Plant-derived hemagglutinin protects ferrets against challenge infection with the A/Indonesia/05/05 strain of avian influenza

The global spread of highly pathogenic avian influenza virus (H5N1 subtype) has promoted efforts to develop human vaccines against potential pandemic outbreaks. However, current platforms for influenza vaccine production are cumbersome, limited in scalability and often require the handling of live infectious virus. We describe the production of hemagglutinin from the A/Indonesia/05/05 strain of H5N1 influenza virus by transient expression in plants, and demonstrate the immunogenicity and protective efficacy of the vaccine candidate in animal models. Immunization of mice and ferrets with plant-derived hemagglutinin elicited serum hemagglutinin-inhibiting antibodies and protected the ferrets against challenge infection with a homologous virus. This demonstrates that plant-derived H5 HA is immunogenic in mice and ferrets, and can induce protective immunity against infection with highly pathogenic avian influenza virus. Plants could therefore be suitable as a platform for the rapid, large-scale production of influenza vaccines in the face of a pandemic.     

Control of pandemic (H1N1) 2009 influenza virus infection of ferret lungs by non-adjuvant-containing pandemic and seasonal vaccines

The pandemic H1N1 2009 influenza virus caused relatively mild disease in most infected people but some suffered extensively from primary lung infection, many more than would have occurred with seasonal influenza infection. Early commercially available pandemic H1N1 vaccines did not contain adjuvant, as did many of the subsequent vaccines, and could not stop infection with the pandemic virus in vaccinated ferrets. Nevertheless, we showed that virus loads in the lungs were greatly diminished in ferrets vaccinated once with an unadjuvanted pandemic vaccine and challenged with 10(6)EID(50) wildtype A/California/07/2009 (H1N1). In addition, a single inoculation with seasonal vaccine showed beneficial reduction in pandemic pulmonary virus loads in the absence of any detectable cross-reactive serological responses. Ferrets primed with either seasonal or pandemic vaccine and then boosted with pandemic vaccine also showed less extensive lung infection when challenged with a tenfold higher dose of pandemic virus. These results implicate non-classical protective mechanisms that prevent severe pulmonary disease but not viral shedding and imply that particular non-adjuvanted vaccines may have retained the ability to induce these responses.     

Protective efficacy of a polyvalent influenza A DNA vaccine against both homologous (H1N1pdm09) and heterologous (H5N1) challenge in the ferret model

This study describes the protective efficacy of a novel influenza plasmid DNA vaccine in the ferret challenge model. The rationally designed polyvalent influenza DNA vaccine encodes haemagglutinin and neuraminidase proteins derived from less glycosylated pandemic H1N1 (2009) and H3N2 (1968) virus strains as well as the nucleoprotein (NP) and matrix proteins (M1 and M2) from a different pandemic H1N1 (1918) strain. Needle-free intradermal immunisation with the influenza DNA vaccine protected ferrets against homologous challenge with an H1N1pdm09 virus strain, demonstrated by restriction of viral replication to the upper respiratory tract and reduced duration of viral shedding post-challenge. Breadth of protection was demonstrated in two heterologous efficacy experiments in which animals immunised with the influenza DNA vaccine were protected against challenge with a highly pathogenic avian influenza H5N1 virus strain with reproducible survival and clinical outcomes.     

Pandemic H1N1 influenza virus-like particles are immunogenic and provide protective immunity to pigs

The outbreak of the 2009 influenza pandemic underscored the important role of swine in influenza virus evolution and the emergence of novel viruses with pandemic potential. Vaccination is the most common practice to control swine influenza in swine industry. Influenza virus-like particle (VLP) vaccines are an alternative approach and have been demonstrated to be immunogenic and confer protection against influenza virus challenge in chickens, mice and ferrets. In this study, we generated VLPs consisting of HA, NA and M1 proteins derived from pandemic virus A/California/04/2009 in insect cells. The immunogenicity and efficacy following vaccination of VLPs were evaluated in swine. Our data showed that vaccination using VLPs elicited robust levels of serum IgG, mucosal IgA, and viral neutralizing antibodies against A/Sw/Manitoba/MAFRI32/2009 H1N1. Following challenge with pandemic H1N1 2009, vaccinated pigs were protected, displaying reduced lung lesions, virus shedding and inhibition of virus replication in the lungs compared to non-vaccinated control pigs. Thus, VLPs can serve as a promising vaccination strategy to control influenza in swine.     

