流感病毒M2蛋白胞外区与铜绿假单胞菌外毒素A融合蛋白的表达及其免疫原性研究

本研究构建了表达甲型流感病毒M2蛋白胞外区与铜绿假单胞菌外毒素A(PEA)融合蛋白的原核表达载体,根据铜绿假单胞菌外毒素A(PEA)核苷酸序列设计突变PCR引物并实施突变PCR,以获得PEA基因编码区第553位氨基酸密码子缺失的突变PEA(ntPE),从而产生无毒性的PEA突变基因,然后用合成的M2e编码区替换ntPE基因中的非必需区Ib,产生ntPE-M2e嵌合基因。将该嵌合基因导入pET表达载体以构建原核表达载体,将表达产物胶回收后与弗氏不完全佐剂联合皮下免疫BALB/c小鼠,终免两周后用5个LD50流感病毒A/PR/34/8株进行攻击。取动物血清作ELISA并取脾脏作ELISPOT试验结果表明,免疫组可以诱导小鼠产生抗M2e特异性抗体反应和细胞免疫反应并能够抑制病毒在肺内的复制。本研究为甲型流感病毒广谱疫苗的进一步研发打下了基础。     

A universal influenza A vaccine based on the extracellular domain of the M2 protein.

The antigenic variation of influenza virus represents a major health problem. However, the extracellular domain of the minor, virus-coded M2 protein is nearly invariant in all influenza A strains. We genetically fused this M2 domain to the hepatitis B virus core (HBc) protein to create fusion gene coding for M2HBc; this gene was efficiently expressed in Escherichia coli. Intraperitoneal or intranasal administration of purified M2HBc particles to mice provided 90-100% protection against a lethal virus challenge. The protection was mediated by antibodies, as it was transferable by serum. The enhanced immunogenicity of the M2 extracellular domain exposed on HBc particles allows broad-spectrum, long-lasting protection against influenza A infections.     

Influenza A virus can undergo multiple cycles of replication without M2 ion channel activity

Ion channel proteins are common constituents of cells and have even been identified in some viruses. For example, the M2 protein of influenza A virus has proton ion channel activity that is thought to play an important role in viral replication. Because direct support for this function is lacking, we attempted to generate viruses with defective M2 ion channel activity. Unexpectedly, mutants with apparent loss of M2 ion channel activity by an in vitro assay replicated as efficiently as the wild-type virus in cell culture. We also generated a chimeric mutant containing an M2 protein whose transmembrane domain was replaced with that from the hemagglutinin glycoprotein. This virus replicated reasonably well in cell culture but showed no growth in mice. Finally, a mutant lacking both the transmembrane and cytoplasmic domains of M2 protein grew poorly in cell culture and showed no growth in mice. Thus, influenza A virus can undergo multiple cycles of replication without the M2 transmembrane domain responsible for ion channel activity, although this activity promotes efficient viral replication.     

Protection against Influenza A Virus Challenge with M2e-Displaying Filamentous Escherichia coli Phages

Human influenza viruses are responsible for annual epidemics and occasional pandemics that cause severe illness and mortality in all age groups worldwide. Matrix protein 2 (M2) of influenza A virus is a tetrameric type III membrane protein that functions as a proton-selective channel. The extracellular domain of M2 (M2e) is conserved in human and avian influenza A viruses and is being pursued as a component for a universal influenza A vaccine. To develop a M2e vaccine that is economical and easy to purify, we genetically fused M2e amino acids 2–16 to the N-terminus of pVIII, the major coat protein of filamentous bacteriophage f88. We show that the resulting recombinant f88−M2e2-16 phages are replication competent and display the introduced part of M2e on the phage surface. Immunization of mice with purified f88−M2e2-16 phages in the presence of incomplete Freund’s adjuvant, induced robust M2e-specific serum IgG and protected BALB/c mice against challenge with human and avian influenza A viruses. Thus, replication competent filamentous bacteriophages can be used as efficient and economical carriers to display conserved B cell epitopes of influenza A.     

