Influenza chimeric hemagglutinin structures in complex with broadly protective antibodies to the stem and trimer interface.

Influenza virus hemagglutinin (HA) has been the primary target for influenza vaccine development. Broadly protective antibodies targeting conserved regions of the HA unlock the possibility of generating universal influenza immunity. Two group 2 influenza A chimeric HAs, cH4/3 and cH15/3, were previously designed to elicit antibodies to the conserved HA stem. Here, we show by X-ray crystallography and negative-stain electron microscopy that a broadly protective antistem antibody can stably bind to cH4/3 and cH15/3 HAs, thereby validating their potential as universal vaccine immunogens. Furthermore, flexibility was observed in the head domain of the chimeric HA structures, suggesting that antibodies could also potentially interact with the head interface epitope. Our structural and binding studies demonstrated that a broadly protective antihead trimeric interface antibody could indeed target the more open head domain of the cH15/3 HA trimer. Thus, in addition to inducing broadly protective antibodies against the conserved HA stem, chimeric HAs may also be able to elicit antibodies against the conserved trimer interface in the HA head domain, thereby increasing the vaccine efficacy.

A transgenic mouse with a humanised B cell repertoire mounts an antibody response to influenza infection and vaccination.

The development of a universal influenza vaccine likely requires an understanding of previous exposure to influenza virus (through vaccination or infection) and how that shapes the antibody repertoire to vaccination, sometimes called Original Antigenic Sin or antigenic imprinting. Whilst animal models can have a much more defined exposure history, they lack a human B cell repertoire. Transgenic mice with the complete human immunoglobulin locus enable studies of controlled infection history leading to human-like antibody evolution. Here we evaluated responses to influenza in the Intelliselect Transgenic mouse (the Kymouse). We show the Kymouse is susceptible to disease following infection with either H1N1, H3N2 or B/Yam influenza viruses and that it induces a robust binding and neutralising antibody response to all three strains of influenza virus. This study demonstrates that human B cell repertoire mice can be used for influenza virus studies, providing a tool for further interrogation of the antibody response.

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

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

Reduction of Influenza A Virus Transmission in Mice by a Universal Intranasal Vaccine Candidate is Long-Lasting and Does Not Require Antibodies.

Vaccination against influenza virus infection can protect the vaccinee and also reduce transmission to contacts. Not all types of vaccines induce sterilizing immunity via neutralizing antibodies; some instead permit low-level, transient infection. There has been concern that infection-permissive influenza vaccines may allow continued spread in the community despite minimizing symptoms in the vaccinee. We have explored that issue for a universal influenza vaccine candidate that protects recipients by inducing T cell responses and nonneutralizing antibodies. Using a mouse model, we have shown previously that an adenoviral vectored vaccine expressing nucleoprotein (NP) and matrix 2 (M2) provides broad protection against diverse strains and subtypes of influenza A viruses and reduces transmission to contacts in an antigen-specific manner. Here, we use this mouse model to further explore the mechanism and features of that reduction in transmission. Passive immunization did not reduce transmission from infected donors to naive contact animals to whom passive serum had been transferred. Vaccination of antibody-deficient mIgTg-JHD-/- mice, which have intact T cell responses and antigen presentation, reduced transmission in an antigen-specific manner, despite the presence of some virus in the lungs and nasal wash, pointing to a role for cellular immunity. Vaccination at ages ranging from 8 to 60 weeks was able to achieve reduction in transmission. Finally, the immune-mediated reduction in transmission persisted for at least a year after a single-dose intranasal vaccination. Thus, this infection-permissive vaccine reduces virus transmission in a long-lasting manner that does not require antibodies. IMPORTANCE Universal influenza virus vaccines targeting antigens conserved among influenza A virus strains can protect from severe disease but do not necessarily prevent infection. Despite allowing low-level infection, intranasal immunization with adenovirus vectors expressing the conserved antigens influenza nucleoprotein (A/NP) and M2 reduces influenza virus transmission from vaccinated to unvaccinated contact mice. Here, we show that antibodies are not required for this transmission reduction, suggesting a role for T cells. We also show that transmission blocking could be achieved in recipients of different ages and remained effective for at least a year following a single-dose vaccination. Such vaccines could have major public health impacts by limiting viral transmission in the community.

A single-shot adenoviral vaccine provides hemagglutinin stalk-mediated protection against heterosubtypic influenza challenge in mice.

