Influenza M2 virus-like particles confer a broader range of cross protection to the strain-specific pre-existing immunity

Immunity in humans with annual vaccination does not provide effective protection against antigenically distinct strains. As an approach to improve cross-protection in the presence of pre-existing strain-specific immunity, we investigated the efficacy of heterologous and heterosubtypic protection in previously vaccinated mice at earlier times after subsequent immunization with conserved-antigenic target influenza M2 ectodomain (M2e) virus-like particle vaccine (M2e5× VLP). Immunization of mice with H1N1 split vaccine induced virus specific antibodies to homologous influenza virus but did not provide heterosubtypic hemagglutination inhibiting antibody responses and cross-protection. However, subsequent M2e5× VLP immunization induced an M2e specific antibody response as well as interferon-γ (IFN-γ) producing cells in systemic and mucosal sites. Upon lethal challenge with H3N2 or H5N1 subtype influenza viruses, subsequently immunized mice with M2e5× VLP were well protected against heterosubtypic influenza viruses. These results provide evidence that non-seasonal immunization with M2e5× VLP, an experimental candidate for universal vaccine, is a promising approach for broadening the cross-protection even in the presence of strain-specific immunity.     

Single dose of oil-adjuvanted inactivated vaccine protects chickens from lethal infections of highly pathogenic H5N1 influenza virus

The highly pathogenic H5N1 influenza viruses are endemic in poultry in many countries, but continuously infect humans and cause human mortality. H5N1 influenza viruses have been regarded as a pandemic candidate. In a pandemic event by this virus, the protection of poultry with an effective vaccine will help to greatly reduce the spread of this virus to humans since it easily infects poultry. Here we showed that immunization with one dose of oil-adjuvanted inactivated H5N1 vaccine could protect chickens from lethal infection by highly pathogenic H5N1 influenza virus until 12 weeks post-immunization. The complete protection of chickens depended on the amount of HA antigens in the vaccine. Complete homologous protection required over 1.25 μg of HA antigens and complete heterologous protection required over 5.0 μg of HA antigens. The bivalent H5N1 inactivated vaccine composed of 1.25 μg of each antigen from clade 1 and clade 2.3.4 H5N1 influenza virus completely protected chickens from the lethal challenge of both viruses. When we determined the induction of antibody subtypes in tissues including nasal cavity, trachea, and lungs, the IgG subtype of antibody was induced more than the IgM or IgA subtype of antibody. Taken together, our results suggest that one dose of oil-adjuvanted inactivated H5N1 vaccine could provide chickens with sterile immunity against the homologous highly pathogenic H5N1 influenza virus.     

Antibody responses and cross protection against lethal influenza A viruses differ between the sexes in C57BL/6 mice

A mouse model was used to determine if protective immunity to influenza A virus infection differs between the sexes. The median lethal dose of H1N1 or H3N2 was lower for naïve females than males. After a sublethal, primary infection with H1N1 or H3N2, females and males showed a similar transient morbidity, but females generated more neutralizing and total anti-influenza A virus antibodies. Immunized males and females showed similar protection against secondary challenge with a homologous virus, but males experienced greater morbidity and had higher lung viral titers after infection with a lethal dose of heterologous virus. Females develop stronger humoral immune responses and greater cross protection against heterosubtypic virus challenge.     

Protection against influenza B virus infection by immunization with DNA vaccines

Protection against a lethal influenza B virus infection was examined in BALB/c mice immunized with plasmid DNAs encoding hemagglutinin (HA), neuraminidase (NA and NB) and nucleoprotein (NP) from the B/Ibaraki/2/85 virus. Each DNA vaccine was administered twice, 3 weeks apart, at a dose of 1 microg per mouse by particle-mediated DNA transfer to the epidermis (gene gun) or at a dose of 30 microg per mouse by electroporation into the muscle. Three weeks after the second vaccination, the mice were challenged with a lethal dose of homologous virus. HA and NA DNAs conferred complete protection against the lethal viral challenge, whereas NB and NP DNAs failed to provide protection against infection. Furthermore, protection in different strains of mice, BALB/c, B10 and C3H, immunized with HA and NA DNAs was compared. Both HA and NA DNAs conferred complete protection against the lethal challenge in all the tested mouse strains. These results suggest that both the HA and NA molecules can be used as vaccine components to provide effective protection against influenza B virus infection.     

