Conserved Influenza Proteins Research Articles

Analysis of polyclonal and monoclonal antibody to the influenza virus nucleoprotein in different oligomeric states.

Influenza virus nucleoprotein (NP) is one of the most conserved influenza proteins. Both NP antigen and anti-NP antibodies are used as reagents in influenza diagnostic kits, with applications in both clinical practice, and influenza zoonotic surveillance programs. Despite this, studies on the biochemical basis of NP diagnostic serology and NP epitopes are not as developed as for hemagglutinin (HA), the fast-evolving antigen which has been the critical component of current influenza vaccines. Here, we characterized the NP serology of mice, ferret, and human sera and the immunogenic effects of NP antigen presented as different structural complexes. Furthermore, we show that a classical anti-NP mouse mAb HB65 could detect NP in some commercial influenza vaccines. MAb HB65 bound a linear epitope with nanomolar affinity. Our analysis suggests that linear NP epitopes paired with their corresponding characterized detection antibodies could aid in designing and improving diagnostic technologies for influenza virus.

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

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. 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) 2weeks after the last vaccination. 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%). Accordingly, this regimen can be considered as an attractive candidate for global vaccination against influenza.

084 T cell vaccination using conserved influenza proteins is highly protective against lethal challenge

A possible future influenza A pandemic similar to the 1918 H1N1 outbreak remains an existential public health threat. A universal influenza A vaccine that protects regardless of hemagluttinin (H) and neuraminidase (N) subgroups remains elusive. The influenza genome encodes for a few proteins highly conserved among all flu strains, including NP and M1. We sought to generate a T cell vaccine broadly protective against all influenza A viruses based on this property. Using Modified Vaccinia Ankara (MVA), we created MVA-NP and MVA-M1 vectors.

Enhancing the Yield and Quality of Influenza Virus-like Particles (VLPs) Produced in Insect Cells by Inhibiting Cytopathic Effects of Matrix Protein M2.

To provide broader protection and eliminate the need for annual update of influenza vaccines, biomolecular engineering of influenza virus-like particles (VLPs) to display more conserved influenza proteins such as the matrix protein M2 has been explored. However, achieving high surface density of full-length M2 in influenza VLPs has been left unrealized. In this study, we show that the ion channel activity of M2 induces significant cytopathic effects in Spodoptera frugiperda (Sf9) insect cells when expressed using M2-encoding baculovirus. These effects include altered Sf9 cell morphology and reduced baculovirus replication, resulting in impaired influenza protein expression and thus VLP production. On the basis of the function of M2, we hypothesized that blocking its ion channel activity could potentially relieve these cytopathic effects, and thus restore influenza protein expression to improve VLP production. The use of the M2 inhibitor amantadine indeed improves Sf9 cellular expression not only of M2 (∼3-fold), but also of hemagglutinin (HA) (∼7-fold) and of matrix protein M1 (∼3-fold) when coexpressed to produce influenza VLPs. This increased cellular expression of all three influenza proteins further leads to ∼2-fold greater VLP yield. More importantly, the quality of the resulting influenza VLPs is significantly improved, as demonstrated by the ∼2-fold, ∼50-fold, and ∼2-fold increase in the antigen density to approximately 53 HA, 48 M1, and 156 M2 per influenza VLP, respectively. Taken together, this study represents a novel approach to enable the efficient incorporation of full-length M2 while enhancing both the yield and quality of influenza VLPs produced by Sf9 cells.

Influenza virus-like particles composed of conserved influenza proteins and GPI-anchored CCL28/GM-CSF fusion proteins enhance protective immunity against homologous and heterologous viruses

Influenza viruses cause significant morbidity and mortality and pose a substantial threat to public health. Vaccination represents the principle means of preventing influenza virus infection. Current vaccine approaches are hindered by the need to routinely reformulate vaccine compositions in an effort to account for the progressive antigenic changes that occur as influenza viruses circulate in the human population. In this study, we evaluated chimeric virus-like particle (cVLP) vaccines containing conserved elements of influenza proteins (HL5M2e (HA stem gene with 5M2e gene inserted) and NP), with or without glycosylphosphatidylinositol-anchored CCL28 (GPI-CCL28) and/or GM-CSF (GPI-GM-CSF) fusion proteins as molecular adjuvants. cVLPs elicited strong humoral and cellular immune responses against homologous and heterologous viruses, and improved survival following lethal challenge with both homologous and heterologous viruses. Inclusion of GPI-anchored adjuvants in cVLP vaccines augmented the generation of influenza-specific humoral and cellular immune responses in mice in comparison to the non-adjuvanted cVLP vaccines. VLPs containing GPI-anchored adjuvants reduced morbidity and improved survival to lethal challenge with homologous and heterologous influenza viruses. This work suggests that VLP vaccines incorporating conserved influenza virus proteins and GPI-anchored molecular adjuvants may serve as a platform for a broadly protective “universal” influenza vaccine.

