Universal Influenza Vaccine Research Articles

Chimeric Virus-like Particles Co-Displaying Hemagglutinin Stem and the C-Terminal Fragment of DnaK Confer Heterologous Influenza Protection in Mice.

Influenza virus hemagglutinin (HA) stem is currently regarded as an extremely promising immunogen for designing universal influenza vaccines. The appropriate antigen-presenting vaccine vector would be conducive to increasing the immunogenicity of the HA stem antigen. In this study, we generated chimeric virus-like particles (cVLPs) co-displaying the truncated C-terminal of DnaK from Escherichia coli and H1 stem or full-length H1 antigen using the baculovirus expression system. Transmission electronic micrography revealed the expression and presentation of H1 stem antigens on the surface of VLPs. Vaccinations of mice with the H1 stem cVLPs induced H1-specific immune responses and provided heterologous immune protection in vivo, which was more effective than vaccinations with VLPs displaying H1 stem alone in protecting mice against weight loss as well as increasing survival rates after lethal influenza viral challenge. The results indicate that the incorporation of the truncated C-terminal of DnaK as an adjuvant protein into the cVLPs significantly enhances the H1-specific immunity and immune protection. We have explicitly identified the VLP platform as an effective way of expressing HA stem antigen and revealed that chimeric VLP is an vaccine vector for developing HA stem-based universal influenza vaccines.

Immune response against DNA- and mRNA vaccines encoding artificial influenza virus immunogens

Constant antigenic drift of circulating influenza viruses leads to inefficiency of seasonal influenza vaccines, thus requiring annual re-design of these vaccines. Therefore, the development of a universal influenza vaccine is of particular relevance. A promising line of research in this area is to design the immunogens consisting of conserved protein fragments from different influenza viral strains. The aim of this work was to assess immunogenicity of DNA vaccines and mRNA vaccines encoding artificial antigens consisting of conserved hemagglutinin stem fragments and conserved M2 protein. We have obtained DNA vaccine constructs encoding artificial immunogens AgH1, AgH3, and AgM2, which contained conserved fragments of the hemagglutinin stalk from the two subtypes of influenza A H1N1 and H3N2, and conserved M2 protein. These DNA vaccines were used as templates for the synthesis of mRNA vaccines. To assess immunogenicity of the obtained constructs, BALB/c mice were immunized with DNA and mRNA vaccines by i/m administration. Assessment of the humoral immune response was carried out by ELISA, using influenza viruses A/Aichi/2/68(H3N2), A/California/07/2009 as antigens and the ULTRIX vaccine containing purified antigens of H1N1 and H3N2 influenza viruses. T cell immune response was assessed using two methods: intracellular cytokine staining (ICS) and ELISpot. ICS was performed to determine CD8+ and CD4+T-lymphocytes producing IFN. ELISpot was carried out using the mouse IFN ELISpot kit (BD). A peptide mixture which included composition of the target antigens, was used for cell stimulation. The results showed that the designed DNA vaccine constructs induce virus-specific humoral and cellular responses in immunized BALB/c mice. Intramuscular administration of the naked mRNA vaccine constructs induced a weak humoral immune response, thus suggesting a need for further work to improve the delivery approaches.

Assessment of Fcγ receptor-dependent binding of influenza hemagglutinin vaccine-induced antibodies in a non-human primate model

SummarySeveral cross-protective antibodies that recognize a broad range of influenza A virus (IAV) strains are known to have functions in virus elimination such as Fcγ receptor (FcγR)-effector function and neutralizing activity against the head region. Although few studies have used primary cells as effector cells, the FcγR-effector function was evaluated after isolating each cell subset. Herein, we established an original assay system to evaluate purified FI6 IgG-mediated binding to hemagglutinin (HA)-expressing cells by flow cytometry using peripheral blood mononuclear cells from cynomolgus macaques. In addition, we evaluated the FcγR-effector function of IAV vaccine-induced anti-HA antibodies in cynomolgus macaques after administering the split vaccine. We found several cell types, mainly classical monocytes, bound to HA-expressing target cells in an FcγR-dependent manner, that were dominant in the binding of the cell population. Thus, this assay system could facilitate the development of a universal influenza vaccine.

