Protection of chickens against highly lethal H5N1 and H7N1 avian influenza viruses with a recombinant fowlpox virus co-expressing H5 haemagglutinin and N1 neuraminidase genes

Inactivated whole avian influenza virus (AIV) vaccine provides protection against homologous haemagglutinin (HA) subtype virus, but poor protection against a heterologous HA virus. Moreover, it induces chickens to produce antibodies to cross-reactive antigens, especially nucleoprotein, which is limits AIV serological surveillance. In this study, a recombinant fowlpox virus co-expressing HA (H5 subtype) and NA (N1 subtype) genes of AIV was evaluated for its ability to protect chickens against intramuscular challenge with a lethal dose of highly pathogenic (HP) AIV. Susceptible chickens were also vaccinated by wing-web puncture with the parent fowlpox vaccine virus. Following challenge 4 weeks later with HPAIV, all chickens vaccinated with recombinant virus were protected, while the chickens vaccinated with either the unaltered parent fowlpox vaccine virus or unvaccinated controls experienced 100% mortality following challenge. This protection was accompanied by the high levels of specific antibody to the respective components of the recombinant vaccine. The above results showed that rFPV-HA-NA could be a potential vaccine to replace current inactivated vaccines for preventing AI.     

Potency of an inactivated avian influenza vaccine prepared from a non-pathogenic H5N1 reassortant virus generated between isolates from migratory ducks in Asia

A reassortant influenza virus, A/duck/Hokkaido/Vac-1/2004 (H5N1) (Dk/Vac-1/04), was generated between non-pathogenic avian influenza viruses isolated from migratory ducks in Asia. Dk/Vac-1/04 (H5N1) virus particles propagated in embryonated chicken eggs were inactivated with formalin and adjuvanted with mineral oil to form a water-in-oil emulsion. The resulting vaccine was injected intramuscularly into chickens. The chickens were challenged with either of the highly pathogenic avian influenza virus strains A/chicken/Yamaguchi/7/2004 (H5N1) or A/swan/Mongolia/3/2005 (H5N1) at 21 days post-vaccination (p. v.), when the geometric mean serum HI titers of the birds was 64 with the challenge virus strains. The vaccinated chickens were protected from manifestation of disease signs upon challenge with either of the highly pathogenic avian influenza viruses. However, challenge virus was recovered at low titers from the birds at 2 and 4 days post-challenge (p.c.). All 3 chickens challenged at 6 days p.v. died, whereas 3 chickens challenged at 8 days p.v. survived. These results indicate that the present vaccine confers clinical protection and reduction of virus shedding against highly pathogenic avian influenza virus challenge and should be useful as an optional tool in emergency cases.     

Inactivated North American and European H5N2 avian influenza virus vaccines protect chickens from Asian H5N1 high pathogenicity avian influenza virus.

High-pathogenicity (HP) avian influenza (AI) virus of the H5N1 subtype has caused an unprecedented epizootic in birds within nine Asian countries/regions since it was first reported in 1996. Vaccination has emerged as a tool for use in managing the infection in view of future eradication. This study was undertaken to determine whether two divergent H5N2 commercial vaccine strains, one based on a European and the other a North American low-pathogenicity AI virus, could protect chickens against a recent Asian H5N1 HPAI virus. The North American and European vaccine viruses had 84 and 91% deduced amino acid sequence similarity to the HA1 segment of haemagglutinin protein of Indonesia H5N1 HPAI challenge virus, respectively. Both vaccine strains provided complete protection from clinical signs and death. The vaccines reduced the number of chickens infected and shedding virus from the respiratory and intestinal tracts at the peak of virus replication. In addition, the quantity of virus shed was reduced by 10(4) to 10(5) median embryo infectious doses. The use of specific neuraminidase inhibition tests allowed identification of infected chickens within the vaccinated groups. These data indicate that the currently available H5 vaccines of European and North American lineages will protect chickens against the Asian H5N1 HPAI virus and reduce environmental contamination by the H5N1 HPAI virus. They will be an adjunct to biosecurity measures to reduce virus transmission.     

