Universal influenza virus neuraminidase vaccine elicits protective immune responses against human seasonal and pre-pandemic strains

The hemagglutinin (HA) surface protein is the primary immune target for most influenza vaccines. The neuraminidase (NA) surface protein is often a secondary target for vaccine designs. In this study, computationally optimized broadly reactive antigen methodology was used to generate the N1-I NA vaccine antigen that was designed to cross-react with avian, swine, and human influenza viruses of N1 NA subtype. The elicited antibodies bound to NA proteins derived from A/California/07/2009 (H1N1)pdm09, A/Brisbane/59/2007 (H1N1), A/Swine/North Carolina/154074/2015 (H1N1) and A/Viet Nam/1203/2004 (H5N1) influenza viruses, with NA-neutralizing activity against a broad panel of HXN1 influenza strains. Mice vaccinated with the N1-I COBRA NA vaccine were protected from mortality and viral lung titers were lower when challenged with four different viral challenges: A/California/07/2009, A/Brisbane/59/2007, A/Swine/North Carolina/154074/2015 and A/Viet Nam/1203/2004. Vaccinated mice had little to no weight loss against both homologous, but also cross-NA genetic clade challenges. Lung viral titers were lower compared to the mock vaccinated mice, and at times, equivalent to the homologous control. Thus, the N1-I COBRA NA antigen has the potential to be a complimentary component in a multi-antigen universal influenza virus vaccine formulation that also contains HA antigens. Importance The development and distribution of a universal influenza vaccines would alleviate global economic and public health stress from annual influenza virus outbreaks. The influenza virus NA vaccine antigen allows for protection from multiple HA subtypes and virus host origins, but it has not been the focus of vaccine development. The N1-I NA antigen described here protected mice from direct challenge of four distinct influenza viruses and inhibited the enzymatic activity of a N1 influenza virus panel. The use of the NA antigen in combination with the HA widens the breadth of protection against various virus strains. Therefore, this research opens the door to the development of a longer lasting vaccine with increased protective breadth.

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Influenza reverse genetics: dissecting immunity and pathogenesis

Expert Reviews in Molecular Medicine ◽

10.1017/erm.2014.4 ◽

2014 ◽

Vol 16 ◽

Cited By ~ 7

Author(s):

Siying Ye ◽

Justin G. Evans ◽

John Stambas

Keyword(s):

Influenza Virus ◽

Protein Function ◽

Reverse Genetics ◽

Influenza Virus Vaccine ◽

Influenza Viruses ◽

Host Pathogen Interactions ◽

H5n1 Influenza ◽

Host Pathogen ◽

Cloned Cdna ◽

Novel Therapeutic Strategies

Reverse genetics systems allow artificial generation of non-segmented and segmented negative-sense RNA viruses, like influenza viruses, entirely from cloned cDNA. Since the introduction of reverse genetics systems over a decade ago, the ability to generate ‘designer’ influenza viruses in the laboratory has advanced both basic and applied research, providing a powerful tool to investigate and characterise host–pathogen interactions and advance the development of novel therapeutic strategies. The list of applications for reverse genetics has expanded vastly in recent years. In this review, we discuss the development and implications of this technique, including the recent controversy surrounding the generation of a transmissible H5N1 influenza virus. We will focus on research involving the identification of viral protein function, development of live-attenuated influenza virus vaccines, host–pathogen interactions, immunity and the generation of recombinant influenza virus vaccine vectors for the prevention and treatment of infectious diseases and cancer.

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Librator, a platform for optimized sequence editing, design, and expression of influenza virus proteins

10.1101/2021.04.29.441999 ◽

2021 ◽

Author(s):

Lei Li ◽

Olivia Stovicek ◽

Jenna J. Guthmiller ◽

Siriruk Changrob ◽

Yanbin Fu ◽

...