The role of cell-mediated immunity against influenza and its implications for vaccine evaluation

Influenza vaccines remain the most effective tools to prevent flu and its complications. Trivalent or quadrivalent inactivated influenza vaccines primarily elicit antibodies towards haemagglutinin and neuraminidase. These vaccines fail to induce high protective efficacy, in particular in older adults and immunocompromised individuals and require annual updates to keep up with evolving influenza strains (antigenic drift). Vaccine efficacy declines when there is a mismatch between its content and circulating strains. Current correlates of protection are merely based on serological parameters determined by haemagglutination inhibition or single radial haemolysis assays. However, there is ample evidence showing that these serological correlates of protection can both over- or underestimate the protective efficacy of influenza vaccines. Next-generation universal influenza vaccines that induce cross-reactive cellular immune responses (CD4+ and/or CD8+ T-cell responses) against conserved epitopes may overcome some of the shortcomings of the current inactivated vaccines by eliciting broader protection that lasts for several influenza seasons and potentially enhances pandemic preparedness. Assessment of cellular immune responses in clinical trials that evaluate the immunogenicity of these new generation vaccines is thus of utmost importance. Moreover, studies are needed to examine whether these cross-reactive cellular immune responses can be considered as new or complementary correlates of protection in the evaluation of traditional and next-generation influenza vaccines. An overview of the assays that can be applied to measure cell-mediated immune responses to influenza with their strengths and weaknesses is provided here.

Vaccination with Deglycosylated Modified Hemagglutinin Broadly Protects against Influenza Virus Infection in Mice and Ferrets.

Recent efforts have been directed toward the development of universal influenza vaccines inducing broadly neutralizing antibodies to conserved antigenic supersites of Hemagglutinin (HA). Although several studies raise the importance of glycosylation in HA antigen design, whether this theory can be widely confirmed remains unclear; which influenza HA with an altered glycosylation profile could impact the amplitude and focus of the host immune response. Here, we evaluated the characteristics and efficacy of deglycosylated modified HA proteins, including monoglycosylated HA (HAmg), unglycosylated HA (HAug), and fully glycosylated HA (HAfg), without treatment with H3N2 Wisconsin/67/2005. Our results showed that HAug could induce a cross-strain protective immune response in mice against both H3N2 and H7N9 subtypes with better antibody-dependent cellular cytotoxicity (ADCC) than the HAmg- and HAfg-immunized groups, which suggested that highly conserved epitopes that were masked by surface glycosylation may be exposed and thus promote the induction of broad antibodies that recognize the hidden epitopes. This strategy may also supplement the direction of deglycosylated modified HA for universal influenza vaccines.

Development and characterization of influenza M2 ectodomain and/or hemagglutinin stalk-based dendritic cell-targeting vaccines.

A universal influenza vaccine is required for broad protection against influenza infection. Here, we revealed the efficacy of novel influenza vaccine candidates based on Ebola glycoprotein dendritic cell (DC)-targeting domain (EΔM) fusion protein technology. The four copies of ectodomain matrix protein of influenza (tM2e) or M2e hemagglutinin stalk (HA stalk) peptides (HM2e) were fused with EΔM to generate EΔM-tM2e or EΔM-HM2e, respectively. We demonstrated that EΔM-HM2e- or EΔM-tM2e-pseudotyped viral particles can efficiently target DC/macrophages in vitro and induced significantly high titers of anti-HA and/or anti-M2e antibodies in mice. Significantly, the recombinant vesicular stomatitis virus (rVSV)-EΔM-tM2e and rVSV-EΔM-HM2e vaccines mediated rapid and potent induction of M2 or/and HA antibodies in mice sera and mucosa. Importantly, vaccination of rVSV-EΔM-tM2e or rVSV-EΔM-HM2e protected mice from influenza H1N1 and H3N2 challenges. Taken together, our study suggests that rVSV-EΔM-tM2e and rVSV-EΔM-HM2e are promising candidates that may lead to the development of a universal vaccine against different influenza strains.

