Development of a candidate influenza vaccine based on virus-like particles displaying influenza M2e peptide into the immunodominant loop region of hepatitis B core antigen: Insertion of multiple copies of M2e increases immunogenicity and protective efficiency

The extracellular domain of the transmembrane protein M2 (M2e) of influenza A virus is a promising target for the development of “universal” vaccines against influenza. M2e is a poor immunogen by itself; however, when M2e is linked to an appropriate carrier, such as hepatitis B virus core (HBc) particles, it becomes highly immunogenic. Insertions of target peptides into the surface-exposed major immunodominant loop region (MIR) of the HBc antigen are especially immunogenic, but such insertions often affect the protein folding and formation of recombinant virus-like particles. To facilitate an appropriate conformation of the M2e insert, we introduced flexible linkers at the junction points between the insert and flanking HBc sequences. This approach allowed the construction of recombinant HBc particles carrying 1, 2 and 4 copies of M2e in the MIR region. These particles were produced in Escherichia coli and purified to homogeneity. The immune response and protective activity of hybrid HBc particles in mice correlated with the number of inserted M2e peptides: the highest immunogenicity and complete protection of mice against the lethal challenge by influenza virus was observed with particles carrying four copies of M2e. The possibility of the simultaneous presentation of M2e peptides from several important influenza strains on a single HBc particle could also facilitate the development of a broad-specificity vaccine efficient not only against influenza A strains of human origin but also for newly emerging strains of animal origin, such as the avian influenza.     

Recombinant baculovirus vaccine containing multiple M2e and adjuvant LTB induces T cell dependent,cross-clade protection against H5N1 influenza virus in mice

H5N1, highly pathogenic avian influenza poses, a threat to animal and human health. Rapid changes in H5N1 viruses require periodic reformulation of the conventional strain-matched vaccines, thus emphasizing the need for a broadly protective influenza vaccine. Here, we constructed BV-Dual-3M2e-LTB, a recombinant baculovirus based on baculovirus display and BacMam technology. BV-Dual-3M2e-LTB harbors a gene cassette expressing three tandem copies of the highly conserved extracellular domain of influenza M2 protein (M2e) and the mucosal adjuvant, LTB. We showed that BV-Dual-3M2e-LTB displayed the target protein (M2e/LTB) on the baculoviral surface and expressed it in transduced mammalian cells. BV-Dual-3M2e-LTB, when delivered nasally in mice, was highly immunogenic and induced superior levels of anti-M2e IgA than the non-adjuvanted baculovirus (BV-Dual-3M2e). Importantly, after challenge with different H5N1 clades (clade 0, 2.3.2.1, 2.3.4 and 4), mice inoculated with BV-Dual-3M2e-LTB displayed improved survival and decreased lung virus shedding compared with mice inoculated with BV-Dual-3M2e. The enhanced protection from BV-Dual-3M2e-LTB is mediated by T cell immunity and is primarily based on CD8⁺ T cells, while mucosal antibodies alone were insufficient for protection from lethal H5N1 challenge. These results suggest that BV-Dual-3M2e-LTB has potential to protect against a broad range of H5N1 strains thereby providing a novel direction for developing broadly protective vaccines based on cellular immunity.     

Influenza virus-like particles comprised of the HA, NA, and M1 proteins of H9N2 influenza virus induce protective immune responses in BALB/c mice

Avian influenza viruses represent a growing threat for an influenza pandemic. To develop recombinant vaccine for avian influenza of the H9N2 subtype, we expressed in insect cells virus-like particles (VLPs) consisting of three structural proteins of influenza A/Hong Kong/1073/99 (H9N2) virus. Upon infection of Sf9 cells with recombinant baculoviruses, the hemagglutinin (HA), neuraminidase (NA), and matrix (M1) proteins were co-expressed in the infected cells, self-assembled, and released into the culture medium as VLPs of 80–120 nm in diameter. VLPs exhibited functional characteristics of influenza virus including hemagglutination and neuraminidase activities. In BALB/c mice, VLPs elicited serum antibodies specific for influenza A/Hong Kong/1073/99 (H9N2) virus and inhibited replication of the influenza virus after challenge. Thus, VLPs represent a potential strategy for the development of human vaccines against avian influenza H9N2 viruses.     