Keyword(s):

Influenza Virus ◽

Vaccine Development ◽

Protein Sequences ◽

Research Field ◽

Influenza Viruses ◽

Vaccine Antigen ◽

Universal Vaccine ◽

Influenza Virus Hemagglutinin ◽

Na Sequences ◽

Mosaic Protein

AbstractArtificial mutagenesis and chimeric/mosaic protein engineering have laid the foundation for antigenic characterization1 and universal vaccine design2–4 for influenza viruses. However, many methods used for influenza research and vaccine development require sequence editing and protein expression, limiting their applicability and the progress of related research to specialists. Rapid tools allowing even novice influenza researchers to properly analyze and visualize influenza protein sequences with accurate nomenclature are needed to expand the research field. To address this need, we developed Librator, a system for analyzing and designing protein sequences of influenza virus Hemagglutinin (HA) and Neuraminidase (NA). With Librator’s graphical user interface (GUI) and built-in sequence editing functions, biologists can easily analyze influenza sequences and phylogenies, automatically port sequences to visualize structures, then readily mutate target residues and design sequences for antigen probes and chimeric/mosaic proteins efficiently and accurately. This system provides optimized fragment design for Gibson Assembly5 of HA and NA expression constructs based on peptide conservation of all historical HA and NA sequences, ensuring fragments are reusable and compatible, allowing for significant reagent savings. Use of Librator will significantly facilitate influenza research and vaccine antigen design.

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Neuraminidase-Inhibiting Antibody Titers Correlate with Protection from Heterologous Influenza Virus Strains of the Same Neuraminidase Subtype

Journal of Virology ◽

10.1128/jvi.01006-18 ◽

2018 ◽

Vol 92 (17) ◽

Cited By ~ 10

Author(s):

Lisa Walz ◽

Sarah-Katharina Kays ◽

Gert Zimmer ◽

Veronika von Messling

Keyword(s):

Influenza Virus ◽

Immune Responses ◽

Vesicular Stomatitis Virus ◽

Influenza Viruses ◽

Antigenic Drift ◽

Influenza Vaccines ◽

Vaccine Antigen ◽

Antibody Titers ◽

Vesicular Stomatitis ◽

Virus Strains

ABSTRACTImmune responses induced by currently licensed inactivated influenza vaccines are mainly directed against the hemagglutinin (HA) glycoprotein, the immunodominant antigen of influenza viruses. The resulting antigenic drift of HA requires frequent updating of the vaccine composition and annual revaccination. On the other hand, the levels of antibodies directed against the neuraminidase (NA) glycoprotein, the second major influenza virus antigen, vary greatly. To investigate the potential of the more conserved NA protein for the induction of subtype-specific protection, vesicular stomatitis virus-based replicons expressing a panel of N1 proteins from prototypic seasonal and pandemic H1N1 strains and human H5N1 and H7N9 isolates were generated. Immunization of mice and ferrets with the replicon carrying the matched N1 protein resulted in robust humoral and cellular immune responses and protected against challenge with the homologous influenza virus with an efficacy similar to that of the matched HA protein, illustrating the potential of the NA protein as a vaccine antigen. The extent of protection after immunization with mismatched N1 proteins correlated with the level of cross-reactive neuraminidase-inhibiting antibody titers. Passive serum transfer experiments in mice confirmed that these functional antibodies determine subtype-specific cross-protection. Our findings illustrate the potential of NA-specific immunity for achieving broader protection against antigenic drift variants or newly emerging viruses carrying the same NA but a different HA subtype.IMPORTANCEDespite the availability of vaccines, annual influenza virus epidemics cause 250,000 to 500,000 deaths worldwide. Currently licensed inactivated vaccines, which are standardized for the amount of the hemagglutinin (HA) antigen, primarily induce strain-specific antibodies, whereas the immune response to the neuraminidase (NA) antigen, which is also present on the viral surface, is usually low. Using NA-expressing single-cycle vesicular stomatitis virus replicons, we show that the NA antigen conferred protection of mice and ferrets against not only the matched influenza virus strains but also viruses carrying NA proteins from other strains of the same subtype. The extent of protection correlated with the level of cross-reactive NA-inhibiting antibodies. This highlights the potential of the NA antigen for the development of more broadly protective influenza vaccines. Such vaccines may also provide partial protection against newly emerging strains with the same NA but a different HA subtype.

