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.     

Generation and evaluation of the trivalent inactivated reassortant vaccine using human, avian, and swine influenza A viruses

Reassortant technology was used to obtain three interspecific reassortant influenza viruses using three influenza viruses of A/Puerto Rico/8/34(H1N1), A/swine/Hebei/1/2005(H3N2) and A/chicken/Guangdong/126/2002(H9N2). The high-growth reassortant strains were H9/PR8, H3/H9N2 and H1/H9N2 that contained hemagglutinin (HA) and neuraminidase (NA) genes from the inactivated parental viruses and the other 6 internal genes from the live parental viruses. The trivalent formalin-inactivated vaccine, containing H1, H3 and H9 subtype antigens from human, swine and avian influenza viruses respectively, was prepared using these reassortant viruses. Animal studies showed that the vaccine was safe and immunogenic. Two-dosing regimen of the influenza vaccine induced high titers of hemagglutination inhibiting (HI) antibodies and influenza-specific IgG antibodies without antigenic cross-interference. It protected 100% chickens from challenge of A/chicken/Guangdong/126/2002 virus and protected 100% mice against challenges with different combinations of the three infective parental viruses. These results indicated that the trivalent vaccine could offer multi-protection against multi-influenza viruses synchronously. This kind of multivalent inactivated reassortant influenza vaccine maybe enlightens the pandemic influenza preparedness as the emergency measure.     

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.     

Partial protection against H5N1 influenza in mice with a single dose of a chimpanzee adenovirus vector expressing nucleoprotein

The development of adenoviral vectors based on non-human serotypes such as the chimpanzee adenovirus simian adenovirus 24 (AdC7) may allow for their utilization in populations harboring neutralizing antibodies to common human serotypes. Because adenoviral vectors can be used to generate potent T cell responses, they may be useful as vaccines against pandemic influenza such as may be caused by the H5N1 strains that are currently endemic in avian populations. The influenza nucleoprotein (NP) is known to provide MHC Class I restricted epitopes that are effective in evoking a cytolytic response. Because there is only low sequence variation in NP sequences between different influenza strains, a T cell vaccine may provide heterosubtypic protection against a spectrum of influenza A strains. An AdC7 vector expressing the influenza A/Puerto Rico/8/34 NP was tested for its efficacy in protecting BALB/c mice against two H5N1 strains and compared to a conventional human adenovirus serotype 5 vaccine. The AdC7 NP vaccine elicited a strong anti-NP T cell response. When tested in a mouse challenge model, there was improved survival following challenge with two strains of H5N1 that have caused human outbreaks, Vietnam/1203/04 and Hong Kong/483/97, although the improved survival reached statistical significance only with the strain from Vietnam.     

Immunization with high epitope density of M2e derived from 2009 pandemic H1N1 elicits protective immunity in mice

As highly conserved amongst human influenza A strains, the extracellular domain of influenza M2 protein (M2e) is considered and proved as a promising candidate for a universal influenza vaccine. However, there are four amino acid variations in the M2e sequence of the 2009 pandemic H1N1 (referred to as "swine flu M2e, SFM2e") compared with the conventional M2e consensus sequence. Whether the sequence variation alters the immunogenicity and protection of SFM2e epitope remains unclear. In our present study, we synthesized SFM2e peptide and constructed a series of GST fusion proteins containing various copies of the SFM2e epitope and immunized mice to evaluate their immunogenicity and protective activity. We found that although the amino acid variations have weakened the immunogenicity of the SFM2e peptide, the SFM2e fusion proteins with high epitope densities induced intense and diverse antibody response as well as T cell response. Moreover, mice immunized with high epitope density of SFM2e were nearly fully protected against a lethal challenge by the mouse-adapted influenza virus A/Porto Rico/8/34. Our study could provide new available data to improve the epitope vaccine strategy against influenza pandemic.     