Hemagglutinin stalk-binding antibodies enhance effectiveness of neuraminidase inhibitors against influenza via Fc-dependent effector functions

SummaryThe conserved hemagglutinin stalk domain is an attractive target for broadly effective antibody-based therapeutics and next-generation universal influenza vaccines. Protection provided by hemagglutinin stalk-binding antibodies is principally mediated through activation of immune effector cells. Titers of stalk-binding antibodies are highly variable on an individual level and tend to increase with age as a result of increasing exposures to influenza virus. In our study, we show that stalk-binding antibodies cooperate with neuraminidase inhibitors to protect against influenza virus infection in an Fc-dependent manner. These data suggest that the effectiveness of neuraminidase inhibitors is likely influenced by an individual’s titers of stalk-binding antibodies and that neuraminidase inhibitors may enhance the effectiveness of future stalk-binding monoclonal antibody-based treatments.

Progress towards the Development of a Universal Influenza Vaccine.

Influenza viruses are responsible for millions of cases globally and significantly threaten public health. Since pandemic and zoonotic influenza viruses have emerged in the last 20 years and some of the viruses have resulted in high mortality in humans, a universal influenza vaccine is needed to provide comprehensive protection against a wide range of influenza viruses. Current seasonal influenza vaccines provide strain-specific protection and are less effective against mismatched strains. The rapid antigenic drift and shift in influenza viruses resulted in time-consuming surveillance and uncertainty in the vaccine protection efficacy. Most recent universal influenza vaccine studies target the conserved antigen domains of the viral surface glycoproteins and internal proteins to provide broader protection. Following the development of advanced vaccine technologies, several innovative strategies and vaccine platforms are being explored to generate robust cross-protective immunity. This review provides the latest progress in the development of universal influenza vaccines.

Formulation of dry powders of vaccines containing MF59 or AddaVax by Thin-Film Freeze-Drying: Towards a dry powder universal flu vaccine

MF59® is an oil-in-water (O/W) nanoemulsion-based vaccine adjuvant that is often used in seasonal and pandemic influenza vaccines. We explored the feasibility of developing dry powders of vaccines adjuvanted with MF59 or AddaVax™, a preclinical grade equivalent of MF59 with the same composition and droplet size as MF59, by thin-film freeze-drying (TFFD). Liquid AddaVax alone was successfully converted to a dry powder by TFFD using trehalose as a stabilizing agent while maintaining the droplet size distribution of AddaVax after it was reconstituted. TFFD was then applied to convert liquid AddaVax-adjuvanted vaccines containing either a model antigen (e.g., ovalbumin) or mono-, bi-, and tri-valent recombinant hemagglutinin (rHA) protein-based H1 and/or H3 (universal) influenza vaccine candidates, as well as the MF59-containing Fluad® Quadrivalent influenza vaccine to dry powders. Both antigens and stabilizing agents affected the physical properties of the vaccines (e.g., mean particle size and particle size distribution) after the vaccines were subjected to TFFD. Importantly, the integrity and hemagglutination activity of the rHA antigens did not significantly change and the immunogenicity of reconstituted influenza vaccine candidates was maintained when evaluated in a mouse model. The vaccine dry powder was not sensitive to repeated freezing-and-thawing, in contrast to its liquid counterpart. It is concluded that TFFD can be applied to convert liquid vaccines containing MF59 or AddaVax to dry powders while maintaining the immunogenicity of the vaccines. Ultimately, TFFD technology may be used to prepare dry powders of multivalent universal influenza vaccines.

