated split-virion influenza vaccine is effective against four influenza virus phenotypes. In this trial, the immunogenicity and safety of the quadrivalent inactivated split-virion influenza vaccine (investigational vaccine) developed by Dalian Aleph Biomedical Co., Ltd. were evaluated. A total of 3,328 participants ≥ 3 years old were included. Participants 3­8 years old were further divided based on the presence or absence of a history of influenza vaccination. Those participants without a vaccination history were administered one or two doses of the investigational vaccine or one dose of a marketed quadrivalent influenza vaccine (control vaccine), and those participants with a vaccination history were administered one dose of the investigational or control vaccine. This study showed for the first time that the immunogenicity and safety of the investigational vaccine were not inferior to those of the control vaccine and that the two-dose procedure induced a good immune effect in the 3­8-year-old group. In conclusion, administration of the investigational vaccine can prevent seasonal influenza in populations aged ≥ 3 years.

Immunological profile of mice immunized with a polyvalent virosome-based influenza vaccine.

BACKGROUND: Influenza A virus (IAV) causes respiratory disease in pigs and is a major concern for public health. Vaccination of pigs is the most successful measure to mitigate the impact of the disease in the herds. Influenza-based virosome is an effective immunomodulating carrier that replicates the natural antigen presentation pathway and has tolerability profile due to their purity and biocompatibility. METHODS: This study aimed to develop a polyvalent virosome influenza vaccine containing the hemagglutinin and neuraminidase proteins derived from the swine IAVs (swIAVs) H1N1, H1N2 and H3N2 subtypes, and to investigate its effectiveness in mice as a potential vaccine for swine. Mice were immunized with two vaccine doses (1 and 15 days), intramuscularly and intranasally. At 21 days and eight months later after the second vaccine dose, mice were euthanized. The humoral and cellular immune responses in mice vaccinated intranasally or intramuscularly with a polyvalent influenza virosomal vaccine were investigated. RESULTS: Only intramuscular vaccination induced high hemagglutination inhibition (HI) titers. Seroconversion and seroprotection (> 4-fold rise in HI antibody titers, reaching a titer of ≥ 1:40) were achieved in 80% of mice (intramuscularly vaccinated group) at 21 days after booster immunization. Virus-neutralizing antibody titers against IAV were detected at 8 months after vaccination, indicating long-lasting immunity. Overall, mice immunized with the virosome displayed greater ability for B, effector-T and memory-T cells from the spleen to respond to H1N1, H1N2 and H3N2 antigens. CONCLUSIONS: All findings showed an efficient immune response against IAVs in mice vaccinated with a polyvalent virosome-based influenza vaccine.

Eficacia, efectividad y seguridad de la vacuna antigripal adyuvada en población de 65 o más años de edad / Efficacy, effectiveness and safety of the adjuvanted influenza vaccine in the population aged 65 or over

La mayoría de las complicaciones y fallecimientos relacionados con la gripe estacional ocurren en población de 65 años o más y con enfermedades de base, y la vacuna frente a la gripe es la forma más efectiva de prevenirlas. La inmunización es menos eficaz en los adultos mayores debido a la inmunosenescencia. Las vacunas adyuvadas con MF59, diseñadas para mejorar la magnitud, persistencia y amplitud de la respuesta inmunitaria en personas de 65 años o más, se vienen utilizando en la práctica clínica desde 1997 en su formulación trivalente y, desde 2020, en formulación tetravalente. Los datos de diversos estudios muestran que estas vacunas son seguras para todos los grupos de edad, con un perfil de reactogenicidad similar al de la vacuna convencional, y que resultan especialmente efectivas para potenciar la respuesta inmunitaria en la población de 65 años o más, al aumentar los títulos de anticuerpos tras la vacunación y reducir significativamente el riesgo de ingreso hospitalario. Las vacunas adyuvadas han demostrado otorgar protección cruzada frente a cepas heterólogas y ser igual de efectivas que la vacuna de alta dosis en población de 65 años o más. En esta revisión se analiza la evidencia científica sobre la eficacia y la efectividad de la vacuna adyuvada con MF59 en la práctica clínica real en personas ≥65 años mediante una revisión narrativa y descriptiva de los datos publicados en ensayos clínicos, estudios observacionales y revisiones sistemáticas o metaanálisis (AU)