Age-related changes in memory and effector T cells responding to influenza A/H3N2 and pandemic A/H1N1 strains in humans

Age-dependent changes in the cellular immune response have been mainly described in CD8+ T cells, with relative sparing of CD4+ T cells. We show that in older compared to young adults, effector memory and effector CD8+ T-cell subsets responding to influenza A/H3N2 challenge have diminished cytolytic activity. In contrast, effector CD4+ T-cell subsets in older adults share similar phenotypic and functional characteristics with those from young adults. Further, we observed a diminished cytolytic T-cell response to both seasonal influenza A/H3N2 and pandemic H1N1 (pH1N1) strains in older compared to young adults who had received seasonal influenza vaccine. These results are consistent with the observed rates of serious complications from seasonal and pandemic influenza infections in different age groups, and suggest that CD4+ T cells may provide a compensatory response to influenza infection when CD8+ T cells become compromised during the aging process.     

Optimization of influenza A vaccine virus by reverse genetic using chimeric HA and NA genes with an extended PR8 backbone

The yield of influenza antigen production may significantly vary between vaccine strains; for example the A/California/07/09 (H1N1)-X179A vaccine virus, prepared during 2009 influenza pandemic, presented a low antigen yield in eggs compared to other seasonal H1N1 reassortants. In this study a bi-chimeric virus expressing HA and NA genes with A/Puerto Rico/8/34 (H1N1) (PR8) and X179A domains was rescued by reverse genetics using a mixture of Vero/CHOK1 cell lines (Medina et al. [7]). The bi-chimeric virus obtained demonstrated to yield much larger amounts of HA than X179A in eggs as measured by single-radial-immunodiffusion (SRID), the reference method to quantify HA protein in influenza vaccine. Such kind of optimized virus using PR8 backbone derived chimeric glycoproteins could be used as improved seed viruses for vaccine production.     

Prior infection with influenza virus but not vaccination leaves a long-term immunological imprint that intensifies the protective efficacy of antigenically drifted vaccine strains

The role of pre-existing immunity for influenza vaccine responses is of great importance for public health, and thus has been studied in various contexts, yet the impact of differential priming on vaccine responses in the midst of antigenic drift remains to be elucidated. To address this with antigenically related viruses, mice were first primed by either infection or immunization with A/Puerto Rico/8/34 (PR8) virus, then immunized with whole-inactivated A/Fort Monmouth/1/47 (FM1) virus. The ensuing vaccine responses and the protective efficacy of FM1 were superior in PR8 infection-primed mice compared to PR8 immunization-primed or unprimed mice. Increased FM1-specific Ab responses of PR8 infection-primed mice also broadened cross-reactivity against contemporary as well as antigenically more drifted strains. Further, prior infection heightened the protective efficacy of antigenically distant strains, such as A/Brisbane/59/2006 infection followed by immunization with split pandemic H1N1 vaccine (A/California/07/2009). Therefore, influenza infection is a significant priming event that intensifies future vaccine responses against drift strains.     

Inactivated trivalent seasonal influenza vaccine induces limited cross-reactive neutralizing antibody responses against 2009 pandemic and 1934 PR8 H1N1 strains

Background

In June 2009, we conducted a prospective study in Singapore on 51 individuals to determine their serologic responses before and following receipt of the 2009 Southern Hemisphere seasonal influenza vaccine.

Materials and methods

Paired serum samples were obtained before and 3–4 weeks after vaccination. Virus microneutralization assays were performed to quantify antibodies against A/Brisbane/59/2007 vaccine, pandemic H1N1-2009 and A/Puerto Rico/08/34 H1N1 strains.

Results

Post-vaccination, 43%, 12% and 24% of subjects displayed a 4-fold or greater rise in neutralizing antibody titers against the three strains, respectively. There was a positive correlation among individuals who showed increased titers to both pandemic H1N1-2009 and A/Puerto Rico/08/34 (p < 0.001). However, this correlation was not observed for A/Brisbane/59/2007 with either strain. The relative conservation and accessibility of predicted B-cell epitopes may explain the limited cross-reactivity of the antibodies directed against common H1N1 epitopes.