Improved design and intranasal delivery of an M2e-based human influenza A vaccine

M2 is the third integral membrane protein of influenza A. M2e, the extracellular, 23 amino acid residues of M2, has been remarkably conserved in all human influenza A strains. This prompted us to evaluate the use of M2e as a potential broad-spectrum immunogen in a mouse model for influenza infection. Genetic fusion of the M2e and hepatitis B virus core (HBc) coding sequences allowed us to obtain highly immunogenic virus-like particles. This M2e-HBc vaccine induced complete protection in mice against a lethal influenza challenge. Protective immunity was obtained regardless of the position of M2e in the M2e-HBc chimera at the amino-terminus or inserted in the immuno-dominant loop of the HBc protein. Increasing the copy number of M2e inserted at the N-terminus from one to three per monomer (240-720 per particle) significantly enhanced the immune response and reduced the number of vaccinations required for complete protection against a lethal challenge with influenza A virus. A series of M2e-HBc constructs was subsequently combined with CTA1-DD, a recombinant cholera toxin A1 derived mucosal adjuvant, to test its efficacy as an intranasally delivered vaccine. All hybrid VLPs tested with CTA1-DD completely protected mice from a potentially lethal infection and, in addition, significantly reduced morbidity. Overall, increased resistance to influenza challenge in the mice correlated with an enhanced Th1-type M2e-specific antibody response induced by vaccination. These results show that M2e is a valid and versatile vaccine candidate to protect against any strain of human influenza A.     

A biomimetic VLP influenza vaccine with interior NP/exterior M2e antigens constructed through a temperature shift-based encapsulation strategy

Here we present a biomimetic strategy towards an influenza vaccine design based on hepatitis B virus core virus-like particles (HBc VLP). To this end, a temperature-shift based encapsulation process based on analysis of the unique thermal-associated structural flexibility of HBc VLP nanocages was proposed and proved efficient for encapsulation of antigen inside the VLP. By displaying a matrix protein 2 ectodomain (M2e) antigen on the exterior of HBc VLP through genetic fusion, and encapsulate a conserved internal nucleoprotein (NP) antigen peptide inside the VLP, a biomimetic dual-antigen influenza vaccine with interior NP/exterior M2e was constructed. For comparison, another non-biomimetic dual-antigen vaccine with interior M2e/exterior NP, and other four VLP-based single-antigen vaccines with NP or M2e either being encapsulated inside or genetically displayed outside the VLP were also constructed. Upon intraperitoneal immunization in mice, the dual-antigen VLP influenza vaccine elicited both NP and M2e-specific antibodies, which were stronger than those elicited by the single-antigen vaccines. Most importantly, after a lethal challenge of H1N1 virus, the biomimetic dual-antigen vaccine conferred the mice 100% protection without noticeable body weight loss in the absence of any adjuvant. While the protective efficacy conferred by the non-biomimetic one was only 62.5%, accompanying 12.5% body weight loss in the immunized mice. Besides the high level of antigen-specific antibodies, more efficient formation of total germinal center (GC) B cells and a higher level of effector memory CD8⁺ T cell population were observed in the biomimetic vaccine group, as compared with the non-biomimetic one. All these results demonstrate that VLP assembly and display of antigens in a biomimetic manner making this a promising strategy for the production of efficient universal vaccines to influenza and other rapidly emerging pathogens.     