Strategies for inducing protection against avian influenza A virus subtypes with DNA vaccines

The cross-species transfer of a H5N1 influenza virus from birds to humans, and the systemic spread of this virus in mice, has accelerated the efforts to devise protective strategies against lethal influenza viruses. DNA vaccination with the highly conserved nucleoprotein gene appears to provide cross protection against influenza A viruses in murine models. Whether such vaccines would protect human hosts against different influenza A viruses, including strains with pandemic potential, is unclear. Our aim in this study is to evaluate the ability of a combination DNA vaccine consisting of two plasmids encoding the HA genes from two different subtypes and a DNA vaccine encoding the viral nucleoprotein gene from a H5 virus to induce protection against highly lethal infection caused by H5 and H7 influenza viruses in chickens. Chickens given a single dose of plasmids expressing H5 and H7 hemagglutinins protected the birds from infection by either subtype. However, birds immunized with nucleoprotein DNA and challenged with either A/Ck/Vic/1/85(H7N7) or A/Ty/Ir/1/83 (H5N8) showed definite signs of infection, suggesting inadequate immunity against viral infection. Fifty percent of the nucleoprotein DNA immunized birds survived infection by influenza A/Ty/Ir/1/83 (H5N8) virus (virus of same subtype) while 42% survived infection by influenza A/Ck/Vic/1/85/(H7N7) virus (virus of a different subtype). These studies demonstrate that immunization with DNA encoding a type-specific gene may not be effective against either homologous or heterologous strains of virus, particularly if the challenge virus causes a highly lethal infection. However, the combination of HA subtype vaccines are effective against lethal infection caused by viruses expressing any of the HA subtypes used in the combination preparation.     

Protection against influenza virus infection in BALB/c mice immunized with a single dose of neuraminidase-expressing DNAs by electroporation

The ability of a single dose of plasmid DNA encoding neuraminidase (NA) or hemagglutinin (HA) from influenza virus A/PR/8/34 (PR8) (H1N1) to protect against homologous virus infection was examined in BALB/c mice. In the present study, mice were immunized once with 30 microg of NA or HA DNA by electroporation. Four weeks or 28 weeks after immunization, mice were challenged with a lethal dose of homologous virus and the ability of NA or HA DNA to protect the mice from influenza was evaluated. We found that a single inoculation of NA DNA could provide protection against influenza virus challenge as well as long-term protection against viral infection. Whereas, the mice immunized with a single dose of HA DNA could not be protected. In addition, neonatal mice immunized with a single dose of 30 microg of NA DNA could be provided with significant protection against viral infection.     

Infection of mice with a human influenza A/H3N2 virus induces protective immunity against lethal infection with influenza A/H5N1 virus

The transmission of highly pathogenic avian influenza (HPAI) A viruses of the H5N1 subtype from poultry to man and the high case fatality rate fuels the fear for a pandemic outbreak caused by these viruses. However, prior infections with seasonal influenza A/H1N1 and A/H3N2 viruses induce heterosubtypic immunity that could afford a certain degree of protection against infection with the HPAI A/H5N1 viruses, which are distantly related to the human influenza A viruses. To assess the protective efficacy of such heterosubtypic immunity mice were infected with human influenza virus A/Hong Kong/2/68 (H3N2) 4 weeks prior to a lethal infection with HPAI virus A/Indonesia/5/05 (H5N1).     

Enhanced protection against a lethal influenza virus challenge by immunization with both hemagglutinin- and neuraminidase-expressing DNAs

The ability of plasmid DNA encoding hemagglutinin (HA), neuraminidase (NA) or matrix protein (M1) from influenza virus A/PR/8/34 (PR8) (H1N1), and mixtures of these plasmid DNAs (HA + NA and HA + NA + M1) to protect against homologous or heterologous virus infection was examined in BALB/c mice. Each DNA was inoculated twice, 3 weeks apart, or four times, 2 weeks apart, at a dose of 1 microg of each component per mouse by particle-mediated DNA transfer to the epidermis (gene gun). Seven days after the last immunization, mice were challenged with a lethal homologous or heterologous virus and the ability of each DNA to protect the mice from influenza was evaluated by observing lung virus titers and survival rates. The administration of a plasmid DNA mixture of either (HA + NA) or (HA + NA + M1) provided almost complete protection against the PR8 virus challenge, and this protection was accompanied by high levels of specific antibody responses to the respective components. The degree of protection afforded in these groups is significantly higher than that in mice given either HA- or NA-expressing DNA alone, which provided only a partial protection against PR8 challenge or that in mice given M1-expressing DNA, which failed to provide any protection. In addition, both of the plasmid DNA mixtures (HA + NA) and (HA + NA + M1) showed a slight tendency to provide cross-protection against an A/Yamagata/120/86 (H1N1) virus challenge, and this was accompanied by a relatively high level of cross-reacting antibodies. Thus, there was no clear difference between the ability of the HA + NA and HA + NA + M1 plasmid DNA mixtures in providing protection against either a PR8 or heterologous virus challenge. These results suggest that in mice immunized by gene gun, a mixture of plasmid DNAs encoding HA and NA can provide the most effective protection against the virus challenge. The addition of the M -expressing plasmid DNA to this mixture does not enhance the degree of protection afforded.     