H9N2禽流感病毒M2 DNA疫苗初免-蛋白加强免疫的保护效果研究

流感病毒M2(基质蛋白2)是A型流感病毒的一个高度保守的蛋白。由于其免疫原性较弱,本研究采用M2 DNA疫苗初免-蛋白加强的策略来考察M2的免疫保护效果。制备A/Chicken/Jiangsu/07/2002(H9N2)流感病毒的M2 DNA疫苗以及经大肠杆菌表达的去除M2跨膜区的M2蛋白即sM2。以SPF级BALB/c小鼠为模型,电击法免疫M2 DNA疫苗,滴鼻法免疫sM2蛋白,免疫间隔三周,并于末次免疫后三周以致死量5LD50流感病毒H9N2攻击小鼠,通过检测小鼠存活率、体重丢失率、肺部病毒滴度及IgG抗体水平等指标来评价免疫的保护效果。实验结果表明,基于M2的疫苗采用DNA疫苗初免蛋白加强免疫二次的免疫程序能诱导较高的特异性抗体,明显减轻小鼠流感病症,提供完全的保护。     

Immunogenicity and protective efficacy of recombinant M2e.Hsp70c（Hsp70359–610） fusion protein against influenza virus infection in mice

New strategies in vaccine development are urgently needed to combat emerging influenza viruses and to reduce the risk of pandemic disease surfacing. Being conserved, the M2 e protein, is a potential candidate for universal vaccine development against influenza A viruses. Mycobacterium tuberculosis Hsp70（mHsp70） is known to cultivate the function of immunogenic antigen-presenting cells, stimulate a strong cytotoxic T lymphocyte（CTL） response, and stop the induction of tolerance. Thus, in this study, a recombinant protein from the extracellular domain of influenza A virus matrix protein 2（M2e）, was fused to the C-terminus of Mycobacterium tuberculosis Hsp70（Hsp70c）, to generate a vaccine candidate. Humoral immune responses, IFN-γ-producing lymphocyte, and strong CTL activity were all induced to confirm the immunogenicity of M2 e.Hsp70c（Hsp70359–610）. And challenge tests showed protection against H1N1 and H9N2 strains in vaccinated groups. Finally these results demonstrates M2 e.Hsp70c fusion protein can be a candidate for a universal influenza A vaccine.     

Studies on the IgA-independent immunological responses in mice to influenza virus challenge after oral vaccination with irradiated whole virus and an erythrocyte complex

Previous studies have described an oral influenza vaccine comprising whole irradiated virus and an erythrocyte complex (IV-EC), which gave broad-based protection against influenza virus challenge in mice. The present study examined the immune responses generated after live virus challenge of vaccinated mice, particularly to determine whether mice vaccinated with IV-EC had enhanced CTL activity to compensate for the previously reported diminution in lung IgA response. Oral vaccine groups examined were IV-EC, live virus alone (LV) or live virus-erythrocyte complex (LV-EC), compared with irradiated virus and erythrocyte alone controls. The antibody responses of IV-EC and LV-EC vaccinated mice showed significantly elevated lung and serum IgG2a levels post live virus challenge, with no comparable increases in IgG1 levels compared to controls. Spleen cells from IV-EC mice showed an enhanced post-challenge proliferative response to antigen compared with mice that had received live oral vaccines, indicating enhanced cellular activity post IV-EC immunization. However, CTL activity was not enhanced for IV-EC mice, and live virus-vaccinated mice had reduced CTL activity compared with controls, indicating that CTL were not important for post-vaccine protection. Cytokine analysis revealed a predominant IFN-gamma response in spleen cells from orally vaccinated mice, whereas IL-4 was not detected in any lung or spleen culture analysed. The results suggest, therefore, that protection from live influenza challenge after IV-EC or LV-EC vaccination was due to an IFN-mediated IgG2a response. Definitive confirmation of the role of these factors in post-vaccine protection can now be tested in IgG2a-depleted or IFN-gamma gene knockout mouse models.     