Layered protein nanoparticles containing influenza B HA stalk induced sustained cross-protection against viruses spanning both viral lineages

The influenza epidemics pose a significant threat to public health. Of them, type B influenza coincided with several severe flu outbreaks. The efficacy of the current seasonal flu vaccine is limited due to the antigenicity changes of circulating strains. In this study, we generated structure-stabilized HA stalk antigens from influenza B and fabricated double-layered protein nanoparticles as universal influenza B vaccine candidates. In vitro studies found that the resulting protein nanoparticles were effectively taken up to activate dendritic cells. Nanoparticle immunization induced broadly reactive immune responses conferring robust and sustained cross-immune protection against influenza B virus strains of both lineages. The results reveal the potential of layered protein nanoparticles incorporated with structure-stabilized constant antigens as a universal influenza vaccine with improved immune protective potency and breadth.

A biepitope, adjuvant-free, self-assembled influenza nanovaccine provides cross-protection against H3N2 and H1N1 viruses in mice.

Currently, the incorporation of multiple epitopes into vaccines is more desirable than the incorporation of a single antigen for universal influenza vaccine development. However, epitopes induce poor immune responses. Although the use of adjuvants can overcome this obstacle, it may raise new problems. Effective antigen delivery vehicles that can function as both antigen carriers and intrinsic adjuvants are highly desired for vaccine development. Here, we report a biepitope nanovaccine that provides complete protection in mice against H3N2 virus as well as partial protection against H1N1 virus. This vaccine (3MCD-f) consists of two conserved epitopes (matrix protein 2 ectodomain (M2e) and CDhelix), and these epitopes were presented on the surface of ferritin in a sequential tandem format. Subcutaneous immunization with 3MCD-f in the absence of adjuvant induces robust humoral and cellular immune responses. These results provide a proof of concept for the 3MCD-f nanovaccine that might be an ideal candidate for future influenza pandemics.

An epitope-optimized human H3N2 influenza vaccine induces broadly protective immunity in mice and ferrets

There is a crucial need for an improved H3N2 influenza virus vaccine due to low vaccine efficacy rates and increased morbidity and mortality associated with H3N2-dominated influenza seasons. Here, we utilize a computational design strategy to produce epitope-optimized, broadly cross-reactive H3 hemagglutinins in order to create a universal H3N2 influenza vaccine. The Epigraph immunogens are designed to maximize the viral population frequency of epitopes incorporated into the immunogen. We compared our Epigraph H3 vaccine to the traditional egg-based inactivated influenza vaccine from 2018–19, FluZone. Epigraph vaccination-induced stronger cross-reactive antibody responses than FluZone against 18 H3N2 viruses isolated from 1968 to 2019 in both mice and ferrets, with protective hemagglutination inhibition titers against 93–100% of the contemporary H3N2 strains compared to only 27% protection measured from FluZone. In addition, Epigraph vaccination-induced strong cross-reactive T-cell immunity which significantly contributes to protection against lethal influenza virus infection. Finally, Epigraph vaccination protected ferrets from influenza disease after challenge with two H3N2 viruses. The superior cross-reactive immunity induced by these Epigraph immunogens supports their development as a universal H3N2 influenza vaccine.

A Hemagglutinin Stem Vaccine Designed Rationally by AlphaFold2 Confers Broad Protection against Influenza B Infection.