EFFECTIVENESS OF CROSS-PROTECTIVE RECOMBINANT INFLUENZA VACCINE BASED ON CONSERVED EPITOPES OF VIRAL PROTEINS М2 AND HEMAGGLUTININ

The influenza virus is the most unique in the level of variability of antigenic and biological properties. Because of constant mutations into genes coding surface viral proteins, in modern vaccines it is necessary to replace 1–2 virus components annually. Traditional influenza vaccines are the strain – specific and have limited efficiency in prevention of new strains of influenza viruses. In this regard, creation of influenza vaccines based on conserved determinants of viral proteins with broad spectrum protection and the short period of production is one of priority tasks which decision will lead to real control of an influenza infection. A current trend in the design of universal flu vaccines is the construction of recombinant proteins based the combination of conserved viral proteins or peptides. The goals of this study: to develop the candidate recombinant flu vaccine based on the two conserved influenza proteins (М2 and НА); to investigate immune response; and to measure the protection activity in an animal model. Results: In this study we investigated the humoral and T-cell response in mice after intranasal immunization with recombinant proteins (Flg-4M2ehs and Flg-HA2-2-4M2ehs). Both proteins induce a robust M2e-specific humoral and CD4+ T-cell response in mice lung. The recombinant protein with two target antigens (M2e and HA2) induces virusspecific CD4+ and CD8+ T-cell response and full protection (100% survival) of mice from lethal challenge human and avian influenza viruses A (A/H3N2, A/H2N2, A/H5N1). In mice immunized with Flg-4M2ehs, the survival after lethal challenge was 60–75%. Conclusion: Our results show an essential role of a conserved fragment of the HA2 in the formation of protective T-cell response and protection of mice from lethal challenge with influenza viruses A of various subtypes. The prospects of the development of vaccine formulation based on two conserved antigenic determinants of influenza virus A are shown.

Qualitative and quantitative effects of 4-1BBL in boosting pre-existing influenza immunity (VAC2P.923)

Abstract Influenza virus induces an acute respiratory infection responsible for up to 500,000 annual deaths, primarily in older persons. CD8+ T cells against conserved influenza proteins confer heterotypic protection, potentially providing a “universal vaccine.” However, live infection only transiently boosts influenza-specific T cell memory, and immunity further declines with age. To improve the longevity of T cell memory, we investigated the effects of stimulating the TNFR-family member 4-1BB. To mimic vaccination of previously flu-immune adults, mice were infected with influenza A/HK-X31 and boosted intranasally one-month later with control replication-deficient adenovirus or adenovirus encoding influenza nucleoprotein (NP) alone (Ad-NP) or NP with 4-1BBL (Ad-NP-4-1BBL). 4-1BBL dramatically enhanced NP-specific CD8+ T cell responses at a dose where Ad-NP had minimal effects. 4-1BBL induced a remarkably long-lived effector-memory population that protects mice against lethal challenge into old age (>1 year old). Increasing the Ad-NP dose did not replicate these effects, and instead caused partial functional exhaustion. Preliminary analysis indicates that the 4-1BBL-induced effector memory T cells have increased expression of IL-7R as well as the transcriptional factor TCF-1 and an increased ratio of T-bet to Eomesodermin. Thus, 4-1BBL in a vaccine vector administered in flu-immune mice induces a potentially unique protective subset of memory CD8+ T cells.