Conclusions

These results suggest that seasonal influenza vaccination confers a certain degree of cross-protection to other H1N1 strains. The correlation in cross-reactive antibody titers to A/Puerto Rico/08/34 and pandemic H1N1-2009 implies that previous exposure to pre-1957 H1N1 strains may confer some protection against the 2009 pandemic strain.     

Characterization of high-growth reassortant influenza A viruses generated in MDCK cells cultured in serum-free medium

In the present study reassortant influenza A viruses of both the H1N1 and H3N2 type were generated in Madin Darby Canine Kidney cells grown in the absence of fetal bovine serum (MDCK-SF1 cells). To this end, MDCK-SF1 cells were simultaneously infected with one of the high-growth laboratory strains A/Puerto Rico/8/34 (H1N1) or A/Hong Kong/2/68 (H3N2) and recent H3N2 and H1N1 vaccine strains, respectively. Reassortant viruses obtained from these mixed infections were genetically characterized by RT-PCR and restriction enzyme analysis and their growth properties were compared to those of the corresponding field strains. Reassortant H3N2 viruses inherited the matrix and polymerase pa gene whilst H1N1 reassortant viruses inherited the matrix and polymerase pbl gene of the high-growth parent. Reassortant viruses generally gave higher viral yields, as measured by a haemagglutination assay, than their wild type counterparts. The procedure followed results in the generation of high-growth reassortant viruses in weeks. The use of MDCK-SF1 cells together with these reassortants for generating influenza virus antigens can significantly speed up the vaccine production procedure.     

Cross-protective immunity against multiple influenza virus subtypes by a novel modified vaccinia Ankara (MVA) vectored vaccine in mice

Development of an influenza vaccine that provides cross-protective immunity remains a challenge. Candidate vaccines based on a recombinant modified vaccinia Ankara (MVA) viral vector expressing antigens from influenza (MVA/Flu) viruses were constructed. A vaccine candidate, designated MVA/HA₁/C13L/NP, that expresses the hemagglutinin from pandemic H1N1 (A/California/04/09) and the nucleoprotein (NP) from highly pathogenic H5N1 (A/Vietnam/1203/04) fused to a secretory signal sequence from vaccinia virus was highly protective. The vaccine elicited strong antibody titers to homologous H1N1 viruses while cross-reactive antibodies to heterologous viruses were not detectable. In mice, this MVA/HA₁/C13L/NP vaccine conferred complete protection against lethal challenge with A/Vietnam/1203/04 (H5N1), A/Norway/3487-2/09 (pandemic H1N1) or A/Influenza/Puerto Rico/8/34 (seasonal H1N1) and partial protection (57.1%) against challenge with seasonal H3N2 virus (A/Aichi/68). The protective efficacy of the vaccine was not affected by pre-existing immunity to vaccinia. Our findings highlight MVA as suitable vector to express multiple influenza antigens that could afford broad cross-protective immunity against multiple subtypes of influenza virus.     

Generation and evaluation of a high-growth reassortant H9N2 influenza A virus as a pandemic vaccine candidate

H9N2 subtype avian influenza viruses (AIVs) are widely distributed in avian species and were isolated from humans in Hong Kong and Guangdong province, China in 1999 raising concern of their potential for pandemic spread. We generated a high-growth reassortant virus (G9/PR8) that contains the hemagglutinin (HA) and neuraminidase (NA) genes from the H9N2 avian influenza virus A/chicken/Hong Kong/G9/97 (G9) and six internal genes from A/Puerto Rico/8/34 (PR8) by genetic reassortment, for evaluation as a potential vaccine candidate in humans. Pathogenicity studies showed that the G9/PR8 reassortant was not highly pathogenic for mice or chickens. Two doses of a formalin-inactivated G9/PR8 virus vaccine induced hemagglutination inhibiting antibodies and conferred complete protection against challenge with G9 and the antigenically distinct H9N2 A/Hong Kong/1073/99 (G1-like) virus in a mouse model. These results indicate that the high growth G9/PR8 reassortant has properties that are desirable in a vaccine seed virus and is suitable for evaluation in humans for use in the event of an H9 pandemic.     

