The response to influenza vaccination is associated with DNA methylation-driven regulation of T cell innate antiviral pathways.

Background: The effect of vaccination on the epigenome remains poorly characterized. In previous research, we identified an association between seroprotection against influenza and DNA methylation at sites associated with the RIG-1 signaling pathway, which recognizes viral double-stranded RNA and leads to a type I interferon response. However, these studies did not fully account for confounding factors including age, gender, and BMI, along with changes in cell type composition. Results: Here, we studied the influenza vaccine response in a longitudinal cohort vaccinated over two consecutive years (2019-2020 and 2020-2021), using peripheral blood mononuclear cells and a targeted DNA methylation approach. To address the effects of multiple factors on the epigenome, we designed a multivariate multiple regression model that included seroprotection levels as quantified by the hemagglutination-inhibition (HAI) assay test. Conclusions: Our findings indicate that 179 methylation sites can be combined as potential signatures to predict seroprotection. These sites were not only enriched for genes involved in the regulation of the RIG-I signaling pathway, as found previously, but also enriched for other genes associated with innate immunity to viruses and the transcription factor binding sites of BRD4, which is known to impact T cell memory. We propose a model to suggest that the RIG-I pathway and BRD4 could potentially be modulated to improve immunization strategies.

Inactivated influenza virus vaccines expressing COBRA hemagglutinin elicited broadly reactive, long-lived protective antibodies.

The influenza viruses cause seasonal respiratory illness that affect millions of people globally every year. Prophylactic vaccines are the recommended method to prevent the breakout of influenza epidemics. One of the current commercial influenza vaccines consists of inactivated viruses that are selected months prior to the start of a new influenza season. In many seasons, the vaccine effectiveness (VE) of these vaccines can be relatively low. Therefore, there is an urgent need to develop an improved, more universal influenza vaccine (UIV) that can provide broad protection against various drifted strains in all age groups. To meet this need, the computationally optimized broadly reactive antigen (COBRA) methodology was developed to design a hemagglutinin (HA) molecule as a new influenza vaccine. In this study, COBRA HA-based inactivated influenza viruses (IIV) expressing the COBRA HA from H1 or H3 influenza viruses were developed and characterized for the elicitation of immediate and long-term protective immunity in both immunologically naïve or influenza pre-immune animal models. These results were compared to animals vaccinated with IIV vaccines expressing wild-type H1 or H3 HA proteins (WT-IIV). The COBRA-IIV elicited long-lasting broadly reactive antibodies that had hemagglutination-inhibition (HAI) activity against drifted influenza variants.

Polymeric cGAMP microparticles affect the immunogenicity of a broadly active influenza mRNA lipid nanoparticle vaccine.

Influenza outbreaks are a major burden worldwide annually. While seasonal vaccines do provide protection against infection, they are limited in that they need to be updated every year to account for the constantly mutating virus. Recently, lipid nanoparticles (LNPs) encapsulating mRNA have seen major success as a vaccine platform for SARS-CoV-2. Herein, we applied LNPs to deliver an mRNA encoding a computationally optimized broadly active (COBRA) influenza immunogen. These COBRA mRNA LNPs induced a broadly active neutralizing antibody response and protection after lethal influenza challenge. To further increase the immunogenicity of the COBRA mRNA LNPs, we combined them with acetalated dextran microparticles encapsulating a STING agonist. Contrary to recent findings, the STING agonist decreased the immunogenicity of the COBRA mRNA LNPs which was likely due to a decrease in mRNA translation as shown in vitro. Overall, this work aids in future selection of adjuvants to use with mRNA LNP vaccines.

COBRA HA and NA vaccination elicits long-live protective immune responses against pre-pandemic H2, H5, and H7 influenza virus subtypes.

Highly pathogenic avian influenza (HPAI) viruses remain a major threat to both the poultry industry and human public health, and these viruses continue to spread worldwide. In this study, mice were vaccinated with COBRA H2, H5, and H7 hemagglutinin (HA) and two neuraminidase (NA) proteins, N1 and N2. Vaccinated mice were fully protected against lethal challenge with H5N6 influenza virus. Sera collected after vaccination showed cross-reactive IgG antibodies against a panel of wild-type H2, H5, and H7 HA proteins, and N1 and N2 NA proteins. Mice with pre-existing immunity to H1N1 and H3N2 influenza viruses that were subsequently vaccinated with COBRA HA/NA vaccines had enhanced anti-HA stem antibodies compared to vaccinated mice without pre-existing immunity. In addition, sera collected after vaccination had hemagglutinin inhibitory activity against a panel of H2Nx, H5Nx, and H7Nx influenza viruses. These protective antibodies were maintained up for up to 4 months after vaccination.

