Prospects of using conservative linear B-cell epitopes of influenza virus A neuraminidase for induction of cross-protective immune response

BACKGROUND: Influenza is a dangerous, widespread infectious disease that takes thousands of lives during annual epidemics, and also causes significant damage to the countrys economy. The most effective means of fighting the influenza virus is vaccination of the population. Due to the variability of influenza viruses, the strain composition of influenza vaccines must be updated annually. In this regard, an urgent task is to improve the existing influenza vaccines in order to expand their spectrum of action. One of the promising approaches is the targeted induction of the humoral immune response to the conservative linear epitopes of influenza A virus neuraminidase. AIM: This project is aimed at assessing the immunogenicity and cross-protective activity of conserved neuraminidase epitopes in order to select promising targets for the targeted design of broad-spectrum influenza vaccines. MATERIALS AND METHODS: Peptides corresponding to linear B-cell epitopes of neuraminidase were chemically synthesized de novo. The peptides were conjugated with keyhole limpet hemocyanin. CBA mice were immunized and challenged with A/PR/8/34 (H1N1) and A/Philippines/2/1982 (H3N2) viruses at a dose of 3 LD50. The survival rate of the animals was assessed within 14 days after infection. The immunogenicity of the peptides was assessed in a standard enzyme-linked immunosorbent assay using the recombinant neuraminidase proteins of the viruses A/California/07/2009 (H1N1) and A/Hong Kong/4801/2014 (H3N2) as antigen. RESULTS: Immunization of neuraminidase with peptides MNPNQKIITIGS and ILRTQESEC, but not DNWKGSNRP, protected mice from lethality caused by the H1N1 and/or H3N2 virus. The protective potential of the peptides correlated with the levels of antineuraminidase antibodies after immunization. CONCLUSIONS: The presence of a cross-protective potential in two conserved linear B-cell epitopes of influenza A neuraminidase (MNPNQKIITIGS and ILRTQESEC) allows them to be recommended as a target for the development of a broad-spectrum influenza vaccine.

Download Full-text

INDUCTION OF CROSS-REACTIVE ANTIBODIES IN MICE IMMUNIZED WITH CONSERVED LINEAR B-CELL EPITOPES DERIVED FROM INFLUENZA A VIRUS NEURAMINIDASE

Russian Journal of Infection and Immunity ◽

10.15789/10.15789/2220-7619-ioc-1343 ◽

2019 ◽

Author(s):

I Sychev ◽

P. Kopeikin ◽

E. Tsvetkova ◽

K. Cheredova ◽

B. Milman ◽

...

Keyword(s):

Weight Loss ◽

Influenza Virus ◽

B Cell ◽

Influenza A Virus ◽

Influenza A ◽

Protective Efficacy ◽

Influenza Viruses ◽

Influenza Vaccines ◽

B Cell Epitopes ◽

Linear B

Introduction. Influenza is a socially considerable infection annually causing profound damage to the populational health and economy. Vaccination is the most effective way to manage influenza and its complications. There are various vaccines against influenza, but their common drawback is the narrow specificity, need for annual virus strain renewal, not always good immunogenicity and effectiveness. In this regard, a close attention is paid to developing universal influenza vaccines aimed to induce cross-reactive factors of the immune response to the most conserved parts of viral proteins. Antibodies against neuraminidase (NA) are able to provide heterosubtypic protection, which is important due to potential threat from influenza viruses, with differed hemagglutinin and neuraminidase compared to the currently circulating viruses. The present study is aimed to search for new and analyze previously predicted linear NA B-cell epitopes, conserved among all subtypes of influenza A virus.Results. there were found out eight conserved linear B-cell epitopes were located around the neuraminidase active site, three of which (MNPNQKIITIGS, ILRTQESEC, and DNWKGSNRP) were synthesized de novo, conjugated with bovine serum albumin to be further used for mouse immunization. Serum IgG antibodies were detected by ELISA in immunized mice. Antibodies specifically bind to various influenza A viruses containing NA subtypes N1, N2, N3, and N9. Immunization with NA peptides provided no protection from profound weight loss after infection with lethal H1N1 influenza virus. However, all immunized mice survived during the observation period, while in control group survival rate was as low as 28.6%. Assessing the viral load in the lungs of mice infected with the H1N1 virus did not reveal differences in titers either on day 4 or 8 post-infection. Nevertheless, the protective effect lacked upon challenge with lethal H7N9 influenza virus: mortality, weight loss, and lung virus titers were comparable both in immunized and control mice.Conclusions. The data obtained uncovered cross-reactivity in anti-NA antibodies induced by immunization with NA peptides as well as protective efficacy against infection caused by the H1N1, but not H7N9 virus. Thus, these data are promising and indicate that linear B-cell NA epitopes can be used for design of epitope-directed influenza vaccines, but a deeper and full examination of specificity for conserved NA epitopes as well as optimized immunization schemes are necessary to achieve higher protective efficacy.