Conventional influenza vaccines fail to confer broad protection against diverse influenza A viruses with pandemic potential. Efforts to develop a universal influenza virus vaccine include refocusing immunity towards the highly conserved stalk domain of the influenza virus surface glycoprotein, hemagglutinin (HA). We constructed a non-replicating adenoviral (Ad) vector, encoding a secreted form of H1 HA, to evaluate HA stalk-focused immunity. The Ad5_H1 vaccine was tested in mice for its ability to elicit broad, cross-reactive protection against homologous, heterologous, and heterosubtypic lethal challenge in a single-shot immunization regimen. Ad5_H1 elicited hemagglutination inhibition (HI+) active antibodies (Abs), which conferred 100% sterilizing protection from homologous H1N1 challenge. Furthermore, Ad5_H1 rapidly induced H1-stalk-specific Abs with Fc-mediated effector function activity, in addition to stimulating both CD4+ and CD8+ stalk-specific T cell responses. This phenotype of immunity provided 100% protection from lethal challenge with a head-mismatched, reassortant influenza virus bearing a chimeric HA, cH6/1, in a stalk-mediated manner. Most importantly, 100% protection from mortality following lethal challenge with a heterosubtypic avian influenza virus, H5N1, was observed following a single immunization with Ad5_H1. In conclusion, Ad-based influenza vaccines can elicit significant breadth of protection in naive animals and could be considered for pandemic preparedness and stockpiling.

Is a Universal Influenza Virus Vaccine Possible?

Influenza viruses remain a severe burden to human health because of their contribution to overall morbidity and mortality. Current seasonal influenza virus vaccines do not provide sufficient protection to alleviate the annual impact of influenza and cannot confer protection against potentially pandemic influenza viruses. The lack of protection is due to rapid changes of the viral epitopes targeted by the vaccine and the often suboptimal immunogenicity of current immunization strategies. Major efforts to improve vaccination approaches are under way. The development of a universal influenza virus vaccine may be possible by combining the lessons learned from redirecting the immune response toward conserved viral epitopes, as well as the use of adjuvants and novel vaccination platforms.

Message in a bottle: mRNA vaccination for influenza.

Current influenza vaccines, while being the best method of managing viral outbreaks, have several major drawbacks that prevent them from being wholly-effective. They need to be updated regularly and require extensive resources to develop. When considering alternatives, the recent deployment of mRNA vaccines for SARS-CoV-2 has created a unique opportunity to evaluate a new platform for seasonal and pandemic influenza vaccines. The mRNA format has previously been examined for application to influenza and promising data suggest it may be a viable format for next-generation influenza vaccines. Here, we discuss the prospect of shifting global influenza vaccination efforts to an mRNA-based system that might allow better control over the product and immune responses and could aid in the development of a universal vaccine.

Evolutionary conservation and positive selection of influenza A nucleoprotein CTL epitopes for universal vaccination.

Influenza (flu) infection is a leading cause of respiratory diseases and death worldwide. Although seasonal flu vaccines are effective at reducing morbidity and mortality, such effects rely on the odds of successful prediction of the upcoming viral strains. Additional threats from emerging flu viruses that we cannot predict and avian flu viruses that can be directly transmitted to humans urge the strategic development of universal vaccination that can protect against flu viruses of different subtypes and across species. Annual flu vaccines elicit mainly humoral responses. Under circumstances when antibodies induced by vaccination fail to recognize and neutralize the emerging virus adequately, virus-specific cytotoxic T lymphocytes (CTLs) are the major contributors to the control of viral replication and elimination of infected cells. Our studies exploited the evolutionary conservation of influenza A nucleoprotein (NP) and the fact that NP-specific CTL responses pose a constant selecting pressure on functional CTL epitopes to screen for NP epitopes that are highly conserved among heterosubtypes but are subjected to positive selection historically. We identified a region on NP that is evolutionarily conserved and historically positively selected (NP137-182 ) and validated that it contains an epitope that is functional in eliciting NP-specific CTL responses and immunity that can partially protect immunized mice against lethal dose infection of a heterosubtypic influenza A virus. Our proof-of-concept study supports the hypothesis that evolutionary conservation and positive selection of influenza NP can be exploited to identify functional CTL epitope to elicit cross-protection against different heterosubtypes, therefore, to help develop strategies to modify flu vaccine formula for a broader and more durable protective immunity.