Immunogenicity and protective efficacy of cold-adapted X-31 live attenuated pre-pandemic H5N1 influenza vaccines

Despite global efforts to control influenza viruses, they have taken a heavy toll on human public health worldwide. Among particular threats is highly pathogenic avian H5N1 influenza virus (HPAI) due to not only its high mortality in humans but also possible human-to-human transmission either through reassortment with other human influenza viruses such as 2009 pandemic H1N1 influenza virus, or by genetic mutations. With the aim of developing effective vaccines against the H5N1 viruses, we generated two live attenuated H5N1 vaccine candidates against A/Indonesia/05/2005 (clade 2.1) and A/chicken/Korea/ES/2003 (clade 2.5) strains, in the genetic background of the cold-adapted donor strain of X-31. In mice, a single dose of immunization with each of the two vaccines was highly immunogenic inducing high titers of serum viral-neutralizing and hemagglutinin-inhibiting antibodies against the homologous H5N1 strain. Furthermore, significant levels of cross-clade antibody responses were induced by the vaccines, suggesting a broad-spectrum cross-reactivity against the heterologous H5N1 strains. The immunizations provided solid protections against heterologous lethal challenges with H5N2 virus, significantly reducing the morbidity and challenge virus replications in the respiratory tracts. The robustness of the antibody responses against both the homologous and heterologous strains, together with efficient protection against the lethal H5N2 challenge, strongly support the protection against wild type H5N1 infections. These results could serve as an experimental basis for the development of safe and effective H5N1 pre-pandemic vaccines while further addressing the biosecurity concerns associated with H5N1 HPAI.     

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.     

Efficacy of inactivated vaccine against H5N1 influenza virus infection in mice with type 1 diabetes

We sought to determine susceptibility to highly pathogenic avian influenza (HPAI) H5N1 virus and to explore immune protection of inactivated H5N1 vaccine in streptozotocin-induced type 1 diabetic mice. Susceptibility of diabetic mice to an H5N1 virus was evaluated by comparing the median lethal dose (LD₅₀) and the lung virus titers with those of the healthy after the viral infection. To evaluate the influence of diabetes on vaccination, diabetic and healthy mice were immunized once with an inactivated H5N1 vaccine and then challenged with a lethal dose of H5N1 virus. The antibody responses, survival rates, lung virus titers and body weight changes were tested. Mice with type 1 diabetes had higher lung virus titers and lower survival rates than healthy mice after H5N1 virus infection. Inactivated H5N1 vaccine induced protective antibody in diabetic mice, but the antibody responses were postponed and weakened. In spite of this, diabetic mice could be protected against the lethal virus challenge by a single dose of immunization when the amount of the antigen increased. These results indicated that type 1 diabetic mice were more susceptible to H5N1 influenza virus infection than healthy mice, and can be effectively protected by inactivated H5N1 vaccine with increased antigen.     

流感病毒神经氨酸酶双表达DNA疫苗的构建及在小鼠中的免疫保护研究

目的研究对多亚型流感病毒流行提供预防的DNA疫苗。方法以pIRES为双表达载体,在其两个多克隆位点中分别插入H1N1（A/PR/8/34）和H3N2（A/Guizhou/54/89）的NA DNA片段,构建双表达质粒pN1-IRES-N2及pN2-IRES-N1。以BALB/c小鼠为模型,采用电穿孔法进行免疫,并用致死量流感病毒感染以检测双表达载体的保护力。结果实验表明pN1-IRES-N2能完全保护小鼠抵御致死量H1N1同源病毒的攻击,部分保护小鼠抵御致死量H3N2同源病毒攻击;pN2-IRES-N1能完全保护小鼠抵御致死量H3N2同源病毒攻击,对致死量H1N1同源病毒的攻击提供部分保护。结论双表达DNA疫苗可望开发为一种提供广泛保护的新型流感疫苗。     