An elastase-dependent attenuated heterologous swine influenza virus protects against pandemic H1N1 2009 influenza challenge in swine

Influenza virus infections continue to cause production losses in the agricultural industry in addition to being a human public health concern. The primary method to control influenza is through vaccination. However, currently used killed influenza virus vaccines must be closely matched to the challenge virus. The ability of an elastase-dependent live attenuated influenza A virus was evaluated to protect pigs against the pandemic H1N1 2009 influenza virus. Pigs vaccinated intranasally or intratracheally with the elastase-dependent swine influenza virus (SIV) vaccine had significantly reduced macroscopic and microscopic lung lesions and lower viral loads in the lung and in nasal swabs. Thus, elastase-dependent SIV mutants can be used as live-virus vaccines against swine influenza in pigs. In addition, low levels of cross-neutralizing antibodies to H1N1 2009 were elicited prior to challenge by the swine adapted H1N1 avian strain vaccine.     

Multi-clade H5N1 virus-like particles: Immunogenicity and protection against H5N1 virus and effects of beta-propiolactone

During the past decade, H5N1 highly pathogenic avian influenza (HPAI) viruses have diversified genetically and antigenically, suggesting the need for multiple H5N1 vaccines. However, preparation of multiple vaccines from live H5N1 HPAI viruses is difficult and economically not feasible representing a challenge for pandemic preparedness. Here we evaluated a novel multi-clade recombinant H5N1 virus-like particle (VLP) design, in which H5 hemagglutinins (HA) and N1 neuraminidase (NA) derived from four distinct clades of H5N1 virus were co-localized within the VLP structure. The multi-clade H5N1 VLPs were prepared by using a recombinant baculovirus expression system and evaluated for functional hemagglutination and neuraminidase enzyme activities, particle size and morphology, as well as for the presence of baculovirus in the purified VLP preparations. To remove residual baculovirus, VLP preparations were treated with beta-propiolactone (BPL). Immunogenicity and efficacy of multi-clade H5N1 VLPs were determined in an experimental ferret H5N1 HPAI challenge model, to ascertain the effect of BPL on immunogenicity and protective efficacy against lethal challenge. Although treatment with BPL reduced immunogenicity of VLPs, all vaccinated ferrets were protected from lethal challenge with influenza A/VietNam/1203/2004 (H5N1) HPAI virus, indicating that multi-clade VLP preparations treated with BPL represent a potential approach for pandemic preparedness vaccines.     

Efficacy of seasonal live attenuated influenza vaccine against virus replication and transmission of a pandemic 2009 H1N1 virus in ferrets

In March 2009, a swine origin influenza A (2009 H1N1) virus was introduced into the human population and quickly spread from North America to multiple continents. Human serologic studies suggest that seasonal influenza virus vaccination or infection would provide little cross-reactive serologic immunity to the pandemic 2009 H1N1 virus. However, the efficacy of seasonal influenza infection or vaccination against 2009 H1N1 virus replication and transmission has not been adequately evaluated in vivo. Here, ferrets received one or two doses of the US licensed 2008-2009 live attenuated influenza vaccine (LAIV) intranasally. An additional group of ferrets were inoculated with the A/Brisbane/59/07 (H1N1) virus to model immunity induced by seasonal influenza virus infection. All vaccinated and infected animals possessed high titer homologous hemagglutination-inhibition (HI) and neutralizing antibodies, with no demonstrable cross-reactive antibodies against 2009 H1N1 virus. However, in comparison to non-immune controls, immunized ferrets challenged with pandemic A/Mexico/4482/09 virus displayed a significant reduction in body temperature and virus shedding. The impact of single-dose LAIV inoculation on 2009 H1N1 disease and virus transmission was also measured in vaccinated ferrets that were challenged with pandemic A/Netherlands/1132/09 virus. Although a single dose of LAIV reduced virus shedding and the frequency of transmission following homologous seasonal virus challenge, it failed to reduce respiratory droplet transmission of 2009 H1N1 virus. The results demonstrate that prior immunization with seasonal LAIV or H1N1 virus infection provides some cross-protection against the 2009 H1N1 virus, but had no significant effect on the transmission efficiency of the 2009 H1N1 virus.     