Download Full-text

Influenza A Virus Vaccination: Immunity, Protection, and Recent Advances Toward A Universal Vaccine

Vaccines ◽

10.3390/vaccines8030434 ◽

2020 ◽

Vol 8 (3) ◽

pp. 434 ◽

Cited By ~ 2

Author(s):

Christopher E. Lopez ◽

Kevin L. Legge

Keyword(s):

Immune Response ◽

Influenza Virus ◽

Virus Infection ◽

Influenza A ◽

Influenza Virus Infection ◽

Influenza Viruses ◽

Antigenic Drift ◽

Influenza Vaccines ◽

Virus Infections ◽

Universal Vaccine

Influenza virus infections represent a serious public health threat and account for significant morbidity and mortality worldwide due to seasonal epidemics and periodic pandemics. Despite being an important countermeasure to combat influenza virus and being highly efficacious when matched to circulating influenza viruses, current preventative strategies of vaccination against influenza virus often provide incomplete protection due the continuous antigenic drift/shift of circulating strains of influenza virus. Prevention and control of influenza virus infection with vaccines is dependent on the host immune response induced by vaccination and the various vaccine platforms induce different components of the local and systemic immune response. This review focuses on the immune basis of current (inactivated influenza vaccines (IIV) and live attenuated influenza vaccines (LAIV)) as well as novel vaccine platforms against influenza virus. Particular emphasis will be placed on how each platform induces cross-protection against heterologous influenza viruses, as well as how this immunity compares to and contrasts from the “gold standard” of immunity generated by natural influenza virus infection.

Download Full-text

Neuraminidase gene homology contributes to the protective activity of influenza vaccines prepared from the influenza virus library

Journal of General Virology ◽

10.1099/vir.0.067488-0 ◽

2014 ◽

Vol 95 (11) ◽

pp. 2365-2371 ◽

Cited By ~ 1

Author(s):

Ahmad M. Haredy ◽

Hiroshi Yamada ◽

Yoshihiro Sakoda ◽

Masatoshi Okamatsu ◽

Naoki Yamamoto ◽

...

Keyword(s):

Influenza Virus ◽

Influenza A ◽

Survival Rates ◽

Influenza Vaccines ◽

H3n2 Virus ◽

Protective Activity ◽

Virus Clearance ◽

Ha Gene ◽

Gene Homology ◽

Antibody Titres

Whole-virus (WV) vaccines from influenza A/duck/Hokkaido/77 (H3N2), and its reassortant strains H3N4, H3N5 and H3N7, which have the same haemagglutinin (HA) gene but different neuraminidase (NA) genes, were prepared from our influenza virus library. Mice were intranasally immunized with equivalent doses of each vaccine (1–0.01 µg per mouse). All of the mice that received the highest dose of each vaccine (1 µg per mouse) showed equivalent high HA-inhibiting (HI) antibody titres and survived the H3N2 challenge viruses. However, mice that received lower doses of vaccine (0.1 or 0.01 µg per mouse) containing a heterologous NA had lower survival rates than those given the H3N2-based vaccine. The lungs of mice challenged with H3N2 virus showed a significantly higher virus clearance rate when the vaccine contained the homologous NA (N2) versus a heterologous NA, suggesting that NA contributed to the protection, especially when the HI antibody level was low. These results suggested that, even if vaccines prepared for a possible upcoming pandemic do not induce sufficient HI antibodies, WV vaccines can still be effective through other matched proteins such as NA.