Protective efficacy in chickens, geese and ducks of an H5N1-inactivated vaccine developed by reverse genetics

We generated a high-growth H5N1/PR8 virus by plasmid-based reverse genetics. The virulence associated multiple basic amino acids of the HA gene were removed, and the resulting virus is attenuated for chickens and chicken eggs. A formalin-inactivated oil-emulsion vaccine was prepared from this virus. When SPF chickens were inoculated with 0.3 ml of the vaccine, the hemagglutinin-inhibition (HI) antibody became detectable at 1 week post-vaccination (p.v.) and reached a peak of 10log2 at 6 weeks p.v. then slowly declined to 4log2 at 43 weeks p.v. Challenge studies performed at 2, 3 and 43 weeks p.v. indicated that all of the chickens were completely protected from disease signs and death. Ducks and geese were completely protected from highly pathogenic H5N1 virus challenge 3 weeks p.v. The duration of protective immunity in ducks and geese was investigated by detecting the HI antibody of the field vaccinated birds, and the results indicated that 3 doses of the vaccine inoculation in geese could induce a 34 weeks protection, while 2 doses induced more than 52 weeks protection in ducks. We first reported that an oil-emulsion inactivated vaccine derived from a high-growth H5N1 vaccine induced approximately 10 months of protective immunity in chickens and demonstrated that the oil-emulsion inactivated avian influenza vaccine is immunogenic for geese and ducks. These results provide useful information for the application of vaccines to the control of H5N1 avian influenza in poultry, including chickens and domestic waterfowl.     

Comprehensive serological analysis of two successive heterologous vaccines against H5N1 avian influenza virus in exotic birds in zoos

In 2005, European Commission directive 2005/744/EC allowed controlled vaccination against avian influenza (AI) virus of valuable avian species housed in zoos. In 2006, 15 Spanish zoos and wildlife centers began a vaccination program with a commercial inactivated H5N9 vaccine. Between November 2007 and May 2008, birds from 10 of these centers were vaccinated again with a commercial inactivated H5N3 vaccine. During these campaigns, pre- and postvaccination samples from different bird orders were taken to study the response against AI virus H5 vaccines. Sera prior to vaccinations with both vaccines were examined for the presence of total antibodies against influenza A nucleoprotein (NP) by a commercial competitive enzyme-linked immunosorbent assay (cELISA). Humoral responses to vaccination were evaluated using a hemagglutination inhibition (HI) assay. In some taxonomic orders, both vaccines elicited comparatively high titers of HI antibodies against H5. Interestingly, some orders, such as Psittaciformes, which did not develop HI antibodies to either vaccine formulation when used alone, triggered notable HI antibody production, albeit in low HI titers, when primed with H5N9 and during subsequent boosting with the H5N3 vaccine. Vaccination with successive heterologous vaccines may represent the best alternative to widely protect valuable and/or endangered bird species against highly pathogenic AI virus infection.     

Preparation of a standardized, efficacious agricultural H5N3 vaccine by reverse genetics

Options for the control of emerging and reemerging H5N1 influenza viruses include improvements in biosecurity and the use of inactivated vaccines. Commercially available H5N2 influenza vaccine prevents disease signs and reduces virus load but does not completely prevent virus shedding after challenge with H5N1 virus. By using reverse genetics, we prepared an H5N3 vaccine whose hemagglutinin is 99.6% homologous to that of A/CK/HK/86.3/02 (H5N1). We used the internal genes of A/PR/8/34 and the H5 of A/Goose/HK/437.4/99 (H5N1) after deletion of basic amino acids from its connecting peptide region. The resulting virus was not lethal to chicken embryos and grew to high HA titers in eggs, allowing preparation of HA protein-standardized vaccine in unconcentrated allantoic fluid. The N3 neuraminidase, derived from A/Duck/Germany/1215/73 (H2N3), permitted discrimination between vaccinated and naturally infected birds. The virus construct failed to replicate in quail and chickens. Similar to parental A/PR/8/34 (H1N1), it replicated in mice and ferrets and spread to the brains of mice; therefore, it should not be used as a live-attenuated vaccine. The H5N3 vaccine, at doses of 1.2 microg HA, induced HI antibodies in chickens and prevented death, signs of disease, and markedly reduced virus shedding after challenge with A/CK/HK/86.3/02 (H5N1) but did not provide sterilizing immunity. Thus, reverse genetics allows the inexpensive preparation of standardized, efficacious H5N3 poultry vaccines that may also reduce the reemergence of H5N1 genotypes.     