MVA Vectors Expressing Conserved Influenza Proteins Protect Mice against Lethal Challenge with H5N1, H9N2 and H7N1 Viruses

BackgroundThe availability of a universal influenza vaccine able to induce broad cross-reactive immune responses against diverse influenza viruses would provide an alternative to currently available strain-specific vaccines. We evaluated the ability of vectors based on modified vaccinia virus Ankara (MVA) expressing conserved influenza proteins to protect mice against lethal challenge with multiple influenza subtypes.MethodsMice were immunized with MVA vectors expressing H5N1-derived nucleoprotein (NP), the stem region of hemagglutinin (HA), matrix proteins 1 and 2 (M1 and M2), the viral polymerase basic protein 1 (PB1), or the HA stem fused to a quadrivalent matrix protein 2 extracellular domain (M2e). Immunized mice were challenged with lethal doses of H5N1, H7N1 or H9N2 virus and monitored for disease symptoms and weight loss. To investigate the influence of previous exposure to influenza virus on protective immune responses induced by conserved influenza proteins, mice were infected with pandemic H1N1 virus (H1N1pdm09) prior to immunization and subsequently challenged with H5N1 virus. Antibody and T cell responses were assessed by ELISA and flow cytometry, respectively.ResultsMVA vectors expressing NP alone, or co-expressed with other conserved influenza proteins, protected mice against lethal challenge with H5N1, H7N1 or H9N2 virus. Pre-exposure to H1N1pdm09 increased protective efficacy against lethal H5N1 challenge. None of the other conserved influenza proteins provided significant levels of protection against lethal challenge. NP-expressing vectors induced high numbers of influenza-specific CD4+ and CD8+ T cells and high titer influenza-specific antibody responses. Higher influenza-specific CD4+ T cell responses and NP-specific CD8+ T cell responses were associated with increased protective efficacy.ConclusionsMVA vectors expressing influenza NP protect mice against lethal challenge with H5N1, H7N1 and H9N2 viruses by a mechanism involving influenza-specific CD4+ and CD8+ T cell responses.

Cooperativity Between CD8+ T Cells, Non-Neutralizing Antibodies, and Alveolar Macrophages Is Important for Heterosubtypic Influenza Virus Immunity

Seasonal epidemics of influenza virus result in ∼36,000 deaths annually in the United States. Current vaccines against influenza virus elicit an antibody response specific for the envelope glycoproteins. However, high mutation rates result in the emergence of new viral serotypes, which elude neutralization by preexisting antibodies. T lymphocytes have been reported to be capable of mediating heterosubtypic protection through recognition of internal, more conserved, influenza virus proteins. Here, we demonstrate using a recombinant influenza virus expressing the LCMV GP33-41 epitope that influenza virus-specific CD8+ T cells and virus-specific non-neutralizing antibodies each are relatively ineffective at conferring heterosubtypic protective immunity alone. However, when combined virus-specific CD8 T cells and non-neutralizing antibodies cooperatively elicit robust protective immunity. This synergistic improvement in protective immunity is dependent, at least in part, on alveolar macrophages and/or other lung phagocytes. Overall, our studies suggest that an influenza vaccine capable of eliciting both CD8+ T cells and antibodies specific for highly conserved influenza proteins may be able to provide heterosubtypic protection in humans, and act as the basis for a potential “universal” vaccine.

Heterosubtypic protective immunity against influenza A virus induced by fusion peptide of the hemagglutinin in comparison to ectodomain of M2 protein

Several types of influenza vaccines are available, but due to the highly unpredictable variability of influenza virus surface antigens (hemagglutinin (HA) and neuraminidase) current vaccines are not sufficiently effective against broad spectrum of the influenza viruses. An innovative approach to extend the vaccine efficacy is based on the selection of conserved influenza proteins with a potential to induce inter-subtype protection against the influenza A viruses. A promising new candidate for the preparation of broadly protective vaccine may be a highly conserved N-terminal part of HA2 glycopolypeptide (HA2 gp) called fusion peptide. To study its capacity to induce a protective immune response, we immunized mice with the fusion peptide (aa 1-38 of HA2 gp). The protective ability of fusion peptide was compared with the ectodomain aa 2-23 of M2 protein (eM2) that is antigenically conserved and its immunogenic properties have already been well documented. Corresponding peptides (both derived from A/Mississippi/1/85 (H3N2) virus) were synthesized and conjugated to the keyhole limpet hemocyanin (KLH) and used for the immunization of mice. Both antigens induced a significant level of specific antibodies. Immunized mice were challenged with the lethal dose of homologous (H3N2) or heterologous A/PR/8/34 (H1N1) influenza A viruses. Immunization with the fusion peptide led to the 100% survival of mice infected with 1 LD50 of homologous as well as heterologous virus. Survival rate decreased when infectious dose was raised to 2 LD50. The immunization with eM2 induced effective cross-protection of mice infected even with 3 LD50 of both challenge viruses. The lower, but still effective protection induced by the fusion peptide of HA2 gp suggested that besides ectodomain of M2, fusion peptide could also be considered as a part of cross-protective influenza vaccine. To our knowledge, this is the first report demonstrating that active immunization with the conjugated fusion peptide of HA2 gp provided the effective production of antibodies, what contributed to the cross-protection against influenza infection.