Pandemic H1N1 vaccine requires the use of an adjuvant to protect against challenge in naïve ferrets

In the context of an A/H1N1 influenza pandemic situation, this study demonstrates that heterologous vaccination with an AS03-adjuvanted 2008/2009 seasonal trivalent and pandemic H5N1 monovalent split vaccine conferred partial protection in influenza-naïve ferrets after challenge with the influenza pandemic H1N1 A/The Netherlands/602/09 virus. Further, unlike saline control and non-adjuvanted vaccine, it was shown that immunization of naïve ferrets with an AS03-adjuvanted pandemic H1N1 A/California/7/09 influenza split vaccine induced increased antibody response and enhanced protection against the challenge strain, including significant reduction in viral shedding in the upper respiratory tract and reduced lung pathology post-challenge. These results show the need for vaccination with the adjuvanted vaccine to fully protect against viral replication and influenza disease in unprimed ferrets.     

An M2 cytoplasmic tail mutant as a live attenuated influenza vaccine against pandemic (H1N1) 2009 influenza virus

The 2009 influenza pandemic brought home the importance of vaccines in infection control. Previously, we demonstrated an M2 cytoplasmic tail mutant H5N1 influenza virus could serve as a live-attenuated vaccine. Here, we adapted that strategy, generating a mutant pandemic (H1N1) 2009 virus that grew well in cell culture, but replicated less well in mice than did wild-type virus. The mutant virus elicited sterile immunity in mice, completely protecting them from challenge with a pandemic (H1N1) 2009 virus. Our results indicate that M2 cytoplasmic tail mutants are suitable for live-attenuated vaccines against pandemic viruses.     

Intranasally administered Endocine™ formulated 2009 pandemic influenza H1N1 vaccine induces broad specific antibody responses and confers protection in ferrets

Influenza is a contagious respiratory disease caused by an influenza virus. Due to continuous antigenic drift of seasonal influenza viruses, influenza vaccines need to be adjusted before every influenza season. This allows annual vaccination with multivalent seasonal influenza vaccines, recommended especially for high-risk groups. There is a need for a seasonal influenza vaccine that induces broader and longer lasting protection upon easy administration. Endocine™ is a lipid-based mucosal adjuvant composed of endogenous lipids found ubiquitously in the human body. Intranasal administration of influenza antigens mixed with this adjuvant has been shown to induce local and systemic immunity as well as protective efficacy against homologous influenza virus challenge in mice. Here we used ferrets, an established animal model for human influenza virus infections, to further investigate the potential of Endocine™ as an adjuvant. Intranasal administration of inactivated pandemic H1N1/California/2009 split antigen or whole virus antigen mixed with Endocine™ induced high levels of serum hemagglutination inhibition (HI) and virus neutralization (VN) antibody titers that were also cross reactive against distant swine viruses of the same subtype. HI and VN antibody titers were already demonstrated after a single nasal immunization. Upon intratracheal challenge with a homologous challenge virus (influenza virus H1N1/The Netherlands/602/2009) immunized ferrets were fully protected from virus replication in the lungs and largely protected against body weight loss, virus replication in the upper respiratory tract and pathological changes in the respiratory tract. Endocine™ formulated vaccines containing split antigen induced higher HI and VN antibody responses and better protection from body weight loss and virus shedding in the upper respiratory tract than the Endocine™ formulated vaccine containing whole virus antigen.     

Toxicological evaluation of live attenuated, cold-adapted H5N1 vaccines in ferrets

Live attenuated influenza vaccines (LAIV) have several attributes related to safety, immunogenicity, cross-protection against antigenic drift strains, high yield and needle-free administration that make them attractive candidates for control of pandemic influenza. H5N1 LAIV vaccine candidates are attenuated in ferrets, chickens and mice. These vaccine candidates were further characterized in the ferret model to evaluate their toxicity at doses comparable to seasonal LAIV and at doses up to 100-fold higher. The results demonstrated that H5N1 LAIV, even when administered at high doses, is restricted in replication in the lower respiratory tract of ferrets. However, intranasal administration of 0.5 mL can result in deposition of H5N1 LAIV in the ferret lung, where it induces a pulmonary inflammatory response in the absence of significant local replication of the vaccine virus. Thus, smaller vaccine dose volumes should be considered for evaluation of LAIV in animal models.     

