Antibody-independent surface plasmon resonance assays for influenza vaccine quality control.

Surface plasmon resonance (SPR)-based biosensors have emerged as a powerful platform for bioprocess monitoring due to their ability to detect biointeractions in real time, without the need for labeling. Paramount for the development of a robust detection platform is the immobilization of a ligand with high specificity and affinity for the in-solution species of interest. Following the 2009 H1N1 pandemic, much effort has been made toward the development of quality control platforms for influenza A vaccine productions, many of which have employed SPR for detection. Due to the rapid antigenic drift of influenza's principal surface protein, hemagglutinin, antibodies used for immunoassays need to be produced seasonally. The production of these antibodies represents a 6-8-week delay in immunoassay and, thus, vaccine availability. This review focuses on SPR-based assays that do not rely on anti-HA antibodies for the detection, characterization, and quantification of influenza A in bioproductions and biological samples. KEY POINTS: • The single radial immunodiffusion assay (SRID) has been the gold standard for the quantification of influenza vaccines since 1979. Due to antigenic drift of influenza's hemagglutinin protein, new antibody reagents for the SRID assay must be produced each year, requiring 6-8 weeks. The resulting delay in immunoassay availability is a major bottleneck in the influenza vaccine pipeline. This review highlights ligand options for the detection and quantification of influenza viruses using surface plasmon resonance biosensors.

Recombinant parainfluenza virus 5 expressing clade 2.3.4.4b H5 hemagglutinin protein confers broad protection against H5Ny influenza viruses.

The global circulation of clade 2.3.4.4b H5Ny highly pathogenic avian influenza viruses (HPAIVs) in poultry and wild birds, increasing mammal infections, continues to pose a public health threat and may even form a pandemic. An efficacious vaccine against H5Ny HPAIVs is crucial for emergency use and pandemic preparedness. In this study, we developed a parainfluenza virus 5 (PIV5)-based vaccine candidate expressing hemagglutinin (HA) protein of clade 2.3.4.4b H5 HPAIV, termed rPIV5-H5, and evaluated its safety and efficacy in mice and ferrets. Our results demonstrated that intranasal immunization with a single dose of rPIV5-H5 could stimulate H5-specific antibody responses, moreover, a prime-boost regimen using rPIV5-H5 stimulated robust humoral, cellular, and mucosal immune responses in mice. Challenge study showed that rPIV5-H5 prime-boost regimen provided sterile immunity against lethal clade 2.3.4.4b H5N1 virus infection in mice and ferrets. Notably, rPIV5-H5 prime-boost regimen provided protection in mice against challenge with lethal doses of heterologous clades 2.2, 2.3.2, and 2.3.4 H5N1, and clade 2.3.4.4h H5N6 viruses. These results revealed that rPIV5-H5 can elicit protective immunity against a diverse clade of highly pathogenic H5Ny virus infection in mammals, highlighting the potential of rPIV5-H5 as a pan-H5 influenza vaccine candidate for emergency use.IMPORTANCEClade 2.3.4.4b H5Ny highly pathogenic avian influenza viruses (HPAIVs) have been widely circulating in wild birds and domestic poultry all over the world, leading to infections in mammals, including humans. Here, we developed a recombinant PIV5-vectored vaccine candidate expressing the HA protein of clade 2.3.4.4b H5 virus. Intranasal immunization with rPIV5-H5 in mice induced airway mucosal IgA responses, high levels of antibodies, and robust T-cell responses. Importantly, rPIV5-H5 conferred complete protection in mice and ferrets against clade 2.3.4.4b H5N1 virus challenge, the protective immunity was extended against heterologous H5Ny viruses. Taken together, our data demonstrate that rPIV5-H5 is a promising vaccine candidate against diverse H5Ny influenza viruses in mammals.

A multivalent nucleoside-modified mRNA vaccine against all known influenza virus subtypes.

Seasonal influenza vaccines offer little protection against pandemic influenza virus strains. It is difficult to create effective prepandemic vaccines because it is uncertain which influenza virus subtype will cause the next pandemic. In this work, we developed a nucleoside-modified messenger RNA (mRNA)-lipid nanoparticle vaccine encoding hemagglutinin antigens from all 20 known influenza A virus subtypes and influenza B virus lineages. This multivalent vaccine elicited high levels of cross-reactive and subtype-specific antibodies in mice and ferrets that reacted to all 20 encoded antigens. Vaccination protected mice and ferrets challenged with matched and mismatched viral strains, and this protection was at least partially dependent on antibodies. Our studies indicate that mRNA vaccines can provide protection against antigenically variable viruses by simultaneously inducing antibodies against multiple antigens.

Universal and Translational Nanoparticulate CpG Adjuvant.

CpG, an agonist of toll-like receptor 9 (TLR9), has become a novel adjuvant that substantially potentiates cellular immunity. However, this agonist may increase systemic toxicity by diffusing into blood after administration and is difficult to be internalized by immune cells to reach TLR9 located in endosomes as a result of the characteristics of negative charge of CpG. Here, we applied a scalable and controllable flash nanocomplexation technology to prepare nanoparticulate CpG adjuvant (npCpG), CpG encapsulated in a physical cross-linking network of protamine and TPP. The nanoadjuvant could redirect CpG into draining lymph nodes to reduce systemic diffusion to improve safety. Further, a combination of npCpG and influenza H1N1 hemagglutinin antigen showed excellent humoral and cellular immunity, evoking high levels of antibodies and cytokines and inducing a great expansion of splenocytes in immunized mice. Also, the nanoadjuvant combined with ovalbumin antigen led to a potent cytotoxic T-cell response, substantially inhibited tumor growth, and improved the survival rate of mice in a melanoma model. This study showed the universal performances of npCpG in infectious disease prevention and tumor immunotherapy to demonstrate the translational potential.

Respiratory Vaccination with Hemagglutinin Nanoliposomes Protects Mice from Homologous and Heterologous Strains of Influenza Virus.

Intranasal vaccination offers the potential advantage of needle-free prevention of respiratory pathogens such as influenza viruses with induction of mucosal immune responses. Optimal design of adjuvants and antigen delivery vehicles for intranasal delivery has not yet been well established. Here, we report that an adjuvant-containing nanoliposome antigen display system that converts soluble influenza hemagglutinin antigens into nanoparticles is effective for intranasal immunization. Intranasal delivery of nanoliposomes in mice delivers the particles to resident immune cells in the respiratory tract, inducing a mucosal response in the respiratory system as evidenced by nasal and lung localized IgA antibody production, while also producing systemic IgG antibodies. Intranasal vaccination with nanoliposome particles decorated with nanogram doses of hemagglutinin protected mice from homologous and heterologous H3N2 and H1N1 influenza virus challenge. IMPORTANCE A self-assembling influenza virus vaccine platform that seamlessly converts soluble antigens into nanoparticles is demonstrated with various H1N1 and H3N2 influenza antigens to protect mice against influenza virus challenge following intranasal vaccination. Mucosal immune responses following liposome delivery to lung antigen-presenting cells are demonstrated.

The Pre-Existing Human Antibody Repertoire to Computationally Optimized Influenza H1 Hemagglutinin Vaccines.

Computationally optimized broadly reactive Ag (COBRA) hemagglutinin (HA) immunogens have previously been generated for several influenza subtypes to improve vaccine-elicited Ab breadth. As nearly all individuals have pre-existing immunity to influenza viruses, influenza-specific memory B cells will likely be recalled upon COBRA HA vaccination. We determined the epitope specificity and repertoire characteristics of pre-existing human B cells to H1 COBRA HA Ags. Cross-reactivity between wild-type HA and H1 COBRA HA proteins P1, X6, and Y2 were observed for isolated mAbs. The mAbs bound five distinct epitopes on the pandemic A/California/04/2009 HA head and stem domains, and most mAbs had hemagglutination inhibition and neutralizing activity against 2009 pandemic H1 strains. Two head-directed mAbs, CA09-26 and CA09-45, had hemagglutination inhibition and neutralizing activity against a prepandemic H1 strain. One mAb, P1-05, targeted the stem region of H1 HA, but did not compete with a known stem-targeting H1 mAb. We determined that mAb P1-05 recognizes a recently discovered HA epitope, the anchor epitope, and we identified similar mAbs using B cell repertoire sequencing. In addition, the trimerization domain distance from HA was critical to recognition of this epitope by mAb P1-05, suggesting the importance of protein design for vaccine formulations. Overall, these data indicate that seasonally vaccinated individuals possess a population of functional H1 COBRA HA-reactive B cells that target head, central stalk, and anchor epitopes, and they demonstrate the importance of structure-based assessment of subunit protein vaccine candidates to ensure accessibility of optimal protein epitopes.

Bivalent H1 and H3 COBRA Recombinant Hemagglutinin Vaccines Elicit Seroprotective Antibodies against H1N1 and H3N2 Influenza Viruses from 2009 to 2019.

Commercial influenza virus vaccines often elicit strain-specific immune responses and have difficulties preventing illness caused by antigenically drifted viral variants. In the last 20 years, the H3N2 component of the annual vaccine has been updated nearly twice as often as the H1N1 component, and in 2019, a mismatch between the wild-type (WT) H3N2 vaccine strain and circulating H3N2 influenza strains led to a vaccine efficacy of ∼9%. Modern methods of developing computationally optimized broadly reactive antigens (COBRAs) for H3N2 influenza viruses utilize current viral surveillance information to design more broadly reactive vaccine antigens. Here, 7 new recombinant hemagglutinin (rHA) H3 COBRA hemagglutinin (HA) antigens were evaluated in mice. Subsequently, two candidates, J4 and NG2, were selected for further testing in influenza-preimmune animals based on their ability to elicit broadly reactive antibodies against antigenically drifted H3N2 viral isolates. In the preimmune model, monovalent formulations of J4 and NG2 elicited broadly reactive antibodies against recently circulating H3N2 influenza viruses from 2019. Bivalent mixtures of COBRA H1 and H3 rHA, Y2 + J4, and Y2 + NG2 outperformed multiple WT H1+H3 bivalent rHA mixtures by eliciting seroprotective antibodies against H1N1 and H3N2 isolates from 2009 to 2019. Overall, the newly generated COBRA HA antigens, namely, Y2, J4, and NG2, had the ability to induce broadly reactive antibodies in influenza-naive and preimmune animals in both monovalent and bivalent formulations, and these antigens outperformed H1 and H3 WT rHA vaccine antigens by eliciting seroprotective antibodies against panels of antigenically drifted historical H1N1 and H3N2 vaccine strains from 2009 to 2019. IMPORTANCE Standard-of-care influenza virus vaccines are composed of a mixture of antigens from different influenza viral subtypes. For the first time, lead COBRA H1 and H3 HA antigens, formulated as a bivalent vaccine, have been investigated in animals with preexisting immunity to influenza viruses. The cocktail of COBRA HA antigens elicited more broadly reactive anti-HA antibodies than those elicited by a comparator bivalent wild-type HA vaccine against H1 and H3 influenza viruses isolated between 2009 and 2019.

Teach 'em young: Influenza vaccines induce broadly neutralizing antibodies in children.

Antibodies against the influenza virus hemagglutinin stalk afford broad protection against antigenically drifted viruses. In this issue of Cell Reports Medicine, Yegorov et al.1 identify that current vaccine formulations induce neutralizing stalk antibodies in children-a highly vulnerable population.

MCMV-based vaccine vectors expressing full-length viral proteins provide long-term humoral immune protection upon a single-shot vaccination.

Global pandemics caused by influenza or coronaviruses cause severe disruptions to public health and lead to high morbidity and mortality. There remains a medical need for vaccines against these pathogens. CMV (cytomegalovirus) is a ß-herpesvirus that induces uniquely robust immune responses in which remarkably large populations of antigen-specific CD8+ T cells are maintained for a lifetime. Hence, CMV has been proposed and investigated as a novel vaccine vector for expressing antigenic peptides or proteins to elicit protective cellular immune responses against numerous pathogens. We generated two recombinant murine CMV (MCMV) vaccine vectors expressing hemagglutinin (HA) of influenza A virus (MCMVHA) or the spike protein of severe acute respiratory syndrome coronavirus 2 (MCMVS). A single injection of MCMVs expressing either viral protein induced potent neutralizing antibody responses, which strengthened over time. Importantly, MCMVHA-vaccinated mice were protected from illness following challenge with the influenza virus, and we excluded that this protection was due to the effects of memory T cells. Conclusively, we show here that MCMV vectors induce not only long-term cellular immunity but also humoral responses that provide long-term immune protection against clinically relevant respiratory pathogens.

Expression and characterization of a recombinant broadly-reactive monoclonal antibody against group 1 and 2 influenza viruses.

Production of broadly-reactive antibodies is critical for universal immunodiagnosis of rapidly-evolving influenza viruses. Most monoclonal antibodies (mAbs) are generated in mice using the hybridoma technology which involves labor- and time-consuming screening and low yield issues. In this study, a recombinant antibody based on a broadly-reactive mAb against the hemagglutinin (HA) stalk of H7N9 avian influenza virus was expressed in CHO cells and its biological characteristics, cross-reactivity and epitope recognition were identified. The variable genes of the parental antibody were amplified and cloned into the antibody-expressing plasmids containing the constant genes of murine IgG1. The recombinant antibody was expressed in high yield and purity in CHO cells and showed similar features to the parental antibody, including negative hemagglutination inhibition activity against H7N9 virus and high binding activity with the H7N9 HA protein. Notably, the recombinant antibody exhibited a broad reactivity with different influenza subtypes belonging to group 1 and group 2, which was associated with its recognition of a highly-conserved epitope in the stalk, as observed for the parental antibody. Our results suggest that cell-based antibody expression system can be utilized as an important alternative to the hybridoma technology for antibody production for influenza virus diagnostics.

Polycationic HA/CpG Nanoparticles Induce Cross-Protective Influenza Immunity in Mice.

The intranasal (i.n.) route is an ideal vaccination approach for infectious respiratory diseases like influenza. Polycationic polyethylenimine (PEI) could form nanoscale complexes with negatively charged viral glycoproteins. Here we fabricated PEI-hemagglutinin (HA) and PEI-HA/CpG nanoparticles and investigated their immune responses and protective efficacies with an i.n. vaccination regimen in mice. Our results revealed that the nanoparticles significantly enhanced HA immunogenicity, providing heterologous cross-protection. The conserved HA stalk region induced substantial antibodies in the nanoparticle immunization groups. In contrast to the Th2-biased, IgG1-dominant antibody response generated by PEI-HA nanoparticles, PEI-HA/CpG nanoparticles generated more robust and balanced IgG1/IgG2a antibody responses with augmented neutralization activity and Fc-mediated antibody-dependent cellular cytotoxicity (ADCC). PEI-HA/CpG nanoparticles also induced enhanced local and systemic cellular immune responses. These immune responses did not decay over six months of observation postimmunization. PEI and CpG synergized these comprehensive immune responses. Thus, the PEI-HA/CpG nanoparticle is a potential cross-protective influenza vaccine candidate. Polycationic PEI nanoplatforms merit future development into mucosal vaccine systems.

Broadly neutralizing antibodies target a haemagglutinin anchor epitope.

Broadly neutralizing antibodies that target epitopes of haemagglutinin on the influenza virus have the potential to provide near universal protection against influenza virus infection1. However, viral mutants that escape broadly neutralizing antibodies have been reported2,3. The identification of broadly neutralizing antibody classes that can neutralize viral escape mutants is critical for universal influenza virus vaccine design. Here we report a distinct class of broadly neutralizing antibodies that target a discrete membrane-proximal anchor epitope of the haemagglutinin stalk domain. Anchor epitope-targeting antibodies are broadly neutralizing across H1 viruses and can cross-react with H2 and H5 viruses that are a pandemic threat. Antibodies that target this anchor epitope utilize a highly restricted repertoire, which encodes two public binding motifs that make extensive contacts with conserved residues in the fusion peptide. Moreover, anchor epitope-targeting B cells are common in the human memory B cell repertoire and were recalled in humans by an oil-in-water adjuvanted chimeric haemagglutinin vaccine4,5, which is a potential universal influenza virus vaccine. To maximize protection against seasonal and pandemic influenza viruses, vaccines should aim to boost this previously untapped source of broadly neutralizing antibodies that are widespread in the human memory B cell pool.

Intranasal delivery of a cDC1 targeted influenza vaccine with poly(I:C) enhances T cell responses and protects against influenza infection.

Targeting antigens to dendritic cells represent a promising method for enhancing immune responses against specific antigens. However, many studies have focused on systemic delivery (intravenous or intraperitoneally) of targeted antigen, approaches that are not easily transferable to humans. Here we evaluate the efficacy of an influenza vaccine targeting Xcr1+ cDC1 administered by intranasal immunization. Intranasal delivery of antigen fused to the chemokine Xcl1, the ligand of Xcr1, resulted in specific uptake by lung CD103+ cDC1. Interestingly, intranasal immunization with influenza A/PR/8/34 haemagglutinin (HA) fused to Xcl1, formulated with poly(I:C), resulted in enhanced induction of antigen-specific IFNγ+ CD4+ and IFNγ+ CD8+ T cell responses in lung compared non-targeted anti-NIP-HA (αNIP-HA). Induction of antibody responses was, however, similar in Xcl1-HA and αNIP-HA immunized mice, but significantly higher than in mice immunized with monomeric HA. Both Xcl1-HA and αNIP-HA vaccines induced full protection when mice were challenged with a lethal dose of influenza PR8 virus, reflecting the strong induction of HA-specific antibodies. Our results demonstrate that i.n. delivery of Xcl1-HA is a promising vaccine strategy for enhancing T cell responses in addition to inducing strong antibody responses.

Enhanced cross protection by hetero prime-boost vaccination with recombinant influenza viruses containing chimeric hemagglutinin-M2e epitopes.

Annual repeat influenza vaccination raises concerns about protective efficacy against mismatched viruses. We investigated the impact of heterologous prime-boost vaccination on inducing cross protection by designing recombinant influenza viruses with chimeric hemagglutinin (HA) carrying M2 extracellular domains (M2e-HA). Heterologous prime-boost vaccination of C57BL/6 mice with M2e-HA chimeric virus more effectively induced M2e and HA stalk specific IgG antibodies correlating with cross protection than homologous prime-boost vaccination. Induction of M2e and HA stalk specific IgG antibodies was compromised in 1-year old mice, indicating significant aging effects on priming subdominant M2e and HA stalk IgG antibody responses. This study demonstrates that a heterologous prime-boost strategy with recombinant influenza virus expressing extra M2e epitopes provides more effective cross protection than homologous vaccination.

Viral surface geometry shapes influenza and coronavirus spike evolution through antibody pressure.

The evolution of circulating viruses is shaped by their need to evade antibody response, which mainly targets the viral spike. Because of the high density of spikes on the viral surface, not all antigenic sites are targeted equally by antibodies. We offer here a geometry-based approach to predict and rank the probability of surface residues of SARS spike (S protein) and influenza H1N1 spike (hemagglutinin) to acquire antibody-escaping mutations utilizing in-silico models of viral structure. We used coarse-grained MD simulations to estimate the on-rate (targeting) of an antibody model to surface residues of the spike protein. Analyzing publicly available sequences, we found that spike surface sequence diversity of the pre-pandemic seasonal influenza H1N1 and the sarbecovirus subgenus highly correlates with our model prediction of antibody targeting. In particular, we identified an antibody-targeting gradient, which matches a mutability gradient along the main axis of the spike. This identifies the role of viral surface geometry in shaping the evolution of circulating viruses. For the 2009 H1N1 and SARS-CoV-2 pandemics, a mutability gradient along the main axis of the spike was not observed. Our model further allowed us to identify key residues of the SARS-CoV-2 spike at which antibody escape mutations have now occurred. Therefore, it can inform of the likely functional role of observed mutations and predict at which residues antibody-escaping mutation might arise.

Monoclonal Antibody Targeting the HA191/199 Region of H1N1 Influenza Virus Mediates the Damage of Neural Cells.

Vaccination is the most effective mean of preventing influenza virus infections. However, vaccination-induced adverse reactions of the nervous system, the causes of which are unknown, lead to concerns on the safety of influenza A vaccine. In this study, we used flow cytometry, cell ELISA, and immunofluorescence to find that H1-84 monoclonal antibody (mAb) against the191/199 region of the H1N1 influenza virus hemagglutinin (HA) protein binds to neural cells and mediates cell damage. Using molecular simulation software, such as PyMOL and PDB viewer, we demonstrated that the HA191/199 region maintains the overall structure of the HA head. Since the HA191/199 region cannot be removed from the HA structure, it has to be altered via introducing point mutations by site-directed mutagenesis. This will provide an innovative theoretical support for the subsequent modification the influenza A vaccine for increasing its safety.

Immune Evaluation of Recombinant Lactobacillus plantarum With Surface Display of HA1-DCpep in Mice.

Avian influenza viruses can be efficiently transmitted through mucous membranes, and conventional vaccines are not effective in protecting against mucosal infection by influenza viruses. To induce multiple immune responses in an organism, we constructed a recombinant Lactobacillus plantarum expressing the influenza virus antigen HA1 with the adjuvant dendritic cell-targeting peptide (DCpep). The recombinant L. plantarum strains NC8Δ-pWCF-HA1 and NC8Δ-pWCF-HA1-DCpep were used to immunize mice via oral administration, and the humoral, cellular and mucosal immune responses were evaluated. In addition, the serum levels of specific antibodies and hemagglutination inhibition (HI) levels were also measured. Our results showed that recombinant L. plantarum activated dendritic cells in Peyer's patches (PPs), increased the numbers of CD4+IFN-γ+ and CD8+IFN-γ+ cells in the spleen and mesenteric lymph nodes (MLNs), and affected the ability of CD4+ and CD8+ cells to proliferate in the spleen and MLNs. Additionally, recombinant L. plantarum increased the number of B220+IgA+ cells in PPs and the level of IgA in the lungs and different intestinal segments. In addition, specific IgG, IgG1 and IgG2a antibodies were induced at high levels in the mice serum, specific IgA antibodies were induced at high levels in the mice feces, and HI potency was significantly increased. Thus, the recombinant L. plantarum strains NC8Δ-pWCF-HA1 and NC8Δ-pWCF-HA1-DCpep have potential as vaccine candidates for avian influenza virus.

Epigallocatechin-3-Gallate as a Novel Vaccine Adjuvant.

Vaccine adjuvants from natural resources have been utilized for enhancing vaccine efficacy against infectious diseases. This study examined the potential use of catechins, polyphenolic materials derived from green tea, as adjuvants for subunit and inactivated vaccines. Previously, catechins have been documented to have irreversible virucidal function, with the possible applicability in the inactivated viral vaccine platform. In a mouse model, the coadministration of epigallocatechin-3-gallate (EGCG) with influenza hemagglutinin (HA) antigens induced high levels of neutralizing antibodies, comparable to that induced by alum, providing complete protection against the lethal challenge. Adjuvant effects were observed for all types of HA antigens, including recombinant full-length HA and HA1 globular domain, and egg-derived inactivated split influenza vaccines. The combination of alum and EGCG further increased neutralizing (NT) antibody titers with the corresponding hemagglutination inhibition (HI) titers, demonstrating a dose-sparing effect. Remarkably, EGCG induced immunoglobulin isotype switching from IgG1 to IgG2a (approximately >64-700 fold increase), exerting a more balanced TH1/TH2 response compared to alum. The upregulation of IgG2a correlated with significant enhancement of antibody-dependent cellular cytotoxicity (ADCC) function (approximately 14 fold increase), providing a potent effector-mediated protection in addition to NT and HI. As the first report on a novel class of vaccine adjuvants with built-in virucidal activities, the results of this study will help improve the efficacy and safety of vaccines for pandemic preparedness.

Safety, Immunogenicity, and Protective Efficacy of an H5N1 Chimeric Cold-Adapted Attenuated Virus Vaccine in a Mouse Model.

H5N1 influenza virus is a threat to public health worldwide. The virus can cause severe morbidity and mortality in humans. We constructed an H5N1 influenza candidate virus vaccine from the A/chicken/Guizhou/1153/2016 strain that was recommended by the World Health Organization. In this study, we designed an H5N1 chimeric influenza A/B vaccine based on a cold-adapted (ca) influenza B virus B/Vienna/1/99 backbone. We modified the ectodomain of H5N1 hemagglutinin (HA) protein, while retaining the packaging signals of influenza B virus, and then rescued a chimeric cold-adapted H5N1 candidate influenza vaccine through a reverse genetic system. The chimeric H5N1 vaccine replicated well in eggs and the Madin-Darby Canine Kidney cells. It maintained a temperature-sensitive and cold-adapted phenotype. The H5N1 vaccine was attenuated in mice. Hemagglutination inhibition (HAI) antibodies, micro-neutralizing (MN) antibodies, and IgG antibodies were induced in immunized mice, and the mucosal IgA antibody responses were detected in their lung lavage fluids. The IFN-γ-secretion and IL-4-secretion by the mouse splenocytes were induced after stimulation with the specific H5N1 HA protein. The chimeric H5N1 candidate vaccine protected mice against lethal challenge with a wild-type highly pathogenic avian H5N1 influenza virus. The chimeric H5 candidate vaccine is thus a potentially safe, attenuated, and reassortment-incompetent vaccine with circulating A viruses.

Designing a multi-epitope vaccine to provoke the robust immune response against influenza A H7N9.

A new strain of Influenza A Virus (IAV), so-called "H7N9 Avian Influenza", is the first strain of this virus in which a human is infected by transmitting the N9 of influenza virus. Although continuous human-to-human transmission has not been reported, the occurrence of various H7N9-associated epidemics and the lack of production of strong antibodies against H7N9 in humans warn of the potential for H7N9 to become a new pandemic. Therefore, the need for effective vaccination against H7N9 as a life-threatening viral pathogen has become a major concern. The current study reports the design of a multi-epitope vaccine against Hemagglutinin (HA) and Neuraminidase (NA) proteins of H7N9 Influenza A virus by prediction of Cytotoxic T lymphocyte (CTL), Helper T lymphocyte (HTL), IFN-γ and B-cell epitopes. Human ß-defensin-3 (HßD-3) and pan HLA DR-binding epitope (PADRE) sequence were considered as adjuvant. EAAAK, AAY, GPGPG, HEYGAEALERAG, KK and RVRR linkers were used as a connector for epitopes. The final construct contained 777 amino acids that are expected to be a recombinant protein of about ~ 86.38 kDa with antigenic and non-allergenic properties after expression. Modeled protein analysis based on the tertiary structure validation, docking studies, and molecular dynamics simulations results like Root-mean-square deviation (RMSD), Gyration, Root-mean-square fluctuation (RMSF) and Molecular Mechanics Poisson-Boltzmann Surface Area (MM/PBSA) showed that this protein has a stable construct and capable of being in interaction with Toll-like receptor 7 (TLR7), TLR8 and m826 antibody. Analysis of the obtained data the demonstrates that suggested vaccine has the potential to induce the immune response by stimulating T and Bcells, and may be utilizable for prevention purposes against Avian Influenza A (H7N9).