Download Full-text

The Analysis of B-Cell Epitopes of Influenza Virus Hemagglutinin

Acta Naturae ◽

10.32607/20758251-2016-8-1-13-20 ◽

2016 ◽

Vol 8 (1) ◽

pp. 13-20 ◽

Cited By ~ 4

Author(s):

D. N. Shcherbinin ◽

S. V. Alekseeva ◽

M. M. Shmarov ◽

Yu. A. Smirnov ◽

B. S. Naroditskiy ◽

...

Keyword(s):

Influenza Virus ◽

B Cell ◽

Broad Spectrum ◽

Neutralizing Antibodies ◽

Passive Immunization ◽

B Cell Epitopes ◽

Humoral Immune ◽

Influenza Virus Hemagglutinin ◽

Long Time ◽

Single Domain Antibodies

Vaccination has been successfully used to prevent influenza for a long time. Influenza virus hemagglutinin (HA), which induces a humoral immune response in humans and protection against the flu, is the main antigenic component of modern influenza vaccines. However, new seasonal and pandemic influenza virus variants with altered structures of HA occasionally occur. This allows the pathogen to avoid neutralization with antibodies produced in response to previous vaccination. Development of a vaccine with the new variants of HA acting as antigens takes a long time. Therefore, during an epidemic, it is important to have passive immunization agents to prevent and treat influenza, which can be monoclonal or single-domain antibodies with universal specificity (broad-spectrum agents). We considered antibodies to conserved epitopes of influenza virus antigens as universal ones. In this paper, we tried to characterize the main B-cell epitopes of hemagglutinin and analyze our own and literature data on broadly neutralizing antibodies. We conducted a computer analysis of the best known conformational epitopes of influenza virus HAs using materials of different databases. The analysis showed that the core of the HA molecule, whose antibodies demonstrate pronounced heterosubtypic activity, can be used as a target for the search for and development of broad-spectrum antibodies to the influenza virus.

Download Full-text

Intranasal immunization with a recombinant protein based on the M2e peptide and second subunit of influenza A viral hemagglutinin fragment induces a cross-protective humoral and Tcell response in mice

Medical Immunology (Russia) ◽

10.15789/1563-0625-iiw-1584 ◽

2020 ◽

Vol 22 (2) ◽

pp. 357-370

Author(s):

M. A Shuklina ◽

L. A Stepanova ◽

A. A. Kovaleva ◽

A. V. Korotkov ◽

A. A. Shaldzhyan ◽

...

Keyword(s):