Serologic status for pandemic (H1N1) 2009 virus, Taiwan

We studied preexisting immunity to pandemic (H1N1) 2009 virus in persons in Taiwan. A total of 18 (36%) of 50 elderly adults in Taiwan born before 1935 had protective antibodies against currently circulating pandemic (H1N1) 2009 virus. Seasonal influenza vaccines induced antibodies that did not protect against pandemic (H1N1) 2009 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.     

NS-based live attenuated H1N1 pandemic vaccines protect mice and ferrets

Although vaccines against influenza A virus are the most effective method to combat infection, it is clear that their production needs to be accelerated and their efficacy improved. We generated live attenuated human influenza A vaccines (LAIVs) by rationally engineering mutations directly into the genome of a pandemic-H1N1 virus. Two LAIVs (NS1-73 and NS1-126) were based on the success of LAIVs for animal influenza A viruses. A third candidate (NSΔ5) is a unique NS-mutant that has never been used as a LAIV. The vaccine potential of each LAIV was determined through analysis of attenuation, interferon production, immunogenicity, and their ability to protect mice and ferrets. This study demonstrates that NSΔ5 is an ideal LAIV candidate, provides important information on the effects that different NS mutations have on the pandemic-H1N1 virus and shows that LAIVs can be engineered directly from the genomes of emerging/circulating influenza A viruses.     

Influenza virus-like particle can accommodate multiple subtypes of hemagglutinin and protect from multiple influenza types and subtypes

Despite existing vaccines and specific therapies, epidemics of seasonal influenza annually claim 200,000-500,000 lives worldwide. Pandemic influenza represents an even greater threat, with numerous potentially pandemic viruses circulating in nature. Development of multi-specific vaccines against multiple pandemic or seasonal strains is important for human health and the global economy. Here we report a novel virus-like particle (VLP) platform that contains three hemagglutinin (HA) subtypes. This recombinant vaccine design resulted in the expression of three HA subtypes co-localized within a VLP. Experimental triple-HA VLPs containing HA proteins derived from H5N1, H7N2, and H2N3 viruses were immunogenic and protected ferrets from challenge from all three potentially pandemic viruses. Similarly, VLPs containing HA subtypes derived from seasonal H1N1, H3N2, and type B influenza viruses protected ferrets from three seasonal influenza viruses. We conclude that this technology may represent a novel strategy for rapid development of trivalent seasonal and pandemic vaccines.     

Cell culture (Vero) derived whole virus (H5N1) vaccine based on wild-type virus strain induces cross-protective immune responses

The rapid spread and the transmission to humans of avian influenza virus (H5N1) have induced world-wide fears of a new pandemic and raised concerns over the ability of standard influenza vaccine production methods to rapidly supply sufficient amounts of an effective vaccine. We report here on a robust and flexible strategy which uses wild-type virus grown in a continuous cell culture (Vero) system to produce an inactivated whole virus vaccine. Candidate vaccines based on clade 1 and clade 2 influenza H5N1 strains were developed and demonstrated to be highly immunogenic in animal models. The vaccines induce cross-neutralising antibodies, highly cross-reactive T-cell responses and are protective in a mouse challenge model not only against the homologous virus but also against other H5N1 strains, including those from another clade. These data indicate that cell culture-grown whole virus vaccines, based on the wild-type virus, allow the rapid high yield production of a candidate pandemic vaccine.     

Heat shock protein gp96 adjuvant induces T cell responses and cross-protection to a split influenza vaccine

The commonly used inactivated or split influenza vaccines induce only induce minimal T cell responses and are less effective in preventing heterologous virus infection. Thus, developing cross-protective influenza vaccines against the spread of a new influenza virus is an important strategy against pandemic emergence. Here we demonstrated that immunization with heat shock protein gp96 as adjuvant led to a dramatic increased antigen-specific T cell response to a pandemic H1N1 split vaccine. Notably, gp96 elicited a cross-protective CD8⁺ T cell response to the internal conserved viral protein NP. Although the split pH1N1vaccine alone has low cross-protective efficiency, adding gp96 as an adjuvant effectively improved the cross-protection against challenge with a heterologous virus in mice. Our study reveals the novel property of gp96 in boosting the T cell response against conserved epitopes of influenza virus and its potential use as an adjuvant for human pre-pandemic inactivated influenza vaccines against different viral subtypes.     