Plant-produced recombinant influenza vaccine based on virus-like HBc particles carrying an extracellular domain of M2 protein

Conventional influenza vaccines are based on a virus obtained in chicken embryos or its components. The high variability of the surface proteins of influenza virus, hemagglutinin and neuraminidase, requires strain-specific vaccines matching the antigenic specificity of newly emerging virus strains to be developed. A recombinant vaccine based on a highly conservative influenza virus protein M2 fused to a nanosized carrier particle can be an attractive alternative to traditional vaccines. We have constructed a recombinant viral vector based on potato X virus that provides for expression in the Nicotiana benthamiana plants of a hybrid protein M2eHBc consisting of an extracellular domain of influenza virus M2 protein (M2e) fused to hepatitis B core antigen (HBc). This vector was introduced into plant cells by infiltrating leaves with agrobacteria carrying the viral vector. The hybrid protein M2eHBc was synthesized in the infected N. benthamiana plants in an amount reaching 1–2% of the total soluble protein and formed virus-like particles with the M2e peptide presented on the surface. Methods of isolation and purification of M2eHBc particles from plant producers were elaborated. Experiments on mice have shown a high immunogenicity of the plant-produced M2eHBc particles and their protective effect against lethal influenza challenge. The developed transient expression system can be used for production of M2e-based candidate influenza vaccine in plants.     

Bacterial HA1 vaccine against pandemic H5N1 influenza virus: evidence of oligomerization, hemagglutination, and cross-protective immunity in ferrets

The impending influenza virus pandemic requires global vaccination to prevent large-scale mortality and morbidity, but traditional influenza virus vaccine production is too slow for rapid responses. We have developed bacterial systems for expression and purification of properly folded functional hemagglutinin as a rapid response to emerging pandemic strains. A recombinant H5N1 (A/Vietnam/1203/2004) hemagglutinin globular domain (HA1) was produced in Escherichia coli under controlled redox refolding conditions. Importantly, the properly folded HA1(1-320), i.e., HA1 lacking amino acids 321 to 330, contained ≥75% functional oligomers without addition of foreign oligomerization sequence. Site-directed mutagenesis mapped the oligomerization signal to the HA1 N-terminal Ile-Cys-Ile residues at positions 3 to 5. The purified HA1 oligomers (but not monomers) bound fetuin and agglutinated red blood cells. Upon immunization of rabbits, the oligomeric HA1(1-320) elicited potent neutralizing antibodies against homologous and heterologous H5N1 viruses more rapidly than HA1(28-320) containing only monomers. Ferrets vaccinated with oligomeric HA1 (but not monomeric HA1 with the N terminus deleted) at 15 and 3 μg/dose were fully protected from lethality and weight loss after challenge with homologous H5N1 (A/Vietnam/1203/2004, clade 1) virus, as well as heterologous clade 2.2 H5N1 (A/WooperSwan/Mongolia/244/2005) virus. Protection was associated with a significant reduction in viral loads in the nasal washes of homologous and heterologous virus challenged ferrets. This is the first study that describes the presence of an N-terminal oligomerization sequence in the globular domain of influenza virus hemagglutinin. Our findings suggest that functional oligomeric rHA1-based vaccines can be produced efficiently in bacterial systems and can be easily upscaled in response to a pandemic influenza virus threat.     

Guiding the Immune Response against Influenza Virus Hemagglutinin toward the Conserved Stalk Domain by Hyperglycosylation of the Globular Head Domain

Influenza virus hemagglutinin consists of a highly variable and immunodominant head domain and a more conserved but immunosubdominant stalk domain. We introduced seven N-linked glycosylation sites in the hemagglutinin head domain to shield the immunodominant antigenic sites. The hyperglycosylated hemagglutinin enhanced stalk-directed seroreactivity while dampening the head response in immunized mice. Upon influenza virus challenge, mice vaccinated with the hyperglycosylated hemagglutinin were better protected against morbidity and mortality than mice receiving the wild-type hemagglutinin.     

Influenza A vaccine based on the extracellular domain of M2: weak protection mediated via antibody-dependent NK cell activity

Vaccination of mice with a peptide corresponding to the extracellular part of M2 protein coupled to the immunodominant domain of hepatitis B core can protect mice from a lethal challenge with influenza A virus. As the extracellular part of M2 protein is highly conserved in all known human influenza A strains, such a vaccine may protect against all human influenza A strains, which would represent a major advantage over current vaccine strategies. The present study demonstrates that protection is mediated exclusively by Abs, a very important feature of a successful preventive vaccine. However, these Abs neither bind efficiently to the free virus nor neutralize virus infection, but bind to M2 protein expressed on the surface of virus-infected cells. The presence of NK cells is important for protection, whereas complement is not, supposing that protection is mediated via Ab-dependent, cell-mediated cytotoxicity. The absence of neutralizing Abs results in much weaker protection than that achieved by vaccination with UV-inactivated influenza virus. Specifically, whereas neutralizing Abs completely eliminate signs of disease even at high viral challenge doses, M2-specific Abs cannot prevent infection, but merely reduce disease at low challenge doses. M2-specific Abs fail to protect from high challenge doses, as vaccinated mice undergo lethal infection under these conditions. In conclusion, protection mediated by M2-hepatitis B core vaccine would be insufficient during the yearly epidemics, for which full protection is desirable, and overall is clearly inferior to protection achieved by immunization with classical inactivated viral preparations.     