Immune Response ◽

T Cells ◽

Influenza Virus ◽

T Cell ◽

Recombinant Protein ◽

Broad Spectrum ◽

Influenza A ◽

Phylogenetic Group ◽

Carrier Protein ◽

Influenza Virus A

Development of vaccines with a broad-spectrum of protection is one of the priorities in the programs of influenza prevention. Recently, the conserved fragments of influenza virus proteins (M1, M2, NP, the second subunit of the hemagglutinin HA2) provoke interest of investigators as the object of the development a broad-spectrum vaccines. Low immunogenicity present a problem when developing vaccines based on such conserved fragments. However, fusion of low immunogenic antigens into the high immunogenic carrier protein may significantly enhance their immunogenicity. The candidate vaccine protein Flg-HA2-2-4M2e was developed which containins two highly conserved viral antigens (the ectodomain of the M2 protein (M2e), 76130 region of the second subunit of HA2), fused with flagellin as a carrier protein. Flagellin (bacterial flagella protein) is a natural ligand of TLR-5, and has a strong adjuvant activity at different ways of its administration. The purpose of this study was to assess development of humoral and T cell immune response, along with broad-spectrum protection after mice immunization with the candidate Flg-HA2-2-4M2e vaccine protein. Mice were immunized intranasally three times with two-week intervals. Two weeks after the final immunization, the mice were challenged at the 5 LD50 dose with influenza viruses A/California/07/09 (H1N1) pdm09 (phylogenetic group I), or A/Shanghai/2/2013 (H7N9) (phylogenetic group II). The results obtained in this study showed induction of strong M2e-specific humoral response (serum IgG and A) in the immunized mice. Immunization with recombinant protein stimulated formation of M2e-specific and virus-specific CD4+ and CD8+T cells in lung which produced TNFα or IFNγ. Production of antigen-specific effector and central memory T cells was also detected in lungs of immunized mice. The formation of cross-protective immunity in immunized mice was demonstrated in a model of lethal influenza infection. The experimental animals were almost completely protected from the high dose of the pandemic virus A/H1N1pdm09, and highly pathogenic avian influenza A/H7N9 (90-100% survival). We also evaluated the changes of antigen-specific immune response in immunized mice after sublethal infection with A/H3N2 influenza virus. Mice of control and experimental groups were infected with MID100 of influenza virus A/Aichi/2/68 (H3N2). It was shown that the M2e-specific response (IgG, IgA) was significantly increased in immunized mice after sublethal infection with influenza virus A/H3N2, and we detected the changes in profile of M2e-specific IgG subclasses. Following sublethal infection in immunized mice, the proportion of M2e-specific IgG2a was increased 10-fold. The results showed that the recombinant protein Flg-HA2-2-4M2e is a promising candidate for development of universal vaccines, which induces a protective humoral and T-cell response to conserved viral epitopes and protects against influenza A viruses of both phylogenetic groups.

Download Full-text

Detailed mapping of the linear B Cell epitopes of the hemagglutinin (HA) protein of swine influenza virus

Virology ◽

10.1016/j.virol.2018.07.013 ◽

2018 ◽

Vol 522 ◽

pp. 131-137 ◽

Cited By ~ 6

Author(s):

Zhao Wang ◽

Bing Huang ◽

Milton Thomas ◽

Chithra C. Sreenivasan ◽

Zizhang Sheng ◽

...

Keyword(s):

Influenza Virus ◽

B Cell ◽

Swine Influenza Virus ◽

Swine Influenza ◽

B Cell Epitopes ◽

Ha Protein ◽

Linear B

Download Full-text

Respiratory and systemic humoral and cellular immune responses of pigs to a heterosubtypic influenza A virus infection

Journal of General Virology ◽

10.1099/0022-1317-82-11-2697 ◽

2001 ◽

Vol 82 (11) ◽

pp. 2697-2707 ◽

Cited By ~ 78

Author(s):

Paul P. Heinen ◽

Els A. de Boer-Luijtze ◽

Andre T. J. Bianchi

Keyword(s):

T Cells ◽

Influenza Virus ◽

Virus Infection ◽

Broad Spectrum ◽

Influenza A ◽

Virus Transmission ◽

Influenza Virus Infection ◽

H3n2 Virus ◽

Control Group ◽

Immune Control

The level of heterosubtypic immunity (Het-I) and the immune mechanisms stimulated by a heterosubtypic influenza virus infection were investigated in pigs. Pigs are natural hosts for influenza virus and, like humans, they host both subtypes H1N1 and H3N2. Marked Het-I was observed when pigs were infected with H1N1 and subsequently challenged with H3N2. After challenge with H3N2, pigs infected earlier with H1N1 did not develop fever and showed reduced virus excretion compared with non-immune control pigs. In addition, virus transmission to unchallenged group-mates could be shown by virus isolation in the non-immune control group but not in the group infected previously with H1N1. Pigs infected previously with homologous H3N2 virus were protected completely. After challenge with H3N2, pigs infected previously with H1N1 showed a considerable increase in serum IgG titre to the conserved extracellular domain of M2 but not to the conserved nucleoprotein. These results suggest that antibodies against external conserved epitopes can have an important role in broad-spectrum immunity. After primary infection with both H1N1 and H3N2, a long-lived increase was observed in the percentage of CD8+ T cells in the lungs and in the lymphoproliferation response in the blood. Upon challenge with H3N2, pigs infected previously with H1N1 again showed an increase in the percentage of CD8+ T cells in the lungs, whereas pigs infected previously with H3N2 did not, suggesting that CD8+ T cells also have a role in Het-I. To confer broad-spectrum immunity, future vaccines should induce antibodies and CD8+ T cells against conserved antigens.