Development of Eurasian H7N7/PR8 high growth reassortant virus for clinical evaluation as an inactivated pandemic influenza vaccine

Avian-to-human transmission of the high pathogenicity (HP) H7N7 subtype avian influenza viruses in the Netherlands during 2003 caused zoonotic infections in 89 people, including a case of acute fatal respiratory distress syndrome. Public health emergency preparedness against H7N7 avian influenza viruses with pandemic potential includes the development of vaccine candidate viruses. In order to develop a high growth reassortant vaccine candidate virus, low pathogenicity (LP) A/mallard/Netherlands/12/2000 (H7N3) and A/mallard/Netherlands/2/2000 (H10N7) strains were selected as donors of the H7 haemagglutinin and N7 neuraminidase genes, respectively. The donor viruses exhibited high amino acid sequence homology with the surface glycoproteins of A/Netherlands/219/03 H7N7 virus (NL219), an isolate recovered from the fatal human case. Adhering to the seasonal influenza vaccine licensure regulations, we generated a H7N7/PR8 reassortant containing desired surface glycoprotein genes from the mallard viruses and internal genes of A/Puerto Rico/8/34 human vaccine strain (H1N1). Antigenic analysis revealed that the vaccine candidate virus confers broad antigenic cross-reactivity against contemporary Eurasian and the North American H7 subtype human isolates. Mice immunized with formalin inactivated (FI) H7N7/PR8 whole virus vaccine with or without aluminum hydroxide adjuvant conferred clinical protection from mortality and reduced pulmonary replication of the NL219 challenge virus. The FI H7N7/PR8 whole virus vaccine also afforded cross-protection in mice at the pulmonary level against antigenically distinct North American LP A/Canada/444/04 (H7N3) human isolate. The vaccine candidate virus satisfied the agricultural safety requirements for chickens, proved safe in mice, and has entered in phase-I human clinical trial in the United States.     

Generation of an attenuated H5N1 avian influenza virus vaccine with all eight genes from avian viruses

In the face of disease outbreaks in poultry and the potential pandemic threat to humans caused by the highly pathogenic avian influenza viruses (HPAIVs) of H5N1 subtype, improvement in biosecurity and the use of inactivated vaccines are two main options for the control of this disease. Vaccine candidates of influenza A viruses of H5N1 subtype have been generated in several laboratories by plasmid-based reverse genetics with hemagglutinin (HA) and neuraminidase (NA) genes from the epidemic strains of avian viruses in a background of internal genes from the vaccine donor strain of human strains, A/Puerto Rico/8/34 (PR8). These reassortant viruses containing genes from both avian and human viruses might impose biosafety concerns, also may be do if C4/F AIV would be a live attenuated vaccine or cold-adaptive strain vaccine. In order to generate better and safer vaccine candidate viruses, we genetically constructed attenuated reassortant H5N1 influenza A virus, designated as C4/F AIV, by plasmid-based reverse genetics with all eight genes from the avian strains. The C4/F AIV virus contained HA and NA genes from an epidemic strain A/Chicken/Huadong/04 (H5N1) (C4/H5N1) in a background of internal genes derived from a low pathogenic strain of A/Chicken/F/98(H9N2). The reassortant virus was attenuated by removal of the multibasic amino acid motif in the HA gene by mutation and deletion (from PQRERRRKKR (downward arrow) G to PQIETR (downward arrow) G). The intravenous pathogenicity index (IVPI) of C4/F AIV virus was 0, whereas that of the donor virus C4/H5N1 was 3.0. The virus HA titer of C4/H5N1 in the allantoic fluid from infected embryonated eggs was as high as 1:2048. The inactivated vaccine prepared from the reassortant virus C4/F AIV-induced high HI titer in vaccinated chickens and gave 100% protection when challenged with highly pathogenic avian influenza virus of H5N1 subtype.     