Characterization of the M protein and nucleoprotein genes of an avian influenza A virus which are involved in host range restriction in monkeys

A reassortant virus possessing RNA segment 7, which codes for the M1 and M2 proteins, of the avian influenza A/Mallard/New York/6750/78 (H2N2) virus and the other seven RNA segments of the human influenza A/Udorn/307/72 (H3N2) virus had been shown previously to be markedly restricted in replication in the respiratory tract of squirrel monkeys. In contrast, a reassortant possessing segment 7 of another avian influenza virus, A/Pintail/Alberta/119/79 (H4N6), and the seven other RNA segments from the A/Udorn/72 virus was not restricted. The nucleotide and deduced amino acid sequence of the RNA segment 7 of each virus was determined to identify the structural basis for the attenuation phenotype specified by RNA segment 7 of the A/Mallard/78 virus. Analysis of the deduced amino acid sequences revealed only two amino acid differences in the M1 protein and one difference in the M2 protein, suggesting that the attenuation phenotype of a reassortant virus possessing segment 7 of the A/Mallard/78 virus may be specified by one to three amino acids. Reassortant viruses possessing RNA segment 6, which codes for the nucleoprotein, of either avian influenza virus and the other seven RNA segments of a human influenza virus were also restricted in replication in squirrel monkeys. A comparison of the deduced amino acid sequences of the two avian nucleopeoteins demonstrated only three amino acid differences indicating that these two avian viruses possess NP genes that are highly related. The high degree of relatedness of both the NP and M proteins of these two avian viruses contrasts with their divergent surface antigens. The structural basis for the attenuation phenotype of the NP gene of the A/Mallard/78 virus is being investigated.     

Complete protection against a H5N2 avian influenza virus by a DNA vaccine expressing a fusion protein of H1N1 HA and M2e

Most influenza vaccines target hemagglutinin (HA) in order to protect the host against infection. However, theses vaccines are strain-specific due to major antigenic variations of HA. Since it is difficult to predict epidemic and pandemic strains of influenza virus, the development of effective vaccines against divergent influenza viruses is urgently needed. Although M2e-based vaccines are associated with weaker protection than HA-based vaccines that induce neutralizing antibodies against challenge virus matched-strain, the extracellular domain of Matrix 2 protein (M2e) is one of a potential broad-spectrum immunogen because it contains highly conserved sequences among influenza A viruses. In this study, M2e sequence was fused to H1N1 HA DNA (M2e-HA) and the immunogenicity and antiviral efficacy of this DNA vaccine was evaluated in response to challenge with a heterosubtypic H5N2 avian influenza virus. Compared to vaccination with HA or M2e DNA alone, vaccination with M2e-HA DNA or combination of M2e DNA and HA DNA (M2e DNA + HA DNA) induced a broad immunity without evidence of immune interference. In addition, HA-specific CD8⁺ and M2e-specific T cell responses elicited by M2e-HA DNA vaccination were significantly higher than those of HA or M2e DNA vaccine alone, respectively. Following challenge with a heterosubtypic influenza virus infection, vaccination with M2e-HA DNA conferred complete protection against mortality. In combination, these results suggest that DNA vaccines expressing a fusion protein, M2e-HA, may provide an attractive approach for the development of broad-spectrum influenza vaccines.     

Evaluation of influenza virus-like particles and Novasome adjuvant as candidate vaccine for avian influenza

The development of safe and effective vaccines for avian influenza viruses is a priority for pandemic preparedness. Adjuvants improve the efficacy of vaccines and may allow antigen sparing during a pandemic. We have previously shown that influenza virus-like particles (VLPs) comprised of HA, NA, and M1 proteins represent a candidate vaccine for avian influenza H9N2 virus [Pushko P, Tumpey TM, Fang Bu, Knell J, Robinson R, Smith G. Influenza virus-like particles comprised of the HA, NA, and M1 proteins of H9N2 influenza virus induce protective immune responses in BALB/c mice. Vaccine 2005;23(50):5751-9]. In this study, an H9N2 VLP vaccine and recombinant HA (rH9) vaccine were evaluated in three animal models. The H9N2 VLP vaccine protected mice and ferrets from challenge with A/Hong Kong/1073/99 (H9N2) virus. Novasome adjuvant improved immunogenicity and protection. Positive effect of the adjuvant was also detected using the rH9 vaccine. The results have implications for the development of safe and effective vaccines for avian influenza viruses with pandemic potential.     