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Cross-Reactive Immunity to Clade 2 Strains of Influenza Virus A Subtype H5N1 Induced in Adults and Elderly Patients by Fluval, a Prototype Pandemic Influenza Virus Vaccine Derived by Reverse Genetics, Formulated with a Phosphate Adjuvant, and Directed to Clade 1 Strains

Clinical and Vaccine Immunology ◽

10.1128/cvi.00327-08 ◽

2008 ◽

Vol 16 (4) ◽

pp. 437-443 ◽

Cited By ~ 14

Author(s):

György Fazekas ◽

Rita Martosne-Mendi ◽

Istvan Jankovics ◽

Istvan Szilvasy ◽

Zoltan Vajo

Keyword(s):

Influenza Virus ◽

Virus Vaccine ◽

Reverse Genetics ◽

Evolutionary Relationship ◽

Influenza Virus Vaccine ◽

Influenza Viruses ◽

Antigenic Structure ◽

Elderly Subjects ◽

H5n1 Influenza ◽

Clade 1

ABSTRACT High fatality rates and multiple cases of transmission of avian H5N1 influenza viruses to humans illustrate the urgent need for an efficacious, cross-protective vaccine against H5N1 strains. Extensive genetic characterization of H5N1 strains has elucidated the natural evolutionary relationship of these strains, linking groups known as clades to a common ancestor. Although the clades and subclades probably differ sufficiently in their antigenic structure to warrant the preparation of different vaccines, there is some evidence that cross-reactive immunity can be afforded. We aimed to assess the immunogenicity of a clade 1 H5N1 (NIBRG-14) whole-virus vaccine with an aluminum phosphate adjuvant and to determine whether it can induce cross-reactive immunity against antigenically drifted clade 2 H5N1 strains, both those derived by reverse genetics and wild-type isolates. A total of 88 (44 adult and 44 elderly) subjects, who received one dose (6 μg) of the vaccine, were studied. As judged by U.S. and European licensing criteria based on hemagglutination inhibition, the subjects developed cross-reactive immunity against all studied H5N1 strains belonging to a clade different from that of the strain utilized to produce the vaccine. Our findings highlight the importance of stockpiling, since cross-immune reactions induced by prepandemic vaccines will likely reduce morbidity and mortality in case of a pandemic.

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The Next Generation of Influenza Vaccines: Towards a Universal Solution

Vaccines ◽

10.3390/vaccines9010026 ◽

2021 ◽

Vol 9 (1) ◽

pp. 26

Author(s):

Christopher L.D. McMillan ◽

Paul R. Young ◽

Daniel Watterson ◽

Keith J. Chappell

Keyword(s):

Influenza Virus ◽

Vaccine Development ◽

Influenza Virus Vaccine ◽

Influenza Viruses ◽

Effective Vaccine ◽

Next Generation ◽

Virus Infections ◽

Virus Surface ◽

Variable Head ◽

Hemagglutinin Protein

Influenza viruses remain a constant burden in humans, causing millions of infections and hundreds of thousands of deaths each year. Current influenza virus vaccine modalities primarily induce antibodies directed towards the highly variable head domain of the hemagglutinin protein on the virus surface. Such antibodies are often strain-specific, meaning limited cross-protection against divergent influenza viruses is induced, resulting in poor vaccine efficacy. To attempt to counteract this, yearly influenza vaccination with updated formulations containing antigens from more recently circulating viruses is required. This is an expensive and time-consuming exercise, and the constant arms race between host immunity and virus evolution presents an ongoing challenge for effective vaccine development. Furthermore, there exists the constant pandemic threat of highly pathogenic avian influenza viruses with high fatality rates (~30–50%) or the emergence of new, pathogenic reassortants. Current vaccines would likely offer little to no protection from such viruses in the event of an epidemic or pandemic. This highlights the urgent need for improved influenza virus vaccines capable of providing long-lasting, robust protection from both seasonal influenza virus infections as well as potential pandemic threats. In this narrative review, we examine the next generation of influenza virus vaccines for human use and the steps being taken to achieve universal protection.

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Contribution of Vaccine-Induced Immunity toward either the HA or the NA Component of Influenza Viruses Limits Secondary Bacterial Complications

Journal of Virology ◽

10.1128/jvi.02621-09 ◽

2010 ◽

Vol 84 (8) ◽

pp. 4105-4108 ◽

Cited By ~ 34

Author(s):

Victor C. Huber ◽

Ville Peltola ◽

Amy R. Iverson ◽

Jonathan A. McCullers

Keyword(s):

Influenza Virus ◽

Influenza Vaccine ◽

Bacterial Infection ◽

Virus Vaccine ◽

Bacterial Infections ◽

Influenza Virus Vaccine ◽

Influenza Viruses ◽

Influenza Vaccines ◽

Secondary Infections ◽

Induced Immunity

ABSTRACT Secondary bacterial infections contribute to morbidity and mortality from influenza. Vaccine effectiveness is typically assessed using prevention of influenza, not secondary infections, as an endpoint. We vaccinated mice with formalin-inactivated influenza virus vaccine preparations containing disparate HA and NA proteins and demonstrated an ability to induce the appropriate anti-HA and anti-NA immune profiles. Protection from both primary viral and secondary bacterial infection was demonstrated with vaccine-induced immunity directed toward either the HA or the NA. This finding suggests that immunity toward the NA component of the virion is desirable and should be considered in generation of influenza vaccines.