Advax-SM™-Adjuvanted COBRA (H1/H3) Hemagglutinin Influenza Vaccines.

Adjuvants enhance immune responses stimulated by vaccines. To date, many seasonal influenza vaccines are not formulated with an adjuvant. In the present study, the adjuvant Advax-SM™ was combined with next generation, broadly reactive influenza hemagglutinin (HA) vaccines that were designed using a computationally optimized broadly reactive antigen (COBRA) methodology. Advax-SM™ is a novel adjuvant comprising inulin polysaccharide and CpG55.2, a TLR9 agonist. COBRA HA vaccines were combined with Advax-SM™ or a comparator squalene emulsion (SE) adjuvant and administered to mice intramuscularly. Mice vaccinated with Advax-SM™ adjuvanted COBRA HA vaccines had increased serum levels of anti-influenza IgG and IgA, high hemagglutination inhibition activity against a panel of H1N1 and H3N2 influenza viruses, and increased anti-influenza antibody secreting cells isolated from spleens. COBRA HA plus Advax-SM™ immunized mice were protected against both morbidity and mortality following viral challenge and, at postmortem, had no detectable lung viral titers or lung inflammation. Overall, the Advax-SM™-adjuvanted COBRA HA formulation provided effective protection against drifted H1N1 and H3N2 influenza viruses.

Structural basis for the broad antigenicity of the computationally optimized influenza hemagglutinin X6.

Influenza causes significant morbidity and mortality. As an alternative approach to current seasonal vaccines, the computationally optimized broadly reactive antigen (COBRA) platform has been previously applied to hemagglutinin (HA). This approach integrates wild-type HA sequences into a single immunogen to expand the breadth of accessible antibody epitopes. Adding to previous studies of H1, H3, and H5 COBRA HAs, we define the structural features of another H1 subtype COBRA, X6, that incorporates HA sequences from before and after the 2009 H1N1 influenza pandemic. We determined structures of this antigen alone and in complex with COBRA-specific as well as broadly reactive and functional antibodies, analyzing its antigenicity. We found that X6 possesses features representing both historic and recent H1 HA strains, enabling binding to both head- and stem-reactive antibodies. Overall, these data confirm the integrity of broadly reactive antibody epitopes of X6 and contribute to design efforts for a next-generation vaccine.

Changes in depressive symptoms and antibody production following influenza vaccination in adolescents and adults.

OBJECTIVE: Psychological distress has been associated with dampened antibody production following vaccination. Questions remain, however, about whether psychological distress influences vaccine response uniformly across the lifespan, and whether changes in distress result in changes in antibody production across the same period. METHODS: Participants (N = 148; Mage = 32.2 years, SD = 19.7, range = 12-80 years) took part in consecutive vaccine studies during the 2017-2018 and 2018-2019 influenza seasons. Each influenza season, they reported on their depressive symptoms, provided blood samples, and received the standard influenza vaccine. Participants then provided a second blood sample one month later. Antibody titers were examined pre- and post-vaccination. RESULTS: Analyses examined both within-season and across-season effects of depressive symptoms, age, and their interaction on vaccine response. Within-season analyses revealed that age predicted antibody response during both seasons (2017-2018 and 2018-2019). Neither depressive symptoms nor the interaction with age were associated with antibody response to vaccination within either season. Across the two seasons, age significantly moderated the association between change in depressive symptoms and change in antibody production. For people who were 48 or older, increases in depressive symptoms across the two seasons were associated with a less robust response to the vaccine in the second season relative to the first season. For people younger than 48, changes in depressive symptoms were not significantly related to changes in antibody production. CONCLUSIONS: These findings highlight the important role of mental health for older adults' vaccine response, which could have clinical relevance for protection against disease.

Repeated vaccination with homologous influenza hemagglutinin broadens human antibody responses to unmatched flu viruses.