Protection against influenza virus infection in mice immunized by administration of hemagglutinin-expressing DNAs with electroporation

Electroporation for the transfer of plasmid DNA encoding influenza virus hemagglutinin (HA) into muscle or nasal mucosa was tried in BALB/c mice to examine the efficacy of this method for inducing anti-HA immune responses and providing protection against homologous A/PR/8/34 (PR8) virus infection. Mice were immunized by two injections, 3 weeks apart, of HA-DNA with electroporation into the muscle wherein a pair of electrode needles was inserted to deliver the electric pulses. One or 3 weeks after the immunization, the mice were infected with a lethal dose of the PR8 virus. Ten micrograms or more of HA-DNA/dose induced strong serum anti-HA IgG antibody (Ab) responses, in which both IgG1 and IgG2a were predominant, and weak cytotoxic T lymphocyte responses. These immune responses were sufficient to provide efficient protection against the lethal infection. In addition, mice were immunized by dropping HA-DNA (12 microg) three times, 2 weeks between each dose into nostrils where each of two electrode needles was placed on the right nostril or the palate. One week after the immunization, the mice were infected with a sublethal dose of the PR8 virus. The DNA immunization by electroporation provided reduced nasal virus titers, in parallel with a relatively high levels of serum anti-HA IgG Ab and a slight nasal anti-HA IgA Ab production. The intranasal administration of cholera toxin before HA-DNA immunization by electroporation enhanced the nasal IgA Ab production together with enhancement of the efficiency of protection. These results suggest that electroporation can be used as one of the efficient gene delivery systems for the transfer of influenza DNA-vaccine into muscle or nasal mucosa to provide protection against influenza virus infection.     

Cross-protection against influenza virus infection by intranasal administration of M1-based vaccine with chitosan as an adjuvant

The antigenic variation of influenza virus represents a major health problem, thus continuous efforts have been made to develop broad-spectrum vaccines against influenza virus. Matrix protein 1 (M1) protein is highly conserved in all influenza A strains. In this study, M1 protein was efficiently expressed in Escherichia coli (E. coli), then purified and used for immunization of BALB/c mice by intranasal drip using chitosan as adjuvant. The M1 protein was administered intranasally to mice in combination with chitosan adjuvant twice at an interval of 3 weeks. Three weeks after the second immunization, the mice were challenged with a lethal dose (5 × LD₅₀) of A/Chicken/Jiangsu/7/2002 (H9N2) virus, PR8 (H1N1) virus and A/Chicken/Henan/12/2004 (H5N1) virus. The protective immunity of the vaccine was evaluated by determining the survival rates, residual lung virus titers, bodyweight, and the serum antibody titers of the mice. The results showed that nasal administration of 100 μg M1 in combination with chitosan could not only completely protect the mice effectively against the challenge of the homologous virus but also protect 70% and 30% of the mice against the heterologous H1N1 and H5N1 viruses, respectively. The study indicated that the M1 protein was a candidate antigen for a broad-spectrum influenza virus vaccine and the adjuvant chitosan significantly improved the efficacy of the M1 vaccine.     

Primary influenza A virus infection induces cross-protective immunity against a lethal infection with a heterosubtypic virus strain in mice

In order to assess the level of protection against a lethal influenza virus infection provided by a primary infection with a virus strain of another subtype, C57BL/6 mice were infected with the sublethal influenza virus X-31 (H3N2) and subsequently challenged with the lethal strain A/PR/8/34 (H1N1). The outcome of the challenge infection was compared with that in mice that did not experience an infection with influenza virus X-31 prior to the challenge infection. The X-31 experienced mice cleared the infection with influenza virus A/PR/8/34 in an accelerated fashion, displayed less clinical signs and a reduction of lesions in the lungs resulting in improved survival rates of these mice compared to the naive mice. The improved outcome of the challenge infection with influenza virus A/PR/8/34 in the X-31 experienced mice correlated with priming for anamnestic virus-specific CD8(+) cytotoxic T lymphocyte (CTL) responses as was demonstrated by the detection of CTL specific for the H-2D(b) restricted NP(366-374) epitope that was shared by the influenza viruses X-31 and A/PR/8/34. Thus previous exposure to influenza A viruses affords partial protection against infection in the absence of virus-neutralizing antibodies specific for the hemagglutinin and the neuraminidase. The implications of these observations are discussed in the light of the current pandemic threat and development of vaccines that aim at the induction of virus-specific CTL.     