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Evaluation of Next-Generation H3 Influenza Vaccines in Ferrets Pre-Immune to Historical H3N2 Viruses

Frontiers in Immunology ◽

10.3389/fimmu.2021.707339 ◽

2021 ◽

Vol 12 ◽

Author(s):

James D. Allen ◽

Ted M. Ross

Keyword(s):

Influenza Virus ◽

Age Groups ◽

Influenza Virus Vaccine ◽

Influenza Viruses ◽

Antigenic Drift ◽

Influenza Vaccines ◽

Virus Infections ◽

Live Virus ◽

Vaccine Antigens ◽

H3n2 Influenza

Each person has a unique immune history to past influenza virus infections. Exposure to influenza viruses early in life establishes memory B cell populations that influence future immune responses to influenza vaccination. Current influenza vaccines elicit antibodies that are typically strain specific and do not offer broad protection against antigenically drifted influenza strains in all age groups of people. This is particularly true for vaccine antigens of the A(H3N2) influenza virus subtype, where continual antigenic drift necessitates frequent vaccine reformulation. Broadly-reactive influenza virus vaccine antigens offer a solution to combat antigenic drift, but they also need to be equally effective in all populations, regardless of prior influenza virus exposure history. This study examined the role that pre-existing immunity plays on influenza virus vaccination. Ferrets were infected with historical A(H3N2) influenza viruses isolated from either the 1970’s, 1980’s, or 1990’s and then vaccinated with computationally optimized broadly reactive antigens (COBRA) or wild-type (WT) influenza virus like particles (VLPs) expressing hemagglutinin (HA) vaccine antigens to examine the expansion of immune breadth. Vaccines with the H3 COBRA HA antigens had more cross-reactive antibodies following a single vaccination in all three pre-immune regimens than vaccines with WT H3 HA antigens against historical, contemporary, and future drifted A(H3N2) influenza viruses. The H3 COBRA HA vaccines also induced antibodies capable of neutralizing live virus infections against modern drifted A(H3N2) strains at higher titers than the WT H3 HA vaccine comparators.

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Hide and seek: interplay between influenza viruses and B cells

International Immunology ◽

10.1093/intimm/dxaa028 ◽

2020 ◽

Vol 32 (9) ◽

pp. 605-611 ◽

Cited By ~ 2

Author(s):

Masayuki Kuraoka ◽

Yu Adachi ◽

Yoshimasa Takahashi

Keyword(s):

B Cells ◽

B Cell ◽

Surface Protein ◽

Influenza Viruses ◽

Influenza Vaccines ◽

Native Structure ◽

Humoral Immune ◽

Protective Antibodies ◽

Major Surface ◽

And Function

Abstract Influenza virus constantly acquires genetic mutations/reassortment in the major surface protein, hemagglutinin (HA), resulting in the generation of strains with antigenic variations. There are, however, HA epitopes that are conserved across influenza viruses and are targeted by broadly protective antibodies. A goal for the next-generation influenza vaccines is to stimulate B-cell responses against such conserved epitopes in order to provide broad protection against divergent influenza viruses. Broadly protective B cells, however, are not easily activated by HA antigens with native structure, because the virus has multiple strategies to escape from the humoral immune responses directed to the conserved epitopes. One such strategy is to hide the conserved epitopes from the B-cell surveillance by steric hindrance. Technical advancement in the analysis of the human B-cell antigen receptor (BCR) repertoire has dissected the BCRs to HA epitopes that are hidden in the native structure but are targeted by broadly protective antibodies. We describe here the characterization and function of broadly protective antibodies and strategies that enable B cells to seek these hidden epitopes, with potential implications for the development of universal influenza vaccines.