Vaccination of chickens against H5N1 avian influenza in the face of an outbreak interrupts virus transmission.

Vaccination of chickens with a commercially available killed H5N2 vaccine was being evaluated as an additional tool to enhanced biosecurity measures and intensive surveillance for control of highly pathogenic avian influenza subtype H5N1 disease in Hong Kong in 2002. In December 2002 to January 2003, there were outbreaks of H5N1 disease in waterfowl in two recreational parks, wild water birds, several poultry markets and five chicken farms. In addition to quarantine, depopulation of the affected sheds and increased biosecurity, vaccination of the unaffected sheds and surrounding unvaccinated farms was undertaken on three farms. In at least two farms, infection spread to the recently vaccinated sheds with low rates of H5N1 mortality in sheds when the chickens were between 9 and 18 days post-vaccination. However, after 18 days post-vaccination no more deaths from H5N1 avian influenza occurred and intensive monitoring by virus culture on these farms showed no evidence of asymptomatic shedding of the virus. This provides evidence that H5 vaccine can interrupt virus transmission in a field setting.     

Genetic characterization of highly pathogenic H5N1 avian influenza virus from live migratory birds in Bangladesh

Since the first outbreak of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 in Bangladesh in 2007, the virus has been circulating among domestic poultry causing severe economic losses. To investigate the presence of HPAIV H5N1 in migratory birds and their potential role in virus spread, 205 pools of fecal samples from live migratory birds were analyzed. Here, the first virus isolation and genome characterization of two HPAIV H5N1 isolates from migratory birds (A/migratory bird/Bangladesh/P18/2010 and A/migratory bird/Bangladesh/P29/2010)are described. Full-length amplification, sequencing, and a comprehensive phylogenetic analysis were performed for HA, NA, M, NS, NP, PA, PB1, and PB2 gene segments. The selected migratory bird isolates belong to clade 2.3.2.1 along with recent Bangladeshi isolates from chickens, ducks, and crows which grouped in the same cluster with contemporary South and South-East Asian isolates. The studied isolates were genetically similar to other H5N1 isolates from different species within the respective clade although some unique amino acid substitutions were observed among them. Migratory birds remain a real threat for spreading pathogenic avian influenza viruses across the continent and introduction of new strains into Bangladesh.     

Efficacy of inactivated vaccines against H5N1 avian influenza infection in ducks

The current Asian H5N1 highly pathogenic avian influenza virus has spread over much of Asia and into Europe and Africa. As well as affecting village and commercial chicken operations in many South East Asian countries, it differs from past H5 avian influenza viruses in that it causes morbidity and mortalities in other domesticated birds, such as ducks and turkeys and in wild water birds. Effective vaccines that can prevent infection, as well as disease, and be used in a variety of avian species are needed for field use. In this report, a bivalent H5N9+H7N1 oil emulsion vaccine is compared, in ducks, to a monovalent H5N3 oil emulsion vaccine that has been derived by reverse genetics with an H5 from A/chicken/Vietnam/C58/04. While both vaccines protected against morbidity, the monovalent vaccine provided effective protection, with no evidence of shedding of the challenge virus and no serological response to the H5N1 challenge virus.     