Two lineages of influenza B viruses (IBV) co-circulating in human beings have been posing a significant public health burden worldwide. A substantial number of broadly neutralizing antibodies (bnAbs) have been identified targeting conserved epitopes on hemagglutinin (HA) stem domain, posing great interest for universal influenza vaccine development. Various strategies to design immunogens that selectively present these conserved epitopes are being explored. However, it has been a challenge to retain native conformation of the HA stem region, especially for soluble expression in prokaryotic systems. Here, using a structure prediction tool AlphaFold2, we rationally designed a stable stem antigen “B60-Stem-8071”, an HA stem vaccine derived from B/Brisbane/60/2006 grafted with a CR8071 epitope as a linker. The B60-Stem-8071 exhibited better solubility and more stable expression in the E. coli system compared to the naïve HA stem antigen. Immunization with B60-Stem-8071 in mice generated cross-reactive antibodies and protected mice broadly against lethal challenge with Yamagata and Victoria lineages of influenza B virus. Notably, soluble expression of B60-stem-8071 in the E. coli system showed the potential to produce the influenza B vaccine in a low-cost way. This study represents a proof of concept for the rational design of HA stem antigen based on structure prediction and analysis.

Thermostable H1 hemagglutinin stem with M2e epitopes provides broad cross-protection against group1 and 2 influenza A viruses

Hemagglutinin (HA) stem-based vaccines have limitations in providing broad and effective protection against cross-group influenza viruses, despite being a promising universal vaccine target. To overcome the limited cross-protection and low efficacy by HA stem vaccination, we genetically engineered a chimeric conjugate of thermostable H1 HA stem and highly conserved M2e repeat (M2e-H1stem), which was expressed at high yields in Escherichia coli. M2e-H1stem protein presented native-like epitopes reactive to antisera of live virus infection. M2e-H1stem protein vaccination of mice induced strong M2e- and HA stem-specific immune responses, conferring broadly effective cross-protection against both antigenically distinct group 1 (H1N1, H5N1, and H9N2 subtypes) and group 2 (H3N2 and H7N9 subtypes) seasonal and pandemic potential influenza viruses. M2e-H1stem vaccination generated CD4+ and CD8+ T cell responses and antibody-dependent cytotoxic cellular and humoral immunity, which contributed to enhancing cross-protection. Furthermore, comparable broad cross-group protection was observed in older aged mice after M2e-H1stem vaccination. This study provides evidence warranting further development of chimeric M2e-stem proteins as a promising universal influenza vaccine candidate in adult and aged populations.

Engineering a Better FLUture with Neuraminidase‐Targeted Monoclonal Antibodies

Each year epidemiologists and public officials predict which influenza strain will be most prevalent and design a vaccine to target those specific strains; unfortunately, they are not always accurate. Traditional vaccines cause our bodies to produce antibodies that target two subtypes of the influenza A, that both bind to the surface protein Hemagglutinin (HA). However, HA mutates quickly, leaving the body susceptible to new mutant strains of influenza. Fortunately, another surface protein, Neuraminidase (NA), mutates significantly slower than HA, which would makeaa vaccine that targets NA potentially more effective against a broader spectrum of influenza A and B subtypes. According to Stadlbauer et al., one H3N2 infected donor naturally created monoclonal antibodies (mAbs) against NA that are protective against various strains of influenza A and B. Our research explored the mAb 1G01 in relation to H3N2 NA using PDB 6Q23. We found that 1G01 targets the active site of NA resulting from attraction due to a difference in charges, thereby blocking binding to its substrate necessary for NA to support virus binding and replication. Therefore, implementing properties of anti‐NA antibodies into current influenza vaccines could be used to improve their protectiveness against more strains of the virus, increasing the predictive reliability of current vaccines, and as a step towards a universal influenza vaccine. The Mahtomedi MSOE Center for BioMolecular Modeling MAPS Team used 3‐D modeling and printing technology to examine structure‐function relationships of antibodies in relation to influenza. The visual model will be a valuable tool in developing our story.

Immunogenic epitope prediction to create a universal influenza vaccine

Influenza virus is one of the most rapidly evolving human pathogens and causes significant morbidity and mortality worldwide. This feature enables the virus to avoid natural or vaccine-induced immunity. For this reason, there is an intensive search for new approaches to create a universal influenza vaccine. Here, we propose pipelines based on modern prediction algorithms that allowed us to select 10 B-cell epitopes, 10 CD8+ T-cell epitopes and 6 CD4+ T-cell epitopes from influenza viruses that were characterized by high conservation and antigenicity. These epitopes could be used to create universal vaccines against influenza viruses. In addition, the scripts used in these pipelines are universal and can be used to select epitopes from other pathogens.