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Host Factors Impact Vaccine Efficacy: Implications for Seasonal and Universal Influenza Vaccine Programs

Journal of Virology ◽

10.1128/jvi.00797-19 ◽

2019 ◽

Vol 93 (21) ◽

Cited By ~ 15

Author(s):

Santosh Dhakal ◽

Sabra L. Klein

Keyword(s):

Influenza Virus ◽

Influenza Vaccine ◽

Virus Vaccine ◽

Vaccine Efficacy ◽

Public Health Problem ◽

Influenza Virus Vaccine ◽

Host Factors ◽

Influenza Vaccines ◽

Vaccine Type ◽

Induced Immunity

ABSTRACT Influenza is a global public health problem. Current seasonal influenza vaccines have highly variable efficacy, and thus attempts to develop broadly protective universal influenza vaccines with durable protection are under way. While much attention is given to the virus-related factors contributing to inconsistent vaccine responses, host-associated factors are often neglected. Growing evidences suggest that host factors including age, biological sex, pregnancy, and immune history play important roles as modifiers of influenza virus vaccine efficacy. We hypothesize that host genetics, the hormonal milieu, and gut microbiota contribute to host-related differences in influenza virus vaccine efficacy. This review highlights the current insights and future perspectives into host-specific factors that impact influenza vaccine-induced immunity and protection. Consideration of the host factors that affect influenza vaccine-induced immunity might improve influenza vaccines by providing empirical evidence for optimizing or even personalizing vaccine type, dose, and use of adjuvants for current seasonal and future universal influenza vaccines.

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Glycine at Position 622 in PB1 Contributes to the Virulence of H5N1 Avian Influenza Virus in Mice

Journal of Virology ◽

10.1128/jvi.02387-15 ◽

2015 ◽

Vol 90 (4) ◽

pp. 1872-1879 ◽

Cited By ~ 12

Author(s):

Xiaoxiao Feng ◽

Zeng Wang ◽

Jianzhong Shi ◽

Guohua Deng ◽

Huihui Kong ◽

...

Keyword(s):

Avian Influenza ◽

Aspartic Acid ◽

H5n1 Virus ◽

Genetic Basis ◽

Vaccine Development ◽

Influenza Viruses ◽

Avian Influenza Viruses ◽

Live Attenuated Vaccine ◽

H5n1 Influenza ◽

H5n1 Avian Influenza

ABSTRACTWe isolated two H5N1 viruses, A/duck/Hunan/S4020/2008 (DK/08) and A/chicken/Guangxi/S2039/2009 (CK/09), from live-bird markets during routine surveillance and found that these two viruses are genetically similar but differ in their replication and virulence in mice. The CK/09 virus is lethal for mice with a 50% mouse lethal dose (MLD50) of 1.6 log1050% egg infectious doses (EID50), whereas the DK/08 virus is nonpathogenic for mice with an MLD50value of 6.2 log10EID50. We explored the genetic basis of the virulence difference of these two viruses by generating a series of reassortant viruses and mutants in the lethal virus CK/09 background and evaluating their virulence in mice. We found that the PB1 gene of the DK/08 virus dramatically attenuated the virulence of the CK/09 virus and that the amino acid at position 622 in PB1 made an important contribution. We further demonstrated that the mutation of glycine (G) to aspartic acid (D) at position 622 in PB1 partially impaired the binding of PB1 to viral RNA, thereby dramatically decreasing the polymerase activity and attenuating H5N1 virus virulence in mice. Our results identify a novel virulence-related marker of H5N1 influenza viruses and provide a new target for live attenuated vaccine development.IMPORTANCEH5N1 avian influenza viruses have caused the deaths of nearly 60% of the humans that they have infected since 1997 and clearly represent a threat to public health. A thorough understanding of the genetic basis of virulence determinants will provide important insights for antiviral drug and live attenuated vaccine development. Several virulence-related markers in the PB2, PA, M1, and NS1 proteins of H5N1 viruses have been identified. In this study, we isolated two H5N1 avian influenza viruses that are genetically similar but differ in their virulence in mice, and we identified a new virulence-related marker in the PB1 gene. We found that the mutation of glycine (G) to aspartic acid (D) at position 622 in PB1 partially impairs the binding of PB1 to viral RNA, thereby attenuating H5N1 virus virulence in mice. This newly identified virulence-related marker could be applied to the development of live attenuated vaccines against H5N1 influenza.

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