Encephalitis in a stone marten (Martes foina) after natural infection with highly pathogenic avian influenza virus subtype H5N1

Recent outbreaks of disease in different avian species, caused by the highly pathogenic avian influenza virus (HPAIV), have involved infection by subtype H5N1 of the virus. This virus has also crossed species barriers and infected felines and humans. Here, we report the natural infection of a stone marten (Martes foina) from an area with numerous confirmed cases of H5N1 HPAIV infection in wild birds. Histopathological examination of tissues from this animal revealed a diffuse nonsuppurative panencephalitis with perivascular cuffing, multifocal gliosis and neuronal necrosis. Additionally, focal necrosis of pancreatic acinar cells was observed. Immunohistochemically, lesions in these organs were associated with avian influenza virus antigen in neurons, glial cells and pancreatic acinar cells. Thus, the microscopical lesions and viral antigen distribution in this stone marten differs from that recently described for cats naturally and experimentally infected with the same virus subtype. This is the first report of natural infection of a mustelid with HPAIV H5N1.     

The immunogenicity and efficacy against H5N1 challenge of reverse genetics-derived H5N3 influenza vaccine in ducks and chickens

H5N1 avian influenza viruses are continuing to spread in waterfowl in Eurasia and to threaten the health of avian and mammalian species. The possibility that highly pathogenic (HP) H5N1 avian influenza is now endemic in both domestic and migratory birds in Eurasia makes it unlikely that culling alone will control H5N1 influenza. Because ducks are not uniformly killed by HP H5N1 viruses, they are considered a major contributor to virus spread. Here, we describe a reverse genetics-derived high-growth H5N3 strain containing the modified H5 of A/chicken/Vietnam/C58/04, the N3 of A/duck/Germany/1215/73, and the internal genes of A/PR/8/34. One or two doses of inactivated oil emulsion vaccine containing 0.015 to 1.2 microg of HA protein provide highly efficacious protection against lethal H5N1 challenge in ducks; only the two dose regimen has so far been tested in chickens with high protective efficacy.     

Susceptibility and transmissibility of pigeons to Asian lineage highly pathogenic avian influenza virus subtype H5N1.

To determine whether or not pigeons are susceptible to infection with Asian lineage highly pathogenic (HP) avian influenza virus (AIV) subtype H5N1 and can serve as a transmission host for H5N1 HPAIV, we experimentally infected 187 young and adult pigeons with five different isolates of H5N1 HPAIV and co-habited some experimentally infected pigeons with susceptible specific pathogen free chickens. Results showed that all infected pigeons remained clinically healthy during the observation period. No gross lesions or histopathological changes were observed in the infected pigeons, and haemagglutination inhibition antibodies were not detected in serum samples of the infected pigeons. Additionally, all chickens placed in contact with AIV H5N1 infected pigeons remained healthy, and no virus or haemagglutination inhibition antibodies were detected in samples from the chickens. Our data suggest that pigeons are not susceptible to Asian lineage H5N1 HPAIV and do not transmit the virus to chickens.     

Prime-Boost Immunization Using a DNA Vaccine Delivered by Attenuated Salmonella enterica Serovar Typhimurium and a Killed Vaccine Completely Protects Chickens from H5N1 Highly Pathogenic Avian Influenza Virus