Development of live attenuated Bordetella pertussis strains expressing the universal influenza vaccine candidate M2e

The attenuated Bordetella pertussis BPZE1 vaccine strain represents an attractive platform for the delivery of heterologous vaccine candidates via the nasal route. The filamentous hemagglutinin (FHA) has been used to secrete or expose the foreign antigens at the bacterial surface. In this study, one, two and three copies of the Cys-containing ectodomain of matrix protein 2 (M2e) from influenza A virus were genetically fused to full length FHA and expressed in BPZE1. The secretion efficacy of the FHA-(M2e)_1,2,3 chimera in the extracellular milieu and the ability of the recombinant bacteria to colonize the mouse lungs inversely correlated with the number of M2e copies fused to FHA. Nevertheless FHA-(M2e)₃-producing bacteria (BPLR3) triggered the highest systemic anti-M2e antibody response upon nasal administration to BALB/c mice. Nasal immunization with BPLR3 bacteria resulted in a significant reduction in the viral loads upon challenge with H1N1/PR8 influenza A virus, but did not improve the survival rate compared to BPZE1-immunized mice. Furthermore, since previous work reported that disulfide bond formation in Cys-containing passenger antigens affects the secretion efficacy of the FHA chimera, the dsbA gene encoding a periplasmic disulfide isomerase was deleted in the FHA-(M2e)₃-producing strain. Despite improving significantly the secretion efficacy of the FHA-(M2e)₃ chimera, the dsbA deletion did not result in higher anti-M2e antibody titers in mice, due to impaired bacterial fitness and colonization ability.     

M2SR,a novel live influenza vaccine,protects mice and ferrets against highly pathogenic avian influenza

The emergence of highly pathogenic avian influenza H5N1 viruses has heightened global concern about the threat posed by pandemic influenza. To address the need for a highly effective universal influenza vaccine, we developed a novel M2-deficient single replication (M2SR) influenza vaccine virus and previously reported that it provided strong heterosubtypic protection against seasonal influenza viruses in mice. In the current study, we assessed M2SR induced protection against H5N1 influenza in mice and ferrets.Mice were intranasally inoculated with M2SR viruses containing the HA and NA from A/Vietnam/1203/2004 (M2SR H5N1) or A/California/07/2009 (M2SR H1N1). All M2SR vaccinated mice survived lethal challenge with influenza A/Vietnam/1203/2004 (H5N1), whereas 40% of mice vaccinated with recombinant H5 HA and none of the naïve controls survived. M2SR H5N1 provided sterile immunity, whereas low levels of virus were detected in the lungs of some M2SR H1N1 vaccinated mice. In contrast, recombinant H5 HA vaccinated mice and naïve controls showed systemic infection.M2SR H5N1 induced strong serum and mucosal antibody responses (IgG and IgA classes) against H5 HA, with high hemagglutination inhibition (HAI) titers. In contrast, while M2SR H1N1 elicited cross-reactive antibodies recognizing the H5 HA2 stalk region or the neuraminidase, no HAI activity against H5N1 virus was detected after M2SR H1N1 immunization.Both M2SR H5N1 and H1N1 also protected ferrets against lethal challenge with A/Vietnam/1203/2004. A prime–boost regimen provided optimal protection with no virus detected in the respiratory tract or brain after challenge. As in the mouse model, only the M2SR H5N1 vaccine induced HAI antibodies against the challenge virus in ferrets, while the M2SR H1N1 was able to provide protection without the induction of HAI antibodies.In summary, effective protection against highly pathogenic H5N1 influenza virus was provided by both homologous H5N1 M2SR and heterologous H1N1 M2SR demonstrating the cross-protective attributes of the M2SR platform.     

Human infections with novel reassortant influenza A(H3N2)v viruses, United States, 2011

During July-December 2011, a variant virus, influenza A(H3N2)v, caused 12 human cases of influenza. The virus contained genes originating from swine, avian, and human viruses, including the M gene from influenza A(H1N1)pdm09 virus. Influenza A(H3N2)v viruses were antigenically distinct from seasonal influenza viruses and similar to proposed vaccine virus A/Minnesota/11/2010.     