Potency of whole virus particle and split virion vaccines using dissolving microneedle against challenges of H1N1 and H5N1 influenza viruses in mice

Transdermal vaccination using a microneedle (MN) confers enhanced immunity compared with subcutaneous (SC) vaccination. Here we developed a novel dissolving MN patch for the influenza vaccine. The potencies of split virion and whole virus particle (WVP) vaccines prepared from A/Puerto Rico/8/1934 (H1N1) and A/duck/Hokkaido/Vac-3/2007 (H5N1), respectively, were evaluated. MN vaccination induced higher neutralizing antibody responses than SC vaccination in mice. Moreover, MN vaccination with a lower dose of antigens conferred protective immunity against lethal challenges of influenza viruses than SC vaccination in mice. These results suggest that the WVP vaccines administered using MN are an effective combination for influenza vaccine to be further validated in humans.     

Analysis of the immunogenicity and bioactivities of a split influenza A/H7N9 vaccine mixed with MF59 adjuvant in BALB/c mice

The H7N9 influenza virus caused significant mortality and morbidity in humans during an outbreak in China in 2013. A recombinant H7N9 influenza seed with hemagglutinin (HA) and neuraminidase (NA) gene segments from A/Zhejiang/DTID-ZJU01/2013(H7N9) and six internal protein gene segments from A/Puerto Rico/8/34(H1N1; PR8) were generated using reverse genetics. We sought to determine the immunogenic, protective properties, and mechanisms of a split avian influenza A/H7N9 vaccine mixed with MF59 adjuvant in comparison to vaccines that included other adjuvant. BALB/c mice were vaccinated with two doses of different amounts and combinations of this novel A/ZJU01/PR8/2013 split vaccine with adjuvant. Mice were subsequently challenged with A/Zhejiang/DTID-ZJU01/2013(H7N9) by intranasal inoculation. We verified that MF59 enhanced the HI, MN, and IgG antibody titers to influenza antigens. Compared with alum, MF59 could more potentially induce humoral immune responses and Th2 cytokine production after virus infection, while both MF59 and alum can slightly increase NK cell activity. This split H7N9 influenza vaccine with MF59 adjuvant could effectively induce antibody production and protect mice from H7N9 virus challenge. We have selected this vaccine for manufacture and future clinical studies to protect humans from H7N9 virus infection.     

Novel G3/DT adjuvant promotes the induction of protective T cells responses after vaccination with a seasonal trivalent inactivated split-virion influenza vaccine

Vaccines used against seasonal influenza are poorly effective against influenza A viruses of novel subtypes that may have pandemic potential. Furthermore, pre(pandemic) influenza vaccines are poorly immunogenic, which can be overcome by the use of adjuvants. A limited number of adjuvants has been approved for use in humans, however there is a need for alternative safe and effective adjuvants that can enhance the immunogenicity of influenza vaccines and that promote the induction of broad-protective T cell responses. Here we evaluated a novel nanoparticle, G3, as an adjuvant for a seasonal trivalent inactivated influenza vaccine in a mouse model. The G3 adjuvant was formulated with or without steviol glycosides (DT, for diterpenoid). The use of both formulations enhanced the virus-specific antibody response to all three vaccine strains considerably. The adjuvants were well tolerated without any signs of discomfort. To assess the protective potential of the vaccine-induced immune responses, an antigenically distinct influenza virus strain, A/Puerto Rico/8/34 (A/PR/8/34), was used for challenge infection. The vaccine-induced antibodies did not cross-react with strain A/PR/8/34 in HI and VN assays. However, mice immunized with the G3/DT-adjuvanted vaccine were partially protected against A/PR/8/34 infection, which correlated with the induction of anamnestic virus-specific CD8⁺ T cell responses that were not observed with the use of G3 without DT. Both formulations induced maturation of human dendritic cells and promoted antigen presentation to a similar extent. In conclusion, G3/DT is a promising adjuvant formulation that not only potentiates the antibody response induced by influenza vaccines, but also induces T cell immunity which could afford broader protection against antigenically distinct influenza viruses.     