Download Full-text

Generation of DelNS1 Influenza Viruses: a Strategy for Optimizing Live Attenuated Influenza Vaccines

mBio ◽

10.1128/mbio.02180-19 ◽

2019 ◽

Vol 10 (5) ◽

Cited By ~ 4

Author(s):

Pui Wang ◽

Min Zheng ◽

Siu-Ying Lau ◽

Pin Chen ◽

Bobo Wing-Yee Mok ◽

...

Keyword(s):

Immune Response ◽

Influenza Virus ◽

T Cell ◽

Protective Immunity ◽

Influenza A ◽

Influenza Viruses ◽

Cross Protection ◽

Influenza Vaccines ◽

Influenza B ◽

Ns1 Protein

ABSTRACT Nonstructural protein 1 (NS1) of influenza virus is a key virulence element with multifunctional roles in virus replication and a potent antagonist of host immune response. Deletion of NS1 (DelNS1) would create a safer and more extensively immunogenic live attenuated influenza virus (LAIV) vaccine. However, DelNS1 viruses are very difficult to grow in regular vaccine-producing systems, which has hampered the application of DelNS1 LAIV vaccines in humans. We have developed two master backbones of deleted-NS1 (DelNS1) viral genomes from influenza A or B viruses which contain novel adaptive mutations to support DelNS1-LAIV replication. These DelNS1-LAIVs are highly attenuated in human cells in vitro and nonpathogenic in mice but replicate well in vaccine-producing cells. Both influenza A and influenza B DelNS1 LAIVs grow better at 33°C than at 37 to 39°C. Vaccination with DelNS1 LAIV performed once is enough to provide potent protection against lethal challenge with homologous virus and strong long-lasting cross protection against heterosubtypic or antigenically distantly related influenza viruses in mice. Mechanistic investigations revealed that DelNS1-LAIVs induce cross protective neutralizing antibody and CD8+ and CD4+ T cell immunities. Importantly, it has been shown that DelNS1-LAIV can be used to enhance specific anti-influenza immunity through expression of additional antigens from the deleted-NS1 site. Generation of DelNS1 viruses which are nonpathogenic and able to grow in vaccine-producing systems is an important strategy for making highly immunogenic LAIV vaccines that induce broad cross protective immunity against seasonal and emerging influenza. IMPORTANCE Current seasonal influenza vaccines are suboptimal and low in immunogenicity and do not provide long-lasting immunity and cross protection against influenza virus strains that have antigenically drifted. More-effective influenza vaccines which can induce both humoral immunity and T cell immunity are needed. The NS1 protein of influenza virus is a virulence element and the critical factor for regulation of the host immune response during virus infection. Deletion of the NS1 protein is a strategy to make an optimal LAIV vaccine. However, DelNS1 viruses are very difficult to grow in regular vaccine-producing systems, hampering the application of DelNS1 LAIV vaccines in humans. We have generated a panel of both influenza A and influenza B DelNS1 LAIVs which are able to grow in regular vaccine-producing cells. These DelNS1 LAIV vaccines are completely nonpathogenic, exhibit potent and long-lasting immunity, and can be used to express extra viral antigen to induce cross protective immunity against seasonal and emerging influenza.

Download Full-text

Diversifying Selection Analysis Predicts Antigenic Evolution of 2009 Pandemic H1N1 Influenza A Virus in Humans

Journal of Virology ◽

10.1128/jvi.03636-14 ◽

2015 ◽

Vol 89 (10) ◽

pp. 5427-5440 ◽

Cited By ~ 13

Author(s):

Alexandra J. Lee ◽

Suman R. Das ◽

Wei Wang ◽

Theresa Fitzgerald ◽

Brett E. Pickett ◽

...