Immunization with M2e-Displaying T7 Bacteriophage Nanoparticles Protects against Influenza A Virus Challenge

Considering the emergence of highly pathogenic influenza viruses and threat of worldwide pandemics, there is an urgent need to develop broadly-protective influenza vaccines. In this study, we demonstrate the potential of T7 bacteriophage-based nanoparticles with genetically fused ectodomain of influenza A virus M2 protein (T7-M2e) as a candidate universal flu vaccine. Immunization of mice with non-adjuvanted T7-M2e elicited M2e-specific serum antibody responses that were similar in magnitude to those elicited by M2e peptide administered in Freund’s adjuvant. Comparable IgG responses directed against T7 phage capsomers were induced following vaccination with wild type T7 or T7-M2e. T7-M2e immunization induced balanced amounts of IgG₁ and IgG_2a antibodies and these antibodies specifically recognized native M2 on the surface of influenza A virus-infected mammalian cells. The frequency of IFN-γ-secreting T cells induced by T7-M2e nanoparticles was comparable to those elicited by M2e peptide emulsified in Freund’s adjuvant. Emulsification of T7-M2e nanoparticles in Freund’s adjuvant, however, induced a significantly stronger T cell response. Furthermore, T7-M2e-immunized mice were protected against lethal challenge with an H1N1 or an H3N2 virus, implying the induction of hetero-subtypic immunity in our mouse model. T7-M2e-immunized mice displayed considerable weight loss and had significantly reduced viral load in their lungs compared to controls. We conclude that display of M2e on the surface of T7 phage nanoparticles offers an efficient and economical opportunity to induce cross-protective M2e-based immunity against influenza A.     

Age Dependence of Immunity Induced by a Candidate Universal Influenza Vaccine in Mice

Influenza has a major impact on the elderly due to increased susceptibility to infection with age and poor response to current vaccines. We have studied universal influenza vaccine candidates based on influenza A nucleoprotein and matrix 2 (A/NP+M2). Long-lasting protection against influenza virus strains of divergent subtypes is induced, especially with mucosal immunization. Here, we tested universal vaccination in BALB/c mice of different ages. Vaccination used intramuscular DNA priming to A/NP+M2 followed by intranasal (i.n.) boosting with recombinant adenoviruses (rAd) expressing the same antigens, or only A/NP+M2-rAd given i.n. Antigen-specific systemic antibody responses were induced in young, middle-aged, and elderly mice (2, 11–17, and 20 months old, respectively), but decreased with age. Antibody responses in bronchoalveolar lavage (BAL) were detected only in young mice. Antigen-specific T cell responses were seen in young and middle-aged but not elderly mice. A/NP+M2 vaccination by the two regimens above protected against stringent challenge in young and middle-aged mice, but not in elderly mice. However, mice vaccinated with A/NP-rAd or A/M2-rAd during their youth were partially protected against challenge 16 months later when they were elderly. In addition, a regimen of two doses of A/NP+M2-rAd given i.n. one month apart beginning in old age protected elderly mice against stringent challenge. This study highlights the potential benefit of cross-protective vaccines through middle age, and suggests that their performance might be enhanced in elderly individuals who had been exposed to influenza antigens early in life, as most humans have been, or by a two-dose rAd regimen given later in life.     