A bivalent influenza VLP vaccine confers complete inhibition of virus replication in lungs

The conventional egg-grown influenza vaccines are trivalent. To test the feasibility of using multivalent influenza virus-like particles (VLPs) as an alternative influenza vaccine, we developed cell-derived influenza VLPs containing the hemagglutinin (HA) of the H1 subtype virus A/PR/8/34 or the H3 subtype virus A/Aichi/2/68 (X31). Mice immunized intramuscularly with bivalent influenza VLPs containing H1 and H3 HAs induced neutralizing activities against the homologous and closely related H1N1 strains A/PR/8/34 and A/WSN/33 as well as the H3N2 strains A/Aichi/2/68 (X31) and A/Hong Kong/68, but not the A/Philippines/2/82 strain isolated 14 years later. HA sequence and structure analysis indicated that antigenic distance could be a major factor in predicting cross-protection by VLP vaccines. The bivalent influenza VLP vaccine demonstrated advantages in broadening the protective immunity after lethal challenge infections when compared to a monovalent influenza VLP vaccine. High levels of the inflammatory cytokine IL-6 were observed in na?ve or unprotected immunized mice but not in protected mice upon lethal challenge. These results indicate that multivalent influenza VLP vaccines can be an effective antigen for developing safe and alternative vaccine to control the spread of influenza viruses.     

Protection against a European H1N2 swine influenza virus in pigs previously infected with H1N1 and/or H3N2 subtypes

A novel swine influenza virus, H1N2, circulates in European swine populations together with H1N1 and H3N2 viruses. This study examines whether post-infection immunity to H1N1 and/or H3N2 viruses provides cross-protection against H1N2 infection. Pigs (n=51) were inoculated intranasally with either Sw/Belgium/1/98 (H1N1) or Sw/Flanders/1/98 (H3N2), or with both viruses at a 5-week interval. Control groups were left uninoculated or inoculated with Sw/Gent/7625/99 (H1N2). Four weeks later, all the pigs were challenged intranasally and intratracheally with a high H1N2 virus dose. The challenge control pigs showed typical influenza symptoms, and all had high H1N2 virus titres in the lungs and nasal virus excretion during 6 or 7 days. The H1N2-immune pigs showed total clinical and virological protection. Pigs immune against H1N1 or H3N2 only were not protected against disease and virus replication in the lungs, but virus excretion was 2 days shorter. By contrast, pigs immune against both H1N1 and H3N2 did not show disease and H1N2 virus replication was either undetectable or markedly reduced. Haemagglutination inhibition (HI) and virus neutralisation (VN) tests indicated that cross-protection against H1N2 was probably not mediated by antibodies against the haemagglutinin (HA). Antibodies inhibiting the neuraminidase (NA) of H1N2 were at minimal levels in H3N2 only-immune pigs, but they were consistently found in (H1N1+H3N2)-immune pigs. The immune response against the internal proteins, which are relatively conserved in H1N1, H3N2 and H1N2 viruses, may play a significant role in protection against H1N2. Given the severe challenge model used here, cross-protection against H1N2 could be more pronounced under natural conditions of infection.     

Onjisaponins, from the root of Polygala tenuifolia Willdenow, as effective adjuvants for nasal influenza and diphtheria-pertussis-tetanus vaccines

Active substances from hot water extracts from 267 different Chinese and Japanese medicinal herbs were screened for mucosal adjuvant activity with influenza HA vaccine in mice. The extract from the root of Polygala tenuifolia was found to contain potent mucosal adjuvant activity. The active substances were purified and identified as onjisaponins A, E, F, and G. When each onjisaponin (10 μg) was intranasally (i.n.) inoculated with influenza vaccine (10 μg) in mice, serum hemagglutination-inhibiting (HI) antibody titers increased 3–14 times over control mice administered vaccine alone after 4 weeks. When each onjisaponin (10 μg) was i.n. inoculated with the vaccine (10 μg) followed by i.n. vaccination of the vaccine alone after 3 weeks, serum HI antibody titers increased 27–50 fold over those mice given i.n. vaccinations without onjisaponins. These same conditions also significantly increased nasal anti-influenza virus IgA antibody titers. Two inoculations with onjisaponin F (1 μg) and influenza HA vaccine (1 μg) at 3 weeks intervals, significantly increased serum HI antibody and nasal anti-influenza virus IgA and IgG antibody titers after only 1 week over mice given HA vaccine alone after the secondary vaccination. Intranasal vaccination with onjisaponin F inhibited proliferation of mouse adapted influenza virus A/PR/8/34 in bronchoalveolar lavages of infected mice. Separate intranasal vaccinations with onjisaponins A, E, F, and G (10 μg) each and diphtheria–pertussis–tetanus (DPT) vaccine (10 μg) of mice followed by i.n. vaccination with DPT vaccine alone after 4 weeks showed significant increases in serum IgG and nasal IgA antibody titers after 2 weeks following secondary vaccination over mice vaccinated with DPT vaccine alone. All onjisaponins showed little hemolytic activity at concentrations up to 100 μg/ml. The results of this study suggest that onjisaponins may provide safe and potent adjuvants for intranasal inoculation of influenza HA and DPT vaccines.     