Keyword(s):

Immune Response ◽

Influenza Virus ◽

Amino Acid ◽

B Cell ◽

Influenza A Virus ◽

Protective Immunity ◽

Influenza A ◽

Acid Sites ◽

Diversifying Selection ◽

Ha Protein

ABSTRACTAlthough a large number of immune epitopes have been identified in the influenza A virus (IAV) hemagglutinin (HA) protein using various experimental systems, it is unclear which are involved in protective immunity to natural infection in humans. We developed a data mining approach analyzing natural H1N1 human isolates to identify HA protein regions that may be targeted by the human immune system and can predict the evolution of IAV. We identified 16 amino acid sites experiencing diversifying selection during the evolution of prepandemic seasonal H1N1 strains and found that 11 sites were located in experimentally determined B-cell/antibody (Ab) epitopes, including three distinct neutralizing Caton epitopes: Sa, Sb, and Ca2 [A. J. Caton, G. G. Brownlee, J. W. Yewdell, and W. Gerhard, Cell 31:417–427, 1982,http://dx.doi.org/10.1016/0092-8674(82)90135-0]. We predicted that these diversified epitope regions would be the targets of mutation as the 2009 H1N1 pandemic (pH1N1) lineage evolves in response to the development of population-level protective immunity in humans. Using a chi-squared goodness-of-fit test, we identified 10 amino acid sites that significantly differed between the pH1N1 isolates and isolates from the recent 2012-2013 and 2013-2014 influenza seasons. Three of these sites were located in the same diversified B-cell/Ab epitope regions as identified in the analysis of prepandemic sequences, including Sa and Sb. As predicted, hemagglutination inhibition (HI) assays using human sera from subjects vaccinated with the initial pH1N1 isolate demonstrated reduced reactivity against 2013-2014 isolates. Taken together, these results suggest that diversifying selection analysis can identify key immune epitopes responsible for protective immunity to influenza virus in humans and thereby predict virus evolution.IMPORTANCEThe WHO estimates that approximately 5 to 10% of adults and 20 to 30% of children in the world are infected by influenza virus each year. While an adaptive immune response helps eliminate the virus following acute infection, the virus rapidly evolves to evade the established protective memory immune response, thus allowing for the regular seasonal cycles of influenza virus infection. The analytical approach described here, which combines an analysis of diversifying selection with an integration of immune epitope data, has allowed us to identify antigenic regions that contribute to protective immunity and are therefore the key targets of immune evasion by the virus. This information can be used to determine when sequence variations in seasonal influenza virus strains have affected regions responsible for protective immunity in order to decide when new vaccine formulations are warranted.

Download Full-text

Evolution in the influenza A H3 stalk – a challenge for broad-spectrum vaccines?

Journal of General Virology ◽

10.1099/vir.0.059410-0 ◽

2014 ◽

Vol 95 (2) ◽

pp. 317-324 ◽

Cited By ~ 8

Author(s):

William D. Lees ◽

David S. Moss ◽

Adrian J. Shepherd

Keyword(s):

B Cell ◽

Broad Spectrum ◽

Influenza A ◽

Vaccine Development ◽

Active Regions ◽

B Cell Epitopes ◽

Human Antibodies ◽

Novel Approach ◽

The Face ◽

Effective Life

Recently, a number of broad-spectrum human antibodies binding to the stalk region of influenza A haemagglutinin (HA) have been isolated. As this region tends to develop substitutions at a slower rate than other regions of HA, a vaccine eliciting such antibodies could have a longer effective life. But this begs a question: is the stalk resistant to change even in the face of evolutionary pressure? In this paper, we analysed the known epitopes in the H3 stalk and, utilizing a collection of 3440 sequences, present a novel approach for detecting putative B-cell epitopes in regions such as this, in which mutations occur infrequently. We concluded that there have been periods of activity in the stalk that are consistent with the evolution of antigenic escape. This work casts light on the presence of stalk-binding antibodies in the population as a whole and, through the analysis of antigenically active regions in the stalk, may contribute to the identification of epitopes that are refractive to change and hence useful for vaccine development.

Download Full-text