Synthetic Long Peptide Influenza Vaccine Containing Conserved T and B Cell Epitopes Reduces Viral Load in Lungs of Mice and Ferrets

Currently licensed influenza vaccines mainly induce antibodies against highly variable epitopes. Due to antigenic drift, protection is subtype or strain-specific and regular vaccine updates are required. In case of antigenic shifts, which have caused several pandemics in the past, completely new vaccines need to be developed. We set out to develop a vaccine that provides protection against a broad range of influenza viruses. Therefore, highly conserved parts of the influenza A virus (IAV) were selected of which we constructed antibody and T cell inducing peptide-based vaccines. The B epitope vaccine consists of the highly conserved HA2 fusion peptide and M2e peptide coupled to a CD4 helper epitope. The T epitope vaccine comprises 25 overlapping synthetic long peptides of 26-34 amino acids, thereby avoiding restriction for a certain MHC haplotype. These peptides are derived from nucleoprotein (NP), polymerase basic protein 1 (PB1) and matrix protein 1 (M1). C57BL/6 mice, BALB/c mice, and ferrets were vaccinated with the B epitopes, 25 SLP or a combination of both. Vaccine-specific antibodies were detected in sera of mice and ferrets and vaccine-specific cellular responses were measured in mice. Following challenge, both mice and ferrets showed a reduction of virus titers in the lungs in response to vaccination. Summarizing, a peptide-based vaccine directed against conserved parts of influenza virus containing B and T cell epitopes shows promising results for further development. Such a vaccine may reduce disease burden and virus transmission during pandemic outbreaks.     

Antibody-forming cell response to virus challenge in mice immunized with DNA encoding the influenza virus hemagglutinin.

Immunization of mice with DNA encoding the influenza virus hemagglutinin (HA) affords complete protection against lethal influenza virus infection and the means to investigate the mechanisms of B-cell responsiveness to virus challenge. Using a single-cell enzyme-linked immunospot assay, we sought to determine the localization of HA-specific antibody-forming cells (AFCs) during the development of humoral immunity in mice given HA DNA vaccine by gene gun. At 33 days postvaccination, populations of AFCs were maintained in the spleen and bone marrow. In response to lethal challenge with influenza virus, the AFCs became localized at the site of antigenic challenge, i.e., within the draining lymph nodes of the lung compartment. Immunoglobulin G (IgG)- and IgA-producing AFCs were detected in lymph nodes of the upper and lower respiratory tracts, underscoring their importance in clearing virus from the lungs. Response to challenge required competent CD4+ T cells, without which no AFCs were generated, even those producing IgM. By contrast, in mice vaccinated with an HA-containing subunit vaccine, fewer AFCs were generated in response to challenge, and these animals were less capable of resisting infection. Our findings demonstrate the comparable localization of AFCs in response to challenge in mice vaccinated with either HA DNA or live virus. Moreover, the former strategy generates both IgG- and IgA-producing plasma cells.     

Cowpea mosaic virus chimeric particles bearing the ectodomain of matrix protein 2 (M2E) of the influenza a virus: Production and characterization

The epitope presentation system for the ectodomain of the M2 protein (M2e) of the influenza A virus was constructed on the basis of the cowpea mosaic virus (CPMV) for expression in the plant Vigna unguiculata. CPMV is widely used as a vector to produce immunogenic chimeric virus particles (CVPs) bearing epitopes of various infectious human and animal pathogens. To produce chimeric CPMV particles in plants, two binary vectors were constructed to bear a modified gene coding for the CPMV S-coat protein with insertions of M2e epitopes of human influenza and bird influenza viruses. Antigenic and immunogenic properties of CVPs were investigated in mice immunization experiments. CVPs were shown to induce anti-M2e IgG production and to partly protect mice against a challenge with low doses of the influenza virus. However, low infectivity and immunogenicity of chimeric CPMV particles indicate that the plant virus-based systems for M2e epitope presentation requires further optimization in order to use plants as a possible source of flu vaccines.     

Monomeric M2e antigen in VesiVax® liposomes stimulates protection against type a strains of influenza comparable to liposomes with multimeric forms of M2e

Abstract

Given the interest in the ectodomain of the matrix 2 (M2e) channel protein as a target for development of a universal influenza vaccine, we examined the role of the antigen configuration of M2e in generating a protective immune response. A series of M2e mutations and a truncated M2e segment were prepared as a means of controlling the formation of monomer, dimer, and higher order multimeric forms of M2e. Each of these M2e peptides was incorporated into a liposome-based vaccine technology platform previously shown to stimulate a protective response to influenza A infection using M2e as a mixture of monomers, dimers and multimers (L-M2e1-HD/MPL). Our results using these modified forms of M2e produced 90–100% survival following lethal challenge with H1N1 (A/PR/8/34) in both inbred BALB/c and outbred Swiss Webster mice vaccinated with a truncated monomeric form of the M2 protein, M2e_1–15 in liposomes. These observations show that a tetrameric configuration is not required to elicit significant protection when the M2e antigen is formulated in immunogenic liposomes and further, that the first 15 amino acids of M2e likely play a primary role in providing the protective immune response.     