Induction of protection against divergent H5N1 influenza viruses using a recombinant fusion protein linking influenza M2e to Onchocerca volvulus activation associated protein-1 (ASP-1) adjuvant

Our previous studies have shown the adjuvanticity of an Onchocerca volvulus recombinant protein, Ov-ASP-1 (ASP-1), when administered in an aqueous formulation with bystander vaccine antigens or commercial vaccines. In this study, we reported a novel formulation that took advantage of the protein nature of the ASP-1 adjuvant by creating recombinant fusion protein vaccines linking the highly conserved extracellular domain of M2 protein (M2e) consensus sequence of H5N1 influenza viruses with the ASP-1 adjuvant. Two recombinant fusion proteins designated M2e-ASP-1 and M2e3-ASP-1 were studied, in which ASP-1 was fused with one or three tandem copies of the M2e antigen. Our results show that these novel recombinant influenza vaccines, particularly M2e3-ASP-1, induced strong anti-M2e-specific humoral and cellular immune responses in the established mouse model. Furthermore, M2e3-ASP-1 was able to provide significant cross-clade protection against divergent H5N1 viruses. Consequently, this study has demonstrated a potential novel vaccine formulation that could provide a complementary prophylactic strategy in preventing the threat of future influenza outbreak resulting from rapid evolution of the H5N1 virus and co-circulation of multiple antigenic variants in various regions.     

基于纳米孔测序技术对新型重组H3N2禽流感病毒的测序鉴定及其基因特征分析

目的 利用纳米孔测序技术对新型重组H3N2禽流感病毒进行测序鉴定并分析病毒的基因特征。方法 对2021年广州市某农贸市场外环境H3N2禽流感阳性样本进行鸡胚培养,联合病毒全基因组序列靶向扩增和纳米孔测序技术进行病毒高通量测序。通过生物信息学软件,分析病毒基因特点。结果 系统发育进化树显示,该毒株各基因片段均属于欧亚进化分支,宿主来源均为禽源。其HA基因与我国H3N6禽流感病毒亲缘关系较近。NA基因与2017-2020年我国H3N2禽流感病毒亲缘关系较近。PB1基因与2016-2022年我国广西壮族自治区、福建省H5N6禽流感病毒亲缘关系较近,与广州市H5N6禽流感流行株PB1基因亲缘关系较远。其余内部基因片段来源复杂,具有明显的遗传多样性。分子特征显示,该毒株具有典型的低致病性禽流感病毒分子特征,倾向结合禽源受体。生物特性相关重要蛋白位点上,该毒株发生PB2-L89V、PB1-L473V、NP-A184K、M1-N30D/T215A、NS1-P42S/N205S等致病性增强突变。结论 本研究通过纳米孔测序鉴定一株新型重组H3N2禽流感病毒,分析发现该病毒与H5N6禽流感病毒发生了基因重组。目前该病毒跨种传播能力较低,但有必要密切关注H3亚型禽流感病毒的流行和变异。     

Transmission of avian influenza virus (H3N2) to dogs

In South Korea, where avian influenza virus subtypes H3N2, H5N1, H6N1, and H9N2 circulate or have been detected, 3 genetically similar canine influenza virus (H3N2) strains of avian origin (A/canine/Korea/01/2007, A/canine/Korea/02/2007, and A/canine/Korea/03/2007) were isolated from dogs exhibiting severe respiratory disease. To determine whether the novel canine influenza virus of avian origin was transmitted among dogs, we experimentally infected beagles with this influenza virus (H3N2) isolate. The beagles shed virus through nasal excretion, seroconverted, and became ill with severe necrotizing tracheobronchitis and bronchioalveolitis with accompanying clinical signs (e.g., high fever). Consistent with histologic observation of lung lesions, large amounts of avian influenza virus binding receptor (SAalpha 2,3-gal) were identified in canine tracheal, bronchial, and bronchiolar epithelial cells, which suggests potential for direct transmission of avian influenza virus (H3N2) from poultry to dogs. Our data provide evidence that dogs may play a role in interspecies transmission and spread of influenza virus.     