Needle-free delivery of DNA: Targeting of hemagglutinin to MHC class II molecules protects rhesus macaques against H1N1 influenza

Conventional influenza vaccines are hampered by slow and limited production capabilities, whereas DNA vaccines can be rapidly produced for global coverage in the event of an emerging pandemic. However, a drawback of DNA vaccines is their generally low immunogenicity in non-human primates and humans. We have previously demonstrated that targeting of influenza hemagglutinin to human HLA class II molecules can increase antibody responses in larger animals such as ferrets and pigs. Here, we extend these observations by immunizing non-human primates (rhesus macaques) with a DNA vaccine encoding a bivalent fusion protein that targets influenza virus hemagglutinin (HA) to Mamu class II molecules. Such immunization induced neutralizing antibodies and antigen-specific T cells. The DNA was delivered by pain- and needle-free jet injections intradermally. No adverse effects were observed. Most importantly, the immunized rhesus macaques were protected against a challenge with influenza virus.     

H5N1 VLP vaccine induced protection in ferrets against lethal challenge with highly pathogenic H5N1 influenza viruses

In this study, recombinant virus-like particles (VLPs) were evaluated as a candidate vaccine against emerging influenza viruses with pandemic potential. The VLPs are composed of the hemagglutinin (HA), neuraminidase (NA), and matrix 1 (M1) proteins of the H5N1 A/Indonesia/05/2005 (clade 2.1; [Indo/05]) virus, which were expressed using baculovirus in Spodoptera frugiperda (Sf9) cells. Ferrets received either 2 injections of the VLP vaccine at escalating doses (based on HA content), recombinant HA, or were mock vaccinated. Vaccinated ferrets were then challenged with either H5N1 Indo/05 or H5N1 A/Viet Nam 1203/2004 (VN/04) wild-type viruses. All ferrets that received the VLP vaccine survived regardless of the VLP dose or challenge strain, whereas seven of eight mock vaccinated ferrets died. The VLP vaccine induced HAI antibodies against the homologous H5N1 clade 2.1 strain, as well as heterologous strains from H5N1 clades 1, 2.2, and 2.3. The magnitude of the HAI titers correlated with VLP dose. Neutralizing antibody responses against the Indo/05 and VN/04 strains showed a similar pattern. Affinity of the anti-HA antibodies raised by the H5N1 Indo/05 VLPs had a higher association rate to the homologous clade 2.1 HA than to the clade 1 (VN/04) HA; however, once bound, antibodies had similar slow disassociation rates. These results provide support for continued development of the H5N1 VLPs as a candidate vaccine against pandemic influenza. Exploration of immunologic correlates of protection for H5N1 vaccines beyond HAI and neutralizing antibody responses is warranted.     

Possible increased pathogenicity of pandemic (H1N1) 2009 influenza virus upon reassortment

Since emergence of the pandemic (H1N1) 2009 virus in April 2009, three influenza A viruses-seasonal (H3N2), seasonal (H1N1), and pandemic (H1N1) 2009-have circulated in humans. Genetic reassortment between these viruses could result in enhanced pathogenicity. We compared 4 reassortant viruses with favorable in vitro replication properties with the wild-type pandemic (H1N1) 2009 virus with respect to replication kinetics in vitro and pathogenicity and transmission in ferrets. Pandemic (H1N1) 2009 viruses containing basic polymerase 2 alone or in combination with acidic polymerase of seasonal (H1N1) virus were attenuated in ferrets. In contrast, pandemic (H1N1) 2009 with neuraminidase of seasonal (H3N2) virus resulted in increased virus replication and more severe pulmonary lesions. The data show that pandemic (H1N1) 2009 virus has the potential to reassort with seasonal influenza viruses, which may result in increased pathogenicity while it maintains the capacity of transmission through aerosols or respiratory droplets.