The on-going diversification of influenza virus necessicates annual vaccine updating. The vaccine antigen, the viral spike protein hemagglutinin (HA), tends to elicit strain-specific neutralizing activity, predicting that sequential immunization with the same HA strain will boost antibodies with narrow coverage. However, repeated vaccination with homologous SARS-CoV-2 vaccine eventually elicits neutralizing activity against highly unmatched variants, questioning this immunological premise. We evaluated a longitudinal influenza vaccine cohort, where each year the subjects received the same, novel H1N1 2009 pandemic vaccine strain. Repeated vaccination gradually enhanced receptor-blocking antibodies (HAI) to highly unmatched H1N1 strains within individuals with no initial memory recall against these historical viruses. An in silico model of affinity maturation in germinal centers integrated with a model of differentiation and expansion of memory cells provides insight into the mechanisms underlying these results and shows how repeated exposure to the same immunogen can broaden the antibody response against diversified targets.

Anti-neuraminidase immunity in the combat against influenza.

INTRODUCTION: Anti-neuraminidase (NA) immunity correlates with the protection against influenza virus infection in both human and animal models. The aim of this review is to better understand the mechanism of anti-NA immunity, and also to evaluate the approaches on developing NA-based influenza vaccines or enhancing immune responses against NA for current influenza vaccines. AREAS COVERED: In this review, the structure of influenza neuraminidase, the contribution of anti-NA immunity to protection, as well as the efforts and challenges of targeting the immune responses to NA were discussed. We also listed some of the newly discovered anti-NA monoclonal antibodies and discussed their contribution in therapeutic as well as the antigen design of a broadly protective NA vaccine. EXPERT OPINION: Targeting the immune response to both HA and NA may be critical for achieving the optimal protection since there are different mechanisms of HA and NA elicited protective immunity. Monoclonal antibodies (mAbs) that target the conserved protective lateral face or catalytic sites are effective therapeutics. The epitope discovery using monoclonal antibodies may benefit NA-based vaccine elicited broadly reactive antibody responses. Therefore, the potential for a vaccine that elicits cross-reactive antibodies against neuraminidase is a high priority for next-generation influenza vaccines.

Immunogenicity of an adjuvanted broadly active influenza vaccine in immunocompromised and diverse populations.

Influenza virus outbreaks are a major burden worldwide each year. Current vaccination strategies are inadequate due to antigenic drift/shift of the virus and the elicitation of low immune responses. The use of computationally optimized broadly reactive antigen (COBRA) hemagglutinin (HA) immunogens subvert the constantly mutating viruses; however, they are poorly immunogenic on their own. To increase the immunogenicity of subunit vaccines such as this, adjuvants can be delivered with the vaccine. For example, agonists of the stimulator of interferon genes (STING) have proven efficacy as vaccine adjuvants. However, their use in high-risk populations most vulnerable to influenza virus infection has not been closely examined. Here, we utilize a vaccine platform consisting of acetalated dextran microparticles loaded with COBRA HA and the STING agonist cyclic GMP-AMP. We examine the immunogenicity of this platform in mouse models of obesity, aging, and chemotherapy-induced immunosuppression. Further, we examine vaccine efficacy in collaborative cross mice, a genetically diverse population that mimics human genetic heterogeneity. Overall, this vaccine platform had variable efficacy in these populations supporting work to better tailor adjuvants to specific populations.

Inactivated recombinant influenza vaccine: the promising direction for the next generation of influenza vaccine.

INTRODUCTION: Vaccination is the most effective method to control the prevalence of seasonal influenza and the most widely used influenza vaccine is the inactivated influenza vaccine (IIV). Each season, the influenza vaccine must be updated to be most effective against current circulating variants. Therefore, developing a universal influenza vaccine (UIV) that can elicit both broad and durable protection is of the utmost importance. AREA COVERED: This review summarizes and compares the available influenza vaccines in the market and inactivation methods used for manufacturing IIVs. Then, we discuss the latest progress of the UIV development in the IIV format and the challenges to address for moving these vaccine candidates to clinical trials and commercialization. The literature search was based on the Centers for Disease Control and Prevention (CDC) and the PubMed databases. EXPERT OPINION: The unmet need for UIV is the primary aim of developing the next generation of influenza vaccines. The IIV has high antigenicity and a refined manufacturing process compared to most other formats. Developing the UIV in IIV format is a promising direction with advanced biomolecular technologies and next-generation adjuvant. It also inspires the development of universal vaccines for other infectious diseases.