Cross-protective immunity in mice induced by live-attenuated or inactivated vaccines against highly pathogenic influenza A (H5N1) viruses

Because of the time required to identify and produce an antigenically well-matched pandemic vaccine, vaccines that offer broader cross-reactive immunity and protection are desirable. We have compared a live attenuated influenza vaccine (LAIV) and inactivated influenza vaccine (IIV) based on a related H5 hemagglutinin (HA) from a nonpathogenic avian influenza virus, A/Duck/Pottsdam/1042-6/86 (H5N2), for the ability to induce cross-reactive immunity and/or cross-protective efficacy against a contemporary highly pathogenic H5N1 viruses. Both LAIV and IIV provided cross-protection from systemic infection, severe disease, and death following lethal challenges with antigenically distinct A/Vietnam/1203/2004 (VN/1203) virus. Substantial levels of serum anti-VN/1203 HA IgG were detected in mice that received either IIV or LAIV, while nasal wash anti-VN/1203 HA IgA was detected in mice that received LAIV. Formulation of IIV with alum adjuvant augmented neutralizing antibody responses and protective efficacy. These results demonstrated that vaccination of mice with H5 IIV or LAIV induced a high degree of cross-protection from illness and death following lethal challenges with a heterologous H5N1 virus.     

Novel influenza vaccine M2SR protects against drifted H1N1 and H3N2 influenza virus challenge in ferrets with pre-existing immunity

Current influenza vaccines do not provide effective protection against heterologous influenza viruses. The ability of the novel M2SR influenza vaccine to protect against drifted influenza viruses was evaluated in naïve ferrets and in ferrets with pre-existing immunity to influenza. In naïve ferrets, M2SR provided similar protection against drifted challenge viruses as the comparator vaccine, FluMist®. However, in ferrets with pre-existing immunity, M2SR provided superior protection than FluMist in two model systems.In the first model, ferrets were infected with influenza A H1N1pdm and influenza B viruses to mimic the diverse influenza exposure in humans. The pre-infected ferrets, seropositive to H1N1pdm and influenza B but seronegative to H3N2, were then vaccinated with H3N2 M2SR or monovalent H3N2 FluMist virus (A/Brisbane/10/2007, clade 1) and challenged 6 weeks later with a drifted H3N2 virus (clade 3C.2a). Antibody titers to Brisbane/10/2007 were higher in M2SR vaccinated ferrets than in FluMist vaccinated ferrets in the pre-infected ferrets whereas the opposite was observed in naïve ferrets. After challenge with drifted H3N2 virus, M2SR provided superior protection than FluMist monovalent vaccine.In the second model, the impact of homologous pre-existing immunity upon vaccine-induced protection was evaluated. Ferrets, pre-infected with H1N1pdm virus, were vaccinated 90 days later with H1N1pdm M2SR or FluMist monovalent vaccine and challenged 6 weeks later with a pre-pandemic seasonal H1N1 virus, A/Brisbane/59/2007 (Bris59). While cross-reactive serum IgG antibodies against the Bris59 HA were detected after vaccination, anti-Bris59 hemagglutination inhibition antibodies were only detected post-challenge. M2SR provided better protection against Bris59 challenge than FluMist suggesting that homologous pre-existing immunity affected FluMist virus to a greater degree than M2SR.These results suggest that the single replication intranasal M2SR vaccine provides effective protection against drifted influenza A viruses not only in naïve ferrets but also in those with pre-existing immunity in contrast to FluMist viruses.