Most of the complications and deaths related to seasonal flu occur in the elderly population (≥65 years) with comorbidities, and the influenza vaccine is the most effective way to prevent them. Immunization is less effective in older adults due to immunosenescence. MF59-adjuvanted vaccines, designed to improve the magnitude, persistence and amplitude of the immune response in elderly people, have been used in clinical practice since 1997 in their trivalent formulation and, since 2020, in their tetravalent formulation. Data from various studies show that these vaccines are not only safe for all age groups, with a reactogenicity profile similar to that of the conventional vaccine, but also that they are especially effective in boosting the immune response in the population aged 65 or over by increasing antibody titers after vaccination and significantly reducing the risk of hospital admission. Adjuvanted vaccines have been shown to provide cross-protection against heterologous strains and to be as effective as the high-dose vaccine in the population aged 65 or over. In this review, the scientific evidence on the efficacy and effectiveness of the MF59-adjuvanted vaccine in real clinical practice in people ≥65 years of age is analyzed through a narrative and descriptive review of the literature with data from clinical trials, observational studies and systematic reviews or meta-analysis (AU)

Evaluación de una intervención comunitaria para incrementar la cobertura vacunal de la gripe en mujeres embarazadas / Evaluation of a community-based intervention to increase influenza vaccination coverage in pregnant women

Objetivo: Conocer el impacto de la intervención educativa realizada sobre los profesionales de una zona básica de salud y su grupo de participación comunitaria, que conforman el grupo intervención (GI), y analizar su repercusión en la cobertura vacunal alcanzada para gripe en el grupo de riesgo (gestantes y puérperas) comparándola con su zona básica vecina, que conforma el grupo control (GC), durante la temporada vacunal 2019/20. Diseño: Estudio cuasiexperimental de intervención comunitaria. Emplazamiento: Dos zonas básicas de salud pertenecientes al departamento de salud Elche-Crevillente, España. Participantes: Gestantes y puérperas de 2 zonas básicas de salud y el grupo de participación comunitaria. Los profesionales de salud directamente relacionados con la campaña vacunal de gripe. Intervenciones: Sesión formativa al GI previa a la campaña de gripe 2019/20. Mediciones principales: Actitudes hacia la vacunación de gripe en profesionales sanitarios mediante el cuestionario validado CAPSVA y la cobertura vacunal de las gestantes y puérperas a través del Registro de Vacunas Nominal y su aceptación a la vacuna en la consulta de la matrona. Resultados: Los datos de cobertura vacunal en gripe registrados en el Registro de Vacunas Nominal para las mujeres gestantes y puérperas fue del 26,4% (n=207) en el GI y del 19,7% (n=144) en el GC (p=0,001), con una razón de incidencia del 1,34, lográndose así un 34% más de vacunación en el GI. La aceptación para la vacunación en las consultas de la matrona también fue elevada, inmunizándose en el GI el 96,5% vs. el 89,0% en el GC, con un RR=1,09 (IC 95% 1,01-1,62).Conclusiones: Estrategias de formación conjunta a profesionales y activos de la comunidad mejoran los resultados de cobertura vacunal.

Objective: To know the impact of the educational intervention carried out on the professionals of a basic health area and their community participation group, which make up the intervention group (IG), and to analyze its repercussion on the vaccination coverage achieved for influenza in the risk group (pregnant and puerperal women) comparing it with its neighboring basic zone, which makes up the control group (CG), during the 2019/20 vaccination season. Design: Quasi-experimental study of community intervention. SiteTwo basic health zones belonging to the Elche-Crevillente health department, Spain. Participants: Pregnant and postpartum women from 2 basic health areas and the community participation group. Health professionals directly related to the flu vaccination campaign. Interventions: Training session for the IG prior to the 2019/20 flu campaign. Main measurements: Attitudes towards influenza vaccination in health professionals through the validated CAPSVA questionnaire and the vaccination coverage of pregnant and postpartum women through the Nominal Vaccine Registry and their acceptance of the vaccine in the midwife's office. Results: The influenza vaccination coverage data recorded in Nominal Vaccine Registry for pregnant and puerperal women was 26.4% (n=207) in the IG and 19.7% (n=144) in the CG (p=0.001), with an incidence ratio of 1.34, thus achieving 34% more vaccination in the IG. Acceptance for vaccination in the midwife's office was also high, with 96.5% immunization in IG vs. 89.0% in CG, with a RR=1.09 (95% CI 1.01-1.62). Conclusions: Joint training strategies for professionals and community assets improve the results of vaccination coverage.(AU)

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.

An ELISA-based assay for determining haemagglutinin potency in egg, cell, or recombinant protein derived influenza vaccines.