Universal Flu mRNA Vaccine: Promises, Prospects, and Problems.

The seasonal flu vaccine is, essentially, the only known way to prevent influenza epidemics. However, this approach has limited efficacy due to the high diversity of influenza viruses. Several techniques could potentially overcome this obstacle. A recent first-in-human study of a chimeric hemagglutinin-based universal influenza virus vaccine demonstrated promising results. The coronavirus pandemic triggered the development of fundamentally new vaccine platforms that have demonstrated their effectiveness in humans. Currently, there are around a dozen messenger RNA and self-amplifying RNA flu vaccines in clinical or preclinical trials. However, the applicability of novel approaches for a universal influenza vaccine creation remains unclear. The current review aims to cover the current state of this problem and to suggest future directions for RNA-based flu vaccine development.

Cost-utility analysis of increasing uptake of universal seasonal quadrivalent influenza vaccine (QIV) in children aged 6 months and older in Germany

ABSTRACT Seasonal influenza causes many cases and related deaths in Europe annually, despite ongoing vaccination programs for older adults and people at high-risk of complications. Children have the highest risk of infection and play a key role in disease transmission. Our cost-utility analysis, based on a dynamic transmission model, estimated the impact of increasing the current vaccination coverage with inactivated quadrivalent influenza vaccine in Germany to all (healthy and high-risk) children under 5 years of age (40% uptake), or under 18 years (40% uptake), or only high-risk children under 18 years (90% uptake). Eight influenza complications were modeled, hospitalization and death rates were based on age and risk status. All three vaccination strategies provided more health benefits than the existing vaccination situation, reducing influenza cases, complications, hospitalizations and deaths across the entire population. The strategy targeting all children under 5 years was highly cost-effective (€6/quality-adjusted life-year gained, payer perspective). The other strategies were cost saving from the payer and societal perspectives. The vaccination strategy targeting all children under 18 years was estimated to provide the most health benefits (preventing on average 1.66 million cases, 179,000 complications, 14,000 hospitalizations and 3,600 deaths due to influenza annually) and the most cost savings (annually €20.5 million and €731.3 million from payer and societal perspectives, respectively). Our analysis provides policy decision-makers with evidence supporting strategies to expand childhood influenza vaccination, to directly protect children, and indirectly all other unvaccinated age groups, in order to reduce the humanistic and economic burden on healthcare systems and society.

The future of haemagglutinin stalk-based universal influenza vaccines

The development of next-generation influenza vaccines with increased breadth and duration of protection is among one of the objectives in WHO's Global Influenza Strategy for 2019–2030, which calls for the development of improved global tools by 2030 to benefit all countries and to instil public confidence and vaccine uptake.1 Over the past two decades, more than 100 universal influenza vaccine prototypes have been proposed; however, only few have progressed beyond animal studies, and even fewer results have been made publicly available.

A single-shot vaccine approach for the universal influenza A vaccine candidate M2e.

SignificanceAlthough the need for a universal influenza vaccine has long been recognized, only a handful of candidates have been identified so far, with even fewer advancing in the clinical pipeline. The 24-amino acid ectodomain of M2 protein (M2e) has been developed over the past two decades. However, M2e-based vaccine candidates have shortcomings, including the need for several administrations and the lack of sustained antibody titers over time. We report here a vaccine targeting strategy that has the potential to confer sustained and strong protection upon a single shot of a small amount of M2e antigen. The current COVID-19 pandemic has highlighted the importance of developing versatile, powerful platforms for the rapid deployment of vaccines against any incoming threat.