Salmonella enterica serovar Choleraesuis vector delivering a dual-antigen expression cassette provides mouse cross-protection against Streptococcus suis serotypes 2, 7, 9, and 1/2.

A universal vaccine protecting against multiple serotypes of Streptococcus suis is urgently needed to improve animal welfare and reduce the consumption of antibiotics. In this study, a dual antigen expression cassette consisting of SS2-SaoA and SS9-Eno was delivered by a recombinant Salmonella Choleraesuis vector to form the vaccine candidate rSC0016(pS-SE). SaoA and Eno were simultaneously synthesized in rSC0016(pS-SE) without affecting the colonization of the recombinant vector in the lymphatic system. In addition, the antiserum of mice immunized with rSC0016(pS-SE) produced a broader and potent opsonophagocytic response against multiple serotypes of S. suis. Finally, rSC0016(pS-SE) provided mice with a 100% protection against a lethal dose of parent S. suis serotype 2 and serotype 9, and provided 90% and 80% protection against heterologous S. suis serotype 7 or 1/2. These values were significantly higher than those obtained with rSC0016(pS-SaoA) or rSC0016(pS-Eno). Together, this study serves as a foundation for developing a universal vaccine against multiple serotypes of S. suis.

Designing AbhiSCoVac - A single potential vaccine for all 'corona culprits': Immunoinformatics and immune simulation approaches.

The coronaviridae family has generated highly virulent viruses, including the ones responsible for three major pandemics in last two decades with SARS in 2002, MERS outbreak in 2012 and the current nCOVID19 crisis that has turned the world breadthless. Future outbreaks are also a plausible threat to mankind. As computational biologists, we are committed to address the need for a universal vaccine that can deter all these pathogenic viruses in a single shot. Notably, the spike proteins present in all these viruses function as credible PAMPs that are majorly sensed by human TLR4 receptors. Our study aims to recognize the amino acid sequence(s) of the viral spike proteins that are precisely responsible for interaction with human TLR4 and to screen the immunogenic epitopes present in them to develop a multi-epitope multi-target chimeric vaccine against the coronaviruses. Molecular design of the constructed vaccine peptide is qualified in silico; additionally, molecular docking and molecular dynamics simulation studies collectively reveal strong and stable interactions of the vaccine construct with TLRs and MHC receptors. In silico cloning is performed for proficient expression in bacterial systems. In silico immune simulation of the vaccine indicates highly immunogenic nature of the vaccine construct without any allergic response. The present biocomputational study hereby innovates a vaccine candidate - AbhiSCoVac hypothesized as a potent remedy to combat all the virulent forms of coronaviruses.

An R848-Conjugated Influenza Virus Vaccine Elicits Robust Immunoglobulin G to Hemagglutinin Stem in a Newborn Nonhuman Primate Model.

Eliciting broadly protective antibodies is a critical goal for the development of more effective vaccines against influenza. Optimizing protection is of particular importance in newborns, who are highly vulnerable to severe disease following infection. An effective vaccination strategy for this population must surmount the challenges associated with the neonatal immune system as well as mitigate the inherent immune subdominance of conserved influenza virus epitopes, responses to which can provide broader protection. Here, we show that prime-boost vaccination with a TLR7/8 agonist (R848)-conjugated influenza A virus vaccine elicits antibody responses to the highly conserved hemagglutinin stem and promotes rapid induction of virus neutralizing stem-specific antibodies following viral challenge. These findings support the efficacy of R848 as an effective adjuvant for newborns and demonstrate its ability to enhance antibody responses to subdominant antigenic sites in this at-risk population.

Computationally Optimized Broadly Reactive H2 HA Influenza Vaccines Elicited Broadly Cross-Reactive Antibodies and Protected Mice from Viral Challenges.