Background: The current compendial assay for haemagglutinin antigen potency in influenza vaccine is the single radial immunodiffusion (SRID) which is time consuming and can lead to delays in release of vaccine. We previously described an alternate capture and detection enzyme linked immunoassay (ELISA) that utilizes sub-type specific, sub-clade cross-reactive monoclonal antibodies (mAbs) that are haemagglutination inhibiting (HAI) and correlate with SRID. The aim of this study is to determine the applicability of ELISA across current platforms for quantitation of seasonal quadrivalent vaccine. Methods: A single mAb capture and detection ELISA was employed to quantitate hemagglutinin (HA) derived from different vaccine platforms and host organisms and compared to SRID and a polyclonal antibody based ELISA. Results: We selected mAbs that displayed appropriate characteristics for a stability indicating potency assay which reacted to avian, insect and mammalian derived HA. Qualification of the homologous mAb assay against egg and cell derived HA demonstrated performance similar to that of the SRID however, superiority in sensitivity and specificity against strains from both influenza B/Victoria and B/Yamagata lineages. Analysis of drifted strains across multiple seasons demonstrated continued utility of this approach, reducing the need to develop reagents each season. With modification of the assay, we were able to accurately measure HA from different platforms and process stages using a single calibrated reference standard. We demonstrated the accuracy of ELISA when testing vaccine formulations containing selected adjuvants at standard and higher concentrations. Accelerated stability analysis indicated a strong correlation in the rate of degradation between the homologous mAb ELISA and SRID but not with ELISA utilizing polyclonal antisera. Further, we demonstrated specificity was restricted to the trimeric and oligomeric forms of HA but not monomeric HA. Conclusion: We believe this homologous mAb ELISA is a suitable replacement for the SRID compendial assay for HA antigen quantitation and stability assessment. Identification of suitable mAbs that are applicable across multiple vaccine platforms with extended sub-type reactivity across a number of influenza seasons, indicate that this assay has broad applicability, leading to earlier availability of seasonal and pandemic vaccines without frequent replacement of polyclonal antisera that is required with SRID.

Superior immunogenicity of high-dose quadrivalent inactivated influenza vaccine versus Standard-Dose vaccine in Japanese Adults ≥ 60 years of age: Results from a phase III, randomized clinical trial.

BACKGROUND: A high-dose, split-virion inactivated quadrivalent influenza vaccine (IIV4-HD; Sanofi) is being used for the prevention of influenza in multiple countries. This study examined the immunogenicity and safety of the IIV4-HD vaccine administered intramuscularly (IM) compared with a locally licensed standard-dose influenza vaccine (IIV4-SD) administered subcutaneously (SC) in Japan. METHODS: This was a phase III, randomized, modified double-blind, active-controlled, multi-center study in older adults ≥ 60 years of age conducted during the Northern Hemisphere (NH) influenza season of 2020-21 in Japan. Participants were randomized in a 1:1 ratio to receive a single IM injection of IIV4-HD or SC injection of IIV4-SD. Hemagglutination inhibition antibody and seroconversion rates were measured at baseline and day 28. Solicited reactions were collected for up to 7 days after vaccination, unsolicited adverse events up to 28 days after vaccination, and serious adverse events throughout the study. RESULTS: The study included 2100 adults ≥ 60 years of age. IIV4-HD given IM induced superior immune responses versus IIV4-SD given SC as assessed by geometric mean titers for all four influenza strains. Superior seroconversion rates were also observed for IIV4-HD compared to IIV4-SD for all influenza strains. The safety profiles of IIV4-HD and IIV4-SD were similar. IIV4-HD was well tolerated in participants, with no safety concerns identified. CONCLUSIONS: IIV4-HD provided superior immunogenicity versus IIV4-SD and was well tolerated in participants ≥ 60 years of age in Japan. With superior immunogenicity based on the multiple randomized controlled trials and real-world evidence of trivalent high-dose formulation, IIV4-HD is expected to be the first differentiated influenza vaccine in Japan that offer a greater protection against influenza and its complications in adults 60 years of age and older. STUDY REGISTRATION: NCT04498832 (clinicaltrials.gov); U1111-1225-1085 (who.int).

Multi-Influenza HA Subtype Protection of Ferrets Vaccinated with an N1 COBRA-Based Neuraminidase.