Potent CD8+ T-Cell Immunogenicity in Humans of a Novel Heterosubtypic Influenza A Vaccine, MVA-NP+M1

Background. Influenza A viruses cause occasional pandemics and frequent epidemics. Licensed influenza vaccines that induce high antibody titers to the highly polymorphic viral surface antigen hemagglutinin must be re-formulated and readministered annually. A vaccine providing protective immunity to the highly conserved internal antigens could provide longer-lasting protection against multiple influenza subtypes.Methods. We prepared a Modified Vaccinia virus Ankara (MVA) vector encoding nucleoprotein and matrix protein 1 (MVA−NP+M1) and conducted a phase I clinical trial in healthy adults.Results. The vaccine was generally safe and well tolerated, with significantly fewer local side effects after intramuscular rather than intradermal administration. Systemic side effects increased at the higher dose in both frequency and severity, with 5 out of 8 volunteers experiencing severe nausea/vomiting, malaise, or rigors. Ex vivo T-cell responses to NP and M1 measured by IFN-γ ELISPOT assay were significantly increased after vaccination (prevaccination median of 123 spot-forming units/million peripheral blood mononuclear cells, postvaccination peak response median 339, 443, and 1443 in low-dose intradermal, low-dose intramuscular, and high-dose intramuscular groups, respectively), and the majority of the antigen-specific T cells were CD8+.Conclusions. We conclude that the vaccine was both safe and remarkably immunogenic, leading to frequencies of responding T cells that appear to be much higher than those induced by any other influenza vaccination approach. Further studies will be required to find the optimum dose and to assess whether the increased T-cell response to conserved influenza proteins results in protection from influenza disease.

Importance of mRNA Secondary Structural Elements for the Expression of Influenza Virus Genes

Development of novel vaccines and therapeutics often requires efficient expression of recombinant viral proteins. Here we show that mutations in essential functional regions of conserved influenza proteins NP and NS1, lead to reduced expression of these genes in vitro. According to in silico analysis, these mRNA regions possess distinct secondary structures sensitive to mutations. We identified a novel structural feature within a region in NS1 mRNA that encodes amino acids essential for NS1 function. Mutations altering this mRNA element lead to significantly reduced protein expression. Conversely, expression was not affected by mutations resulting in amino acid substitutions, when they were designed to preserve this secondary RNA structural element. Furthermore, altering this structure significantly reduced RNA transcription without affecting mRNA stability. Therefore, distinct internal secondary structures of viral mRNA may be important for viral gene expression. If such elements encode amino acids essential for the protein function, then early selection against mutations in this region will be beneficial for the virus. This might point at yet another mechanism of viral evolution, especially for RNA viruses. Finally, introducing mutations into viral genes while preserving their secondary RNA structure, suggests a new method for the generation of efficiently expressed recombinants of viral proteins.

A humanized anti-M2 scFv shows protective in vitro activity against influenza

M2 is one of the most conserved influenza proteins, and has been widely prospected as a potential universal vaccine target, with protection predominantly mediated by antibodies. In this paper we describe the creation of a humanized single chain Fv from 14C2, a potent monoclonal antibody against M2. We show that the humanized scFv demonstrates similar activity to the parental mAb: it is able to recognize M2 in its native context on cell surfaces and is able to show protective in vitro activity against influenza, and so represents a potential lead antibody candidate for universal prophylactic or therapeutic intervention in influenza.