Characterization of the murine Th2 response to immunization with liposomal M2e influenza vaccine

While the current influenza vaccine strategy is dependent on eliciting neutralizing antibodies to the hemagglutinin (H or HA) surface glycoprotein, antigenic drifts and occasional antigenic shifts necessitate constant surveillance and annual updates to the vaccine components. The ectodomain of the matrix 2 (M2e) channel protein has been proposed as a universal vaccine candidate, although it has not yet been shown to elicit neutralizing antibodies. Utilizing a liposome-based vaccine technology, an M2e vaccine (L-M2e-HD/MPL) was tested and shown to stimulate the production of anti-M2e antibodies which precipitated with whole virus and inhibited viral cell lysis by multiple type A strains of influenza virus using a novel in vitro assay. The anti-M2e antibodies also conferred complete protection following passive transfer from L-M2e-HD/MPL vaccinated mice to naïve mice challenged with H1N1 virus. Significantly higher levels of IL-4 compared to IFN-γ were secreted by the splenocytes of L-M2e-HD/MPL vaccinated mice incubated with M2e. In addition, depletion of CD4 cells or CD4 cells plus CD8 cells from L-M2e-HD/MPL vaccinated mice using monoclonal antibodies markedly decreased the level of protection of the vaccine when compared to just CD8 depletion of L-M2e-HD/MPL vaccinated mice. These results suggest that the protective immune response elicited by this vaccine is mediated primarily by a Th2 mechanism.     

长沙市首例人感染H9N2禽流感病毒的分离及基因特征分析

2015年9月从来源于国家级流感样病例监测哨点医院的标本中分离出一株H9N2亚型禽流感病毒。为了了解病毒的来源、重组、变异及耐药等分子特征,本研究对这株病毒的神经氨酸酶(Neuraminidase,NA)和基质蛋白(Matrix protein,M)基因进行测序及序列特征分析。结果显示长沙市人感染H9N2毒株的NA基因与A/duck/jiangxi/20147/2013(H9N2)禽源关系最近,遗传进化分支属于欧亚系中A/Duck/Hong Kong/Y280/97(H9N2)分支。M基因与A/duck/wenzhou/YHQL64/2014 (H9N2)禽源关系最近,属于A/Quail/Hong Kong/G1/97(H9N2)分支。该病毒分离株的NA和M基因与2014年长沙市活禽环境中的H9N2病毒高度同源。影响NA蛋白功能的关键氨基酸位点相对保守,未产生与抗神经氨酸酶抑制剂相关的氨基酸突变;M2蛋白的第31位氨基酸由S突变为N导致病毒对金刚烷胺类药物耐药。因此,长沙市的人感染H9N2禽流感病毒有可能是来源于长沙市活禽环境中的H9N2病毒,它们是通过家禽贸易传播并发生病毒重配而产生。     

Cross-subtype immunity against avian influenza in persons recently vaccinated for influenza

Avian influenza virus (H5N1) can be transmitted to humans, resulting in a severe or fatal disease. The aim of this study was to evaluate the immune cross-reactivity between human and avian influenza (H5N1) strains in healthy donors vaccinated for seasonal influenza A (H1N1)/(H3N2). A small frequency of CD4 T cells specific for subtype H5N1 was detected in several persons at baseline, and seasonal vaccine administration enhanced the frequency of such reactive CD4 T cells. We also observed that seasonal vaccination is able to raise neutralizing immunity against influenza (H5N1) in a large number of donors. No correlation between influenza-specific CD4 T cells and humoral responses was observed. N1 may possibly be a target for both cellular and humoral cross-type immunity, but additional experiments are needed to clarify this point. These findings highlight the possibility of boosting cross-type cellular and humoral immunity against highly pathogenic avian influenza A virus subtype H5N1 by seasonal influenza vaccination.     

Serological studies of influenza viruses in pigs in Great Britain 1991-2.