The influenza neuraminidase (NA) is a promising target for next-generation vaccines. Protection induced by vaccination with the computationally optimized broadly reactive NA antigen (N1-I COBRA NA) was characterized in both influenza serologically naive and pre-immune ferret models following H1N1 (A/California/07/2009, CA/09) or H5N1 (A/Vietnam/1203/2004, Viet/04) influenza challenges. The N1-I COBRA NA vaccine elicited antibodies with neutralizing ELLA activity against both seasonal and pandemic H1N1 influenza, as well as the H5N1 influenza virus. In both models, N1-I COBRA NA-vaccinated ferrets that were challenged with CA/09 virus had similar morbidity (weight loss and clinical symptoms) as ferrets vaccinated with the CA/09 HA control vaccine. There were significantly reduced viral titers compared to the mock-vaccinated control animals. Ferrets vaccinated with N1-I COBRA NA or Viet/04 NA vaccines were protected against the H5N1 virus infection with minimal clinical symptoms and negligible weight loss. In contrast, ferrets vaccinated with the CA/09 NA vaccine lost ~10% of their original body weight with 25% mortality. Vaccination with either HA or NA vaccines did not inhibit contact transmission of CA/09 virus to naïve cage mates. Overall, the N1-I COBRA vaccine elicited protective immune responses against both H1N1 and H5N1 infections and partially mitigated disease in contact-transmission receiving ferrets. These results indicate that the N1-I COBRA NA performed similarly to the CA/09 HA and NA positive controls. Therefore, the N1-I COBRA NA alone induces protection against viruses from both H5N1 and H1N1 subtypes, indicating its value as a vaccine component in broadly protective influenza vaccines.

Influenza vaccination reveals sex dimorphic imprints of prior mild COVID-19.

Acute viral infections can have durable functional impacts on the immune system long after recovery, but how they affect homeostatic immune states and responses to future perturbations remain poorly understood1-4. Here we use systems immunology approaches, including longitudinal multimodal single-cell analysis (surface proteins, transcriptome and V(D)J sequences) to comparatively assess baseline immune statuses and responses to influenza vaccination in 33 healthy individuals after recovery from mild, non-hospitalized COVID-19 (mean, 151 days after diagnosis) and 40 age- and sex-matched control individuals who had never had COVID-19. At the baseline and independent of time after COVID-19, recoverees had elevated T cell activation signatures and lower expression of innate immune genes including Toll-like receptors in monocytes. Male individuals who had recovered from COVID-19 had coordinately higher innate, influenza-specific plasmablast, and antibody responses after vaccination compared with healthy male individuals and female individuals who had recovered from COVID-19, in part because male recoverees had monocytes with higher IL-15 responses early after vaccination coupled with elevated prevaccination frequencies of 'virtual memory'-like CD8+ T cells poised to produce more IFNγ after IL-15 stimulation. Moreover, the expression of the repressed innate immune genes in monocytes increased by day 1 to day 28 after vaccination in recoverees, therefore moving towards the prevaccination baseline of the healthy control individuals. By contrast, these genes decreased on day 1 and returned to the baseline by day 28 in the control individuals. Our study reveals sex-dimorphic effects of previous mild COVID-19 and suggests that viral infections in humans can establish new immunological set-points that affect future immune responses in an antigen-agnostic manner.

Molecular fate-mapping of serum antibody responses to repeat immunization.

The protective efficacy of serum antibodies results from the interplay of antigen-specific B cell clones of different affinities and specificities. These cellular dynamics underlie serum-level phenomena such as original antigenic sin (OAS)-a proposed propensity of the immune system to rely repeatedly on the first cohort of B cells engaged by an antigenic stimulus when encountering related antigens, in detriment to the induction of de novo responses1-5. OAS-type suppression of new, variant-specific antibodies may pose a barrier to vaccination against rapidly evolving viruses such as influenza and SARS-CoV-26,7. Precise measurement of OAS-type suppression is challenging because cellular and temporal origins cannot readily be ascribed to antibodies in circulation; its effect on subsequent antibody responses therefore remains unclear5,8. Here we introduce a molecular fate-mapping approach with which serum antibodies derived from specific cohorts of B cells can be differentially detected. We show that serum responses to sequential homologous boosting derive overwhelmingly from primary cohort B cells, while later induction of new antibody responses from naive B cells is strongly suppressed. Such 'primary addiction' decreases sharply as a function of antigenic distance, allowing reimmunization with divergent viral glycoproteins to produce de novo antibody responses targeting epitopes that are absent from the priming variant. Our findings have implications for the understanding of OAS and for the design and testing of vaccines against evolving pathogens.