A Vero-cell-adapted vaccine donor strain of influenza A virus generated by serial passages

A cell culture-based vaccine production system is preferred for the large-scale production of influenza vaccines and has advantages for generating vaccines against highly pathogenic influenza A viruses. Vero cells have been widely used in human vaccine manufacturing, and the safety of these cells has been well demonstrated. However, the most commonly used influenza-vaccine donor virus, A/Puerto Rico/8/1934 (PR8) virus, does not grow efficiently in Vero cells. Therefore, we adapted the PR8 virus to Vero cells by continuous passaging, and a high-growth strain was obtained after 20 passages. Sequence analysis and virological assays of the adapted strain revealed that mutations in four viral internal genes (NP, PB1, PA and NS1) were sufficient for adaptation. The recombinant virus harboring these mutations (PR8-4mut) displayed accelerated viral transport into the nucleus and increased RNP activity. Importantly, the PR8-4mut could serve as a backbone donor virus to support the growth of the H7N1, H9N2 and H5N1 avian viruses and the H1N1 and H3N2 human viruses in Vero cells without changing its pathogenicity in either chicken embryos or mice. Thus, our work describes the generation of a Vero-adapted, high-yield PR8-4mut virus that may serve as a promising candidate for an influenza-vaccine donor virus.     

Induction of heterosubtypic immunity to influenza A virus using a DNA vaccine expressing hemagglutinin-C3d fusion proteins

Cross-protection between different subtypes of influenza A virus has been attributed to heterosubtypic immunity (HSI). Although, HSI can occur in the absence of anti-HA or anti-NA antibodies, HSI seems to be mediated, in part, by cross-reactive antibodies. In this study, we examined the effects of a DNA vaccine expressing an influenza HA fused to three copies of murine C3d of complement (HA-mC3d(3)). HA-mC3d(3) elicited heterosubtypic immunity more efficiently than non-fused forms of HA and protected mice from lethal challenge of influenza with different subtypes. Plasmid encoding for various forms of HA were constructed from two influenza strains, A/Puerto Rico/8/34 (H1N1) or A/Aichi/2/68-x31 (H3N2). Vaccinated mice were analyzed for enhancement of anti-HA titers, affinity maturation of antibody, hemagglutinin-inhibition activity, and altered cytokine profiles. The HA-mC3d(3)-DNA vaccinated mice were protected from heterologous influenza challenge, even though sera from these mice had no viral-neutralizing activity against heterologous virus.     

Preclinical study of influenza virus A M2 peptide conjugate vaccines in mice, ferrets, and rhesus monkeys

A universal influenza virus vaccine that does not require frequent updates and/or annual immunizations will offer significant advantages over current seasonal flu vaccines. The highly conserved influenza virus A M2 membrane protein has been previously suggested as a potential antigen target for such a vaccine. Here, we report systematic evaluation of M2 peptide conjugate vaccines (synthetic peptides of M2 extracellular domain conjugated to keyhole limpet hemocyanin (KLH) or Neisseria meningitidis outer membrane protein complex (OMPC)) in mice, ferrets, and rhesus monkeys. The conjugate vaccines were highly immunogenic in all species tested and were able to confer both protection against lethal challenge of either H1N1 or H3N1 virus in mice and reduce viral shedding in the lower respiratory tracts of mice and ferrets. The protection against lethal challenge in mice could also be achieved by passive transfer of monkey sera containing high M2 antibody titers. In addition, we showed that M2 antisera were cross reactive with M2 peptides derived from a wide range of human influenza A strains, but they failed to react with M2 peptides of the pathogenic H5N1 virus (A/Hong Kong/97). The data presented here will permit better understanding of the potential of an M2-based vaccine approach.