Samples from a sow serum bank representative of the pig population of Great Britain collected during 1991-2, were examined for antibodies to influenza A, B and C viruses, using viruses which had been isolated from a variety of hosts. For influenza A viruses there was evidence of the continued circulation of classical swine H1N1 virus (26%) seroprevalence), and human H3N2 viruses (39%) which are antigenically most closely-related to A/Port Chalmers/1/73 virus. In addition antibodies were detected to A/swine/England/201635/92 (8%), a strain of H3N2 virus which appears to have arisen by antigenic drift from conventional H3N2 swine strains. Specific antibodies (2%) were detected to an H1N1 virus (A/swine/England/195852/92) related most closely to avian H1N1 strains. In tests with human H1N1 and H3N2 viruses, excluding isolates from pigs, the highest seroprevalence was detected to the prevailing strains from the human population. Serological tests with avian H4 and H10, human H2, equine 1 and 2 influenza A viruses were all negative. Seven pigs seropositive by haemagglutination-inhibition, virus neutralization and immunoblotting assays for antibody to influenza B virus, were randomly distributed geographically suggesting that influenza B viruses may be transmitted to pigs but fail to spread. The seroprevalence to influenza C viruses was 9.9% indicating that these viruses are widespread in pigs. These results provide further evidence that the pig can be infected by a number of influenza viruses, some of which may have significance in the epidemiology of human influenza.     

Protection against H1, H5, H6 and H9 influenza A infection with liposomal matrix 2 epitope vaccines

The recent emergence of multiple avian influenza A subtypes that cause human disease (i.e., H5N1, H9N2 and H7N7), coupled with the fear that one of these strains might precipitate a new pandemic, underscores the need to develop new technological approaches to immunization which elicit protective immune responses against multiple subtypes of influenza A. In response to this demand, several matrix 2 protein ectodomain segments (M2eA) corresponding to the H1N1, H5N1 and H9N2 influenza strains were formulated using a novel liposome-based vaccine technology and evaluated as potential immunogens for developing a "universal" influenza vaccine. Mice immunized with liposomal M2eA survived homologous challenges with H1N1 (100% survival) or H9N2 (80% survival) influenza strains. There were significant reductions in their lung viral load as well as in immunized mice challenged with the H5N1 subtype. The mice vaccinated with an M2eA segment corresponding to the H1N1 and H6N2 (a reassortant influenza A virus carrying the M2eA from PR8/34) strains elicited elevated IgG ELISA antibody titers to this M2eA epitope segment and antiserum from these immunized mice provided passive protection (100% survival) to na?ve mice receiving a lethal dose of H6N2 influenza virus. These results provide the first evidence that recombinant M2eA epitopes to multiple subtypes elicited immune protection against a homologous challenge and provides further evidence in favor of the development of a "universal" influenza vaccine based on M2eA.     

Protective immunity against influenza virus challenge by norovirus P particle-M2e and HA2-AtCYN vaccines in chickens

Development of a broadly reactive influenza vaccine that can provide protection against emerging type A influenza viruses is a big challenge. We previously demonstrated that a vaccine displaying the extracellular domain of the matrix protein 2 (M2e) on the surface loops of norovirus P-particle (M2eP) can partially protect chickens against several subtypes of avian influenza viruses. In the current study, a chimeric vaccine containing a conserved peptide from the subunit 2 of hemagglutinin (HA) glycoprotein (HA2) and Arabidopsis thaliana cyanase protein (AtCYN) (HA2-AtCYN vaccine) was evaluated in 2-weeks-old chickens. Depending on the route of administration, the HA2-AtCYN vaccine was shown to induce various levels of HA2-specific IgA in tears as well as serum IgG, which were associated with partial protection of chickens against tracheal shedding of a low pathogenicity H5N2 challenge virus. Furthermore, intranasal administration with a combination of HA2-AtCYN and M2eP vaccines resulted in enhanced protection compared to each vaccine alone. Simultaneous intranasal administration of the vaccines did not interfere with secretory IgA induction by each vaccine. Additionally, significantly higher M2eP-specific proliferative responses were observed in peripheral blood mononuclear cells of all M2eP-vaccinated groups when compared with the mock-vaccinated group. Although tripling the number of M2e copies did not enhance the protective efficacy of the chimeric vaccine, it significantly reduced immunodominance of P-particle epitopes without affecting the robustness of M2e-specific immune responses. Taken together, our data suggests that mucosal immunization of chickens with combinations of mechanistically different cross-subtype-conserved vaccines has the potential to enhance the protective efficacy against influenza virus challenge.