A Novel Subnucleocapsid Nanoplatform for Mucosal Vaccination against Influenza Virus That Targets the Ectodomain of Matrix Protein 2

In this study, subnucleocapsid nanorings formed by the recombinant nucleoprotein (N) of the respiratory syncytial virus were evaluated as a platform to anchor heterologous antigens. The ectodomain of the influenza virus A matrix protein 2 (M2e) is highly conserved and elicits protective antibodies when it is linked to an immunogenic carrier, making it a promising target to develop universal influenza vaccines. In this context, one or three M2e copies were genetically linked to the C terminus of N to produce N-M2e and N-3M2e chimeric recombinant nanorings. Mice were immunized intranasally with N-M2e or N-3M2e or with M2e or 3M2e control peptides. N-3M2e-vaccinated mice showed the strongest mucosal and systemic antibody responses. These mice presented a reduced viral load and minor weight loss, and all survived upon challenge with influenza virus A/PR8/34 (H1N1) (PR8). We compared the intranasal route to the subcutaneous route of N-3M2e immunization. Only the intranasal route induced a strong local IgA response and led to the protection of mice upon challenge. Finally, we demonstrated that the induction of anti-M2e antibodies by N-3M2e is not impaired by preexisting anti-N immunity. Overall, these results show that the N nanoring is a potent carrier for mucosal delivery of vaccinal antigens.     

Soluble recombinant influenza vaccines.

Soluble, recombinant forms of influenza A virus haemagglutinin and neuraminidase have been produced in cells of lower eukaryotes, and shown in a mouse model to induce complete protective immunity against a lethal virus challenge. Soluble neuraminidase, produced in a baculovirus system, consisted of tetramers, dimers and monomers. Only the tetramers were enzymatically active. The immunogenicity decreased very considerably in the order tetra > di > mono. Therefore, we fused the head part of the neuraminidase gene to a tetramerizing leucine zipper sequence; the resulting product was enzymatically active, tetrameric neuraminidase. The protective immunity induced by this engineered neuraminidase, however, remained fairly strain-specific. A third influenza A virus protein, the M2 protein, has only 23 amino acids exposed on the outer membrane surface. This extracellular part, M2e, has been remarkably conserved in all human influenza A strains since 1933. By fusing the M2e sequence to hepatitis B virus core protein, we could obtain highly immunogenic particles that induced complete, strain-independent, long-lasting protection in mice against a lethal viral challenge. Native M2 is a tetrameric protein and this conformation of the M2e part can also be mimicked by fusing this sequence to a tetramerizing leucine zipper. The potential of the resulting protein as a vaccine candidate remains to be evaluated.     

Protection against lethal lymphocytic choriomeningitis virus (LCMV) infection by immunization of mice with an influenza virus containing an LCMV epitope recognized by cytotoxic T lymphocytes.

The reverse genetics system has made it possible to modify the influenza virus genome. By this method, we were able to assess influenza virus as a vaccine vector for protecting BALB/c mice against otherwise lethal lymphocytic choriomeningitis virus (LCMV) infection. A single dose of influenza virus [A/WSN/33 (H1N1)] bearing a cytotoxic T-lymphocyte-specific epitope of the LCMV nucleoprotein (residues 116 to 127) in the neuraminidase stalk protected mice against LCMV challenge for at least 4 months. The immunity was mediated by cytotoxic T lymphocytes and was haplotype specific, indicating that the observed protective response was solely a consequence of prior priming with the H-2d LCMV nucleoprotein epitope expressed in the recombinant influenza virus. We also found that as many as 58 amino acids could be inserted into the neuraminidase stalk without loss of viral function. These findings demonstrate the potential of influenza virus as a vaccine vector, with the neuraminidase stalk as a repository for foreign epitopes.