H5N1 highly pathogenic avian influenza virus (HPAIV) has posed a great threat not only for the poultry industry but also for human health. However, an effective vaccine to provide a full spectrum of protection is lacking in the poultry field. In the current study, a novel prime-boost vaccination strategy against H5N1 HPAIV was developed: chickens were first orally immunized with a hemagglutinin (HA) DNA vaccine delivered by attenuated Salmonella enterica serovar Typhimurium, and boosting with a killed vaccine followed. Chickens in the combined vaccination group but not in single vaccination and control groups were completely protected against disease following H5N1 HPAIV intranasal challenge, with no clinical signs and virus shedding. Chickens in the prime-boost group also generated significantly higher serum hemagglutination inhibition (HI) titers and intestinal mucosal IgA titers against avian influenza virus (AIV) and higher host immune cellular responses than those from other groups before challenge. These results demonstrated that the prime-boost vaccination strategy provides an effective way to prevent and control H5N1 highly pathogenic avian influenza virus.The highly pathogenic avian influenza virus (HPAIV) H5N1 strain can cause severe clinical signs in poultry, which may result in a mortality rate up to 100% (1, 2). The first outbreak of the H5N1 HPAIV was in Hong Kong in 1997. Subsequently, the virus spread to several other countries in Asia, Europe, and Africa (2). Not only did infections with H5N1 strains result in production losses and high mortality in poultry, but this virus also infected humans, causing severe public health problems. Since 2003, there have been 467 confirmed human cases of H5N1 virus infection, and its fatality rate reached 60.4% (as of 30 December 2009) (http://www.who.int/csr/disease/avian_influenza/country/cases_table_2009_12_30/en/index.html).Vaccination is an important measure to prevent and control H5N1 HPAIV infections in the poultry industry. Killed vaccines have been used to control the spread of highly pathogenic H5 avian influenza viruses (AIV) in some countries (9). In China, a killed vaccine derived from A/turkey/England/N-28/73 (H5N2) was first used for buffer zone vaccination during H5N1 outbreaks in 2004. Killed vaccines against H5N1 AIV can eliminate clinical signs of illness, but they do not completely prevent infection and virus shedding (5, 29). DNA vaccination has been explored as an alternative approach to protect chickens against H5N1 HPAIV (15, 19, 28). However, to our knowledge, there is no known poultry vaccine that can provide a full spectrum of protection against H5N1 HPAIV.The lack of an effective vaccine against H5N1 HPAIV is most likely due to H5N1 HPAIV transmission through mucosal sites (12); the conventional killed vaccines and DNA vaccines are administered usually via parenteral pathways, leading to a weak mucosal immunity. Recently, a variety of Gram-positive and Gram-negative bacteria (such as Salmonella, Shigella, and Listeria) have been used as carriers for efficient delivery of either DNA vaccine constructs or vaccine antigens (10). In particular, attenuated Salmonella enterica serovar Typhimurium has been used to deliver DNA vaccines encoding immunogens of pathogenic microorganisms, including AIV, infectious bursal disease virus (IBDV), porcine reproductive and respiratory syndrome virus (PRRSV), and transmissible gastroenteritis virus (TGEV) (14, 22, 31, 38). This strategy allows administration of DNA vaccines via mucosal surfaces as well as delivery of the plasmid DNA directly to professional antigen-presenting cells (APC), which can elicit humoral and cellular responses against the protective antigens at both mucosal and systemic levels (4, 27, 37).In a previous study, we have reported that Salmonella carrying hemagglutinin (HA) DNA vaccine could provide partial protection from H5N1 HPAIV challenge in chickens (31). To seek a more effective method of vaccination against H5N1 HPAIV, in the current study we tested the ability of different vaccination schedules to suppress viral shedding and resist homologous avian influenza virus challenge. As a number of studies have reported the effects of a DNA prime-protein or killed vaccine boost immunization against protozoal, bacterial, and viral infections (8, 20, 23, 24, 36), we determined whether priming with a DNA vaccine delivered by attenuated Salmonella Typhimurium and boosting with a killed vaccine could enhance the immune response and the protective efficacy against the challenge by H5N1 HPAIV.     

Induction of cytotoxic T-lymphocyte and antibody responses against highly pathogenic avian influenza virus infection in mice by inoculation of apathogenic H5N1 influenza virus particles inactivated with formalin

We investigated whether a vaccine derived from an apathogenic reassortant type A H5N1 influenza strain could induce immune responses in vivo that mediated protection from highly pathogenic avian influenza virus infection in mice. After two subcutaneous immunizations with formalin-inactivated H5N1 whole virus particles (whole particle vaccine), significant killing specific for cells presenting a nucleoprotein peptide from the vaccine strain of the virus was observed. Similar vaccination with viruses treated with ether and formalin, which are commonly used for humans as ether-split vaccines, induced little or no cytotoxic T-cell response. Furthermore, whole particle vaccines of the apathogenic H5N1 strain were more effective than ether-split vaccines at inducing antibody production able to neutralize a highly pathogenic H5N1