Influenza viruses have caused numerous pandemics throughout human history. The 1957 influenza pandemic was initiated by an H2N2 influenza virus. This H2N2 influenza virus was the result of a reassortment event between a circulating H2N2 avian virus and the seasonal H1N1 viruses in humans. Previously, our group has demonstrated the effectiveness of hemagglutinin (HA) antigens derived using computationally optimized broadly reactive antigen (COBRA) methodology against H1N1, H3N2, and H5N1 viruses. Using the COBRA methodology, H2 HA COBRA antigens were designed using sequences from H2N2 viruses isolated from humans in the 1950s and 1960s, as well as H2Nx viruses isolated from avian and mammalian species between the 1950s and 2016. In this study, the effectiveness of H2 COBRA HA antigens (Z1, Z3, Z5, and Z7) was evaluated in DBA/2J mice and compared to that of wild-type H2 HA antigens. The COBRA HA vaccines elicited neutralizing antibodies to the majority of viruses in our H2 HA panel and across all three clades as measured by hemagglutination inhibition (HAI) and neutralization assays. Comparatively, several wild-type HA vaccines elicited antibodies against a majority of the viruses in the H2 HA panel. DBA/2J mice vaccinated with COBRA vaccines showed increase survival for all three viral challenges compared to the wild-type H2 vaccines. In particular, the Z1 COBRA is a promising candidate for future work toward a pandemic H2 influenza vaccine.IMPORTANCE H2N2 influenza has caused at least one pandemic in the past. Given that individuals born after 1968 have not been exposed to H2N2 influenza viruses, a future pandemic caused by H2 influenza is likely. An effective H2 influenza vaccine would need to elicit broadly cross-reactive antibodies to multiple H2 influenza viruses. Choosing a wild-type virus to create a vaccine may elicit a narrow immune response and not protect against multiple H2 influenza viruses. COBRA H2 HA vaccines were developed and evaluated in mice along with wild-type H2 HA vaccines. Multiple COBRA H2 HA vaccines protected mice from all three viral challenges and produced broadly cross-reactive neutralizing antibodies to H2 influenza viruses.

A Multi-Targeting, Nucleoside-Modified mRNA Influenza Virus Vaccine Provides Broad Protection in Mice.

Influenza viruses are respiratory pathogens of public health concern worldwide with up to 650,000 deaths occurring each year. Seasonal influenza virus vaccines are employed to prevent disease, but with limited effectiveness. Development of a universal influenza virus vaccine with the potential to elicit long-lasting, broadly cross-reactive immune responses is necessary for reducing influenza virus prevalence. In this study, we have utilized lipid nanoparticle-encapsulated, nucleoside-modified mRNA vaccines to intradermally deliver a combination of conserved influenza virus antigens (hemagglutinin stalk, neuraminidase, matrix-2 ion channel, and nucleoprotein) and induce strong immune responses with substantial breadth and potency in a murine model. The immunity conferred by nucleoside-modified mRNA-lipid nanoparticle vaccines provided protection from challenge with pandemic H1N1 virus at 500 times the median lethal dose after administration of a single immunization, and the combination vaccine protected from morbidity at a dose of 50 ng per antigen. The broad protective potential of a single dose of combination vaccine was confirmed by challenge with a panel of group 1 influenza A viruses. These findings support the advancement of nucleoside-modified mRNA-lipid nanoparticle vaccines expressing multiple conserved antigens as universal influenza virus vaccine candidates.

Combination of conserved recombinant proteins (NP & 3M2e) formulated with Alum protected BALB/c mice against influenza A/PR8/H1N1 virus challenge.

PURPOSE: Influenza is one of the most important agents of pandemic outbreak causing substantial morbidity and mortality. Vaccination strategies of influenza must be adapted annually due to constant antigenic changes in various strains. Therefore, the present study was conducted to evaluate protective immunity of the conserved influenza proteins. METHODS: For this purpose, three tandem repeats of M2e (3M2e) and NP were separately expressed in E. coli and were purified using column chromatography. Female Balb/c mice were injected intradermally with a combination of the purified 3M2e and NP alone or formulated with Alum (AlOH3) adjuvant in three doses. The mice were challenged by intranasal administration of H1N1 (A/PR/8/34) 2 weeks after the last vaccination. RESULTS: The results demonstrated that recombinant NP and M2e proteins are immunogenic and could efficiently elicit immune responses in mice compared to non-immunized mice. The combination of 3M2e and NP supplemented with Alum stimulated both NP and M2e-specific antibodies, which were higher than those stimulated by each single antigen plus Alum. In addition, the secretion of IFN-γ and IL-4 as well as the induction of lymphocyte proliferation in mice received the mixture of these proteins with Alum was considerably higher than other groups. Moreover, the highest survival rate (86%) with the least body weight change was observed in the mice immunized with 3M2e and NP supplemented with Alum followed by the mice received NP supplemented with Alum (71%). CONCLUSION: Accordingly, this regimen can be considered as an attractive candidate for global vaccination against influenza.

Better influenza vaccines: an industry perspective.

Vaccination is the most effective measure at preventing influenza virus infections. However, current seasonal influenza vaccines are only protective against closely matched circulating strains. Even with extensive monitoring and annual reformulation our efforts remain one step behind the rapidly evolving virus, often resulting in mismatches and low vaccine effectiveness. Fortunately, many next-generation influenza vaccines are currently in development, utilizing an array of innovative techniques to shorten production time and increase the breadth of protection. This review summarizes the production methods of current vaccines, recent advances that have been made in influenza vaccine research, and highlights potential challenges that are yet to be overcome. Special emphasis is put on the potential role of glycoengineering in influenza vaccine development, and the advantages of removing the glycan shield on influenza surface antigens to increase vaccine immunogenicity. The potential for future development of these novel influenza vaccine candidates is discussed from an industry perspective.

One step closer to influenza vaccine inclusiveness.

Flu virus infection is a common cause of acute respiratory illness, with the major incidence in pediatric age, high morbidity, and mortality. The flu vaccine is recommended for all people aged ≥6 months, unless specific contraindications are present. Younger and older age, pregnancy, chronic diseases like asthma, and immunodeficiency are risk factors for severe complications following flu infection. Thus, these categories represent the target for flu vaccine strategies in most countries. Inactivated influenza vaccine (IIV), recombinant influenza vaccine (RIV) or live-attenuated influenza virus (LAIV) are currently available, with specific precautions and contraindications. We aim to resume the current indications for vaccines in the vulnerable populations to support flu vaccination inclusiveness, in anticipation of a "universal vaccine" strategy.

Multi-Epitope Peptide-Based and Vaccinia-Based Universal Influenza Vaccine Candidates Subjected to Clinical Trials.

In light of the limited protection conferred by current influenza vaccines, immunisation using universal influenza vaccines has been proposed for protection against all or most influenza sub-types. The fundamental principle of universal influenza vaccines is based on conserved antigens found in most influenza strains, such as matrix 2, nucleocapsid, matrix 1 and stem of hemagglutinin proteins. These antigens trigger cross-protective immunity against different influenza strains. Many researchers have attempted to produce the conserved epitopes of these antigens in the form of peptides in the hope of generating universal influenza vaccine candidates that can broadly induce cross-reactive protection against influenza viral infections. However, peptide vaccines are poorly immunogenic when applied individually owing to their small molecular sizes. Hence, strategies, such as combining peptides as multi-epitope vaccines or presenting peptides on vaccinia virus particles, are employed. This review discusses the clinical and laboratory findings of several multi-epitope peptide vaccine candidates and vaccinia-based peptide vaccines. The majority of these vaccine candidates have reached the clinical trial phase. The findings in this study will indeed shed light on the applicability of universal influenza vaccines to prevent seasonal and pandemic influenza outbreaks in the near future.

Alternative Strategy for a Quadrivalent Live Attenuated Influenza Virus Vaccine.

Influenza virus infections continue to pose a major public health threat worldwide associated with seasonal epidemics and sporadic pandemics. Vaccination is considered the first line of defense against influenza. Live attenuated influenza virus vaccines (LAIVs) may provide superior responses compared to inactivated vaccines because the former can better elicit a combination of humoral and cellular responses by mimicking a natural infection. Unfortunately, during the 2013-2014, 2014-2015, and 2015-2016 seasons, concerns emerged about the effectiveness of the only LAIV approved in the United States that prevented the Advisory Committee on Immunization Practices (ACIP) from recommending its use. Such drawbacks open up the opportunity for alternative LAIV strategies that could overcome such concerns. Previously, we developed a combined strategy of temperature-sensitive mutations in the PB2 and PB1 segments and an epitope tag in the C terminus of PB1 that effectively attenuates influenza A viruses of avian and mammalian origin. More recently, we adopted a similar strategy for influenza B viruses. The resulting attenuated (att) influenza A and B viruses were safe, immunogenic, and protective against lethal influenza virus challenge in a variety of animal models. In this report, we provide evidence of the potential use of our att strategy in a quadrivalent LAIV (QIV) formulation carrying H3N2 and H1N1 influenza A virus subtype viruses and two antigenic lineages of influenza B viruses. In naive DBA/2J mice, two doses of the QIV elicited hemagglutination inhibition (HI) responses with HI titers of ≥40 and effectively protected against lethal challenge with prototypical pandemic H1N1 influenza A and influenza B virus strains.IMPORTANCE Seasonal influenza viruses infect 1 billion people worldwide and are associated with ∼500,000 deaths annually. In addition, the never-ending emergence of zoonotic influenza viruses associated with lethal human infections and of pandemic concern calls for the development of better vaccines and/or vaccination strategies against influenza virus. Regardless of the strategy, novel influenza virus vaccines must aim at providing protection against both seasonal influenza A and B viruses. In this study, we tested an alternative quadrivalent live attenuated influenza virus vaccine (QIV) formulation whose individual components have been previously shown to provide protection. We demonstrate in proof-of principle studies in mice that the QIV provides effective protection against lethal challenge with either influenza A or B virus.

Intranodal administration of mRNA encoding nucleoprotein provides cross-strain immunity against influenza in mice.

BACKGROUND: Current human influenza vaccines lack the adaptability to match the mutational rate of the virus and therefore require annual revisions. Because of extensive manufacturing times and the possibility that antigenic alterations occur during viral vaccine strain production, an inherent risk exists for antigenic mismatch between the new influenza vaccine and circulating viruses. Targeting more conserved antigens such as nucleoprotein (NP) could provide a more sustainable vaccination strategy by inducing long term and heterosubtypic protection against influenza. We previously demonstrated that intranodal mRNA injection can induce potent antigen-specific T-cell responses. In this study, we investigated whether intranodal administration of mRNA encoding NP can induce T-cell responses capable of protecting against a heterologous influenza virus challenge. METHODS: BALB/c mice were immunized in the inguinal lymph nodes with different vaccination regimens of mRNA encoding NP. Immune responses were compared with NP DNA vaccination via IFN-γ ELISPOT and in vivo cytotoxicity. For survival experiments, mice were prime-boost vaccinated with 17 µg NP mRNA and infected with 1LD50 of H1N1 influenza virus 8 weeks after boost. Weight was monitored and viral titers, cytokines and immune cell populations in the bronchoalveolar lavage, and IFN-γ responses in the spleen were analyzed. RESULTS: Our results demonstrate that NP mRNA induces superior systemic T-cell responses against NP compared to classical DNA vaccination. These responses were sustained for several weeks even at low vaccine doses. Upon challenge infection, vaccination with NP mRNA resulted in reduced lung viral titers and improved recovery from infection. Finally, we show that vaccination with NP mRNA affects the immune response in infected lungs by lowering immune cell infiltration while increasing the fraction of T cells, monocytes and MHC II+ alveolar macrophages within immune infiltrates. This change was associated with altered levels of both pro- and anti-inflammatory cytokines. CONCLUSIONS: These findings suggest that intranodal vaccination with NP mRNA induces cross-strain immunity against influenza, but also highlight a paradox of influenza immunity, whereby robust immune responses can provide protection, but can also transiently exacerbate symptoms during infection.

Chimeric protein consisting of 3M2e and HSP as a universal influenza vaccine candidate: from in silico analysis to preliminary evaluation.

The 23-amino acid ectodomain of influenza virus M2 protein (M2e) is highly conserved among human influenza virus variants and represents an attractive target for developing a universal vaccine. Although this peptide has limited potency and low immunogenicity, the degree of M2e density has been shown to be a critical factor influencing the magnitude of epitope-specific responses. The aim of this study was to design a chimer protein consisting of three tandem repeats of M2e peptide sequence fused to the Leishmania major HSP70 gene and evaluate its characteristics and immunogenicity. The structure of the deduced protein and its stability, aliphatic index, biocomputed half-life and the anticipated immunogenicity were analyzed by bioinformatics software. The oligonucleotides encoding 3M2e and chimer 3M2e-HSP70 were expressed in Escherichia coli and affinity purified. The immunogenicity of the purified recombinant proteins was preliminary examined in mouse model. It was predicted that fusion of HSP70 to the C-terminal of 3M2e peptide led to increased stability, hydropathicity, continuous B cell epitopes and antigenic propensity score of chimer protein. Also, the predominant 3M2e epitopes were not hidden in the chimer protein. The initial in vivo experiment showed that 3M2e-HSP chimer protein stimulates specific immune responses. In conclusion, the results of the current study suggest that 3M2e-HSP chimer protein would be an effective universal subunit vaccine candidate against influenza infection.