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.

Multi-COBRA hemagglutinin formulated with cGAMP microparticles elicit protective immune responses against influenza viruses.

Influenza viruses cause a common respiratory disease known as influenza. In humans, seasonal influenza viruses can lead to epidemics, with avian influenza viruses of particular concern because they can infect multiple species and lead to unpredictable and severe disease. Therefore, there is an urgent need for a universal influenza vaccine that provides protection against seasonal and pre-pandemic influenza virus strains. The cyclic GMP-AMP (cGAMP) is a promising adjuvant for subunit vaccines that promotes type I interferons production through the stimulator of interferon genes (STING) pathway. The encapsulation of cGAMP in acetalated dextran (Ace-DEX) microparticles (MPs) enhances its intracellular delivery. In this study, the Computationally Optimized Broadly Reactive Antigen (COBRA) methodology was used to generate H1, H3, and H5 vaccine candidates. Monovalent and multivalent COBRA HA vaccines formulated with cGAMP Ace-DEX MPs were evaluated in a mouse model for antibody responses and protection against viral challenge. Serological analysis showed that cGAMP MPs adjuvanted monovalent and multivalent COBRA vaccines elicited robust antigen-specific antibody responses after a prime-boost vaccination and antibody titers were further enhanced after second boost. Compared to COBRA vaccine groups with no adjuvant or blank MPs, the cGAMP MPs enhanced HAI antibody responses against COBRA vaccination. The HAI antibody titers were not significantly different between cGAMP MPs adjuvanted monovalent and multivalent COBRA vaccine groups for most of the viruses tested in panels. The cGAMP MPs adjuvanted COBRA vaccines groups had higher antigen-specific IgG2a binding titers than the COBRA vaccine groups with no adjuvant or blank MPs. The COBRA vaccines formulated with cGAMP MPs mitigated disease caused by influenza viral challenge and decreased pulmonary viral titers in mice. Therefore, the formulation of COBRA vaccines plus cGAMP MPs is a promising universal influenza vaccine that elicits protective immune responses against human seasonal and pre-pandemic strains.

Patient Subtyping Analysis of Baseline Multi-omic Data Reveals Distinct Pre-immune States Predictive of Vaccination Responses.

Understanding the molecular mechanisms that underpin diverse vaccination responses is a critical step toward developing efficient vaccines. Molecular subtyping approaches can offer valuable insights into the heterogeneous nature of responses and aid in the design of more effective vaccines. In order to explore the molecular signatures associated with the vaccine response, we analyzed baseline transcriptomics data from paired samples of whole blood, proteomics and glycomics data from serum, and metabolomics data from urine, obtained from influenza vaccine recipients (2019-2020 season) prior to vaccination. After integrating the data using a network-based model, we performed a subtyping analysis. The integration of multiple data modalities from 62 samples resulted in five baseline molecular subtypes with distinct molecular signatures. These baseline subtypes differed in the expression of pre-existing adaptive or innate immunity signatures, which were linked to significant variation across subtypes in baseline immunoglobulin A (IgA) and hemagglutination inhibition (HAI) titer levels. It is worth noting that these significant differences persisted through day 28 post-vaccination, indicating the effect of initial immune state on vaccination response. These findings highlight the significance of interpersonal variation in baseline immune status as a crucial factor in determining vaccine response and efficacy. Ultimately, incorporating molecular profiling could enable personalized vaccine optimization.

Complex changes in serum protein levels in COVID-19 convalescents.

The COVID-19 pandemic, triggered by severe acute respiratory syndrome coronavirus 2, has affected millions of people worldwide. Much research has been dedicated to our understanding of COVID-19 disease heterogeneity and severity, but less is known about recovery associated changes. To address this gap in knowledge, we quantified the proteome from serum samples from 29 COVID-19 convalescents and 29 age-, race-, and sex-matched healthy controls. Samples were acquired within the first months of the pandemic. Many proteins from pathways known to change during acute COVID-19 illness, such as from the complement cascade, coagulation system, inflammation and adaptive immune system, had returned to levels seen in healthy controls. In comparison, we identified 22 and 15 proteins with significantly elevated and lowered levels, respectively, amongst COVID-19 convalescents compared to healthy controls. Some of the changes were similar to those observed for the acute phase of the disease, i.e. elevated levels of proteins from hemolysis, the adaptive immune systems, and inflammation. In contrast, some alterations opposed those in the acute phase, e.g. elevated levels of CETP and APOA1 which function in lipid/cholesterol metabolism, and decreased levels of proteins from the complement cascade (e.g. C1R, C1S, and VWF), the coagulation system (e.g. THBS1 and VWF), and the regulation of the actin cytoskeleton (e.g. PFN1 and CFL1) amongst COVID-19 convalescents. We speculate that some of these shifts might originate from a transient decrease in platelet counts upon recovery from the disease. Finally, we observed race-specific changes, e.g. with respect to immunoglobulins and proteins related to cholesterol metabolism.

Differential interferon responses to influenza A and B viruses in primary ferret respiratory epithelial cells.

Influenza B viruses (IBV) cocirculate with influenza A viruses (IAV) and cause periodic epidemics of disease, yet antibody and cellular responses following IBV infection are less well understood. Using the ferret model for antisera generation for influenza surveillance purposes, IAV resulted in robust antibody responses following infection, whereas IBV required an additional booster dose, over 85% of the time, to generate equivalent antibody titers. In this study, we utilized primary differentiated ferret nasal epithelial cells (FNECs) which were inoculated with IAV and IBV to study differences in innate immune responses which may result in differences in adaptive immune responses in the host. FNECs were inoculated with IAV (H1N1pdm09 and H3N2 subtypes) or IBV (B/Victoria and B/Yamagata lineages) and assessed for 72 h. Cells were analyzed for gene expression by quantitative real-time PCR, and apical and basolateral supernatants were assessed for virus kinetics and interferon (IFN), respectively. Similar virus kinetics were observed with IAV and IBV in FNECs. A comparison of gene expression and protein secretion profiles demonstrated that IBV-inoculated FNEC expressed delayed type-I/II IFN responses and reduced type-III IFN secretion compared to IAV-inoculated cells. Concurrently, gene expression of Thymic Stromal Lymphopoietin (TSLP), a type-III IFN-induced gene that enhances adaptive immune responses, was significantly downregulated in IBV-inoculated FNECs. Significant differences in other proinflammatory and adaptive genes were suppressed and delayed following IBV inoculation. Following IBV infection, ex vivo cell cultures derived from the ferret upper respiratory tract exhibited reduced and delayed innate responses which may contribute to reduced antibody responses in vivo.IMPORTANCEInfluenza B viruses (IBV) represent nearly one-quarter of all human influenza cases and are responsible for significant clinical and socioeconomic impacts but do not pose the same pandemic risks as influenza A viruses (IAV) and have thus received much less attention. IBV accounts for greater severity and deaths in children, and vaccine efficacy remains low. The ferret can be readily infected with human clinical isolates and demonstrates a similar course of disease and immune responses. IBV, however, generates lower antibodies in ferrets than IAV following the challenge. To determine whether differences in initial innate responses following infection may affect the development of robust adaptive immune responses, ferret respiratory tract cells were isolated, infected with IAV/IBV, and compared. Understanding the differences in the initial innate immune responses to IAV and IBV may be important in the development of more effective vaccines and interventions to generate more robust protective immune responses.

Multivalent next generation influenza virus vaccines protect against seasonal and pre-pandemic viruses.

Each year, new influenza virus vaccine formulations are generated to keep up with continuously circulating and mutating viral variants. A next-generation influenza virus vaccine would provide long-lasting, broadly-reactive immune protection against current and future influenza virus strains for both seasonal and pre-pandemic viruses. Next generation immunogens were designed using computationally optimized broadly reactive antigen (COBRA) methodology to protect against a broad range of strains over numerous seasons. Novel HA and NA amino acid sequences were derived from multilayered consensus sequence alignment for multiple subtypes of influenza. This multivalent formulation was hypothesized to elicit broadly protective immune responses against both seasonal and pre-pandemic influenza viruses. Mice were vaccinated with multivalent mixtures of HA and NA (H1, H2, H3, H5, H7, N1, N2) proteins. Multivalent COBRA vaccinations elicited antibodies that recognized a broad panel of strains and vaccinated mice were protected against viruses representing multiple subtypes. This is a promising candidate for a universal influenza vaccine that elicits protective immune responses against seasonal and pre-pandemic strains over multiple seasons.