The proteosomal degradation of fusion proteins cannot be predicted from the proteosome susceptibility of their individual components

It is assumed that the proteosome-processing characteristics of fusion constructs can be predicted from the sum of the proteosome sensitivity of their components. In the present study, we observed that a fusion construct consisting of proteosome-degradable proteins does not necessarily result in a proteosome-degradable chimera. Conversely, fusion of proteosome-resistant proteins may result in a proteosome-degradable composite. We previously demonstrated that conserved influenza proteins can be unified into a single fusion antigen that is protective, and that vaccination with combinations of proteosome-resistant and proteosome-degradable antigens resulted in an augmented T-cell response. In the present study we constructed proteosome-degradable mutants of conserved influenza proteins NP, M1, NS1, and M2. These were then fused into multipartite proteins in different positions. The stability and degradation profiles of these fusion constructs were demonstrated to depend on the relative position of the individual proteins within the chimeric molecule. Combining unstable sequences of either NP and M1 or NS1 and M2 resulted in either rapidly proteosome degraded or proteosome-resistant bipartite fusion mutants. However, further unification of the proteosome-degradable forms into a single four-partite fusion molecule resulted in relatively stable chimeric proteins. Conversely, the addition of proteosome-resistant wild-type M2 to proteosome-resistant NP-M1-NS1 fusion protein lead to the decreased stability of the resulting four-partite multigene products, which in one case was clearly proteosome dependent. Additionally, a highly destabilized form of M1 failed to destabilize the wild-type NP. Collectively, we did not observe any additive effect leading to proteosomal degradation/nondegradation of a multigene construct.

Protection against mouse and avian influenza A strains via vaccination with a combination of conserved proteins NP, M1 and NS1

Experimental data accumulated over more than a decade indicate that cross-strain protection against influenza may be achieved by immunization with conserved influenza proteins. At the same time, the efficacy of immunization schemes designed along these lines and involving internal influenza proteins, mostly NP and M1, has not been sufficient. To test the immunogenicity and protective efficacy of DNA vaccination with a combination of NP, M1 and NS1 genes of influenza virus. The immunogenicity and protective efficacy of DNA vaccination with NP, M1 and NS1 was tested in mice and chickens. Mice were challenged with mouse-adapted viral strains H3N2 and H5N2 and chicken challenged with avian H5N3 virus. In these settings, wild-type NS1 did not impede the cellular and humoral response to NP/M1 immunization in vivo. Moreover, addition of NS1-encoding plasmid to the NP/M1 immunization protocol resulted in a significantly increased protective efficacy in vivo. The addition of NS1 to an influenza immunization regimen based on conserved proteins bears promise. It is feasible that upon further genetic modification of these and additional conserved influenza proteins, providing for their higher safety, expression and immunogenicity, a recombinant vaccine based on several structural and non-structural proteins or their epitopes will offer broad anti-influenza protection in a wide range of species.

Immundominante Peptide von konservierten Influenzaproteinen – Eine Möglichkeit für eine wirksamere Impfung älterer Personen?

Influenza-specific CD8+ T cells are important for the clearance of infection especially in high risk groups such as elderly persons. Activation of these cells by immunization might therefore be a useful tool for a better protection of this specific age group. We therefore analyzed the frequency, phenotype and function of CD8+ T cells with specificity to the influenza M1(58-66) peptide in young, middle-aged and elderly persons ex vivo and after in vitro stimulation. Significantly lower numbers of M1(58-66)-specific CD8+ T cells were detected in the middle-aged and elderly compared to young donors. M1(58-66)-specific CD8+ T cells were either CD45RA(low)CD45RO(low) or CD45RA-CD45RO+, expressed CD28 and CD62L and did not produce perforin. There was no difference in the phenotype of influenza-specific CD8+ T cells between the three age groups. Despite the initially low numbers of M1(58-66)-specific CD8+ T cells in the older age groups, these cells could be expanded in vitro following peptide stimulation. They also acquired a CD45RO+CD28+ CD62L(+/-) phenotype and produced perforin. Our results demonstrate that although initially low in number, M1(58-66)-specific CD8+ T cells from elderly persons can be propagated and differentiated into perforin producing effector cells upon appropriate stimulation. M1(58-66) peptide or other immunodominant peptides derived from conserved influenza proteins could therefore be useful in future influenza vaccines in order to render elderly persons better protected against disease, in particular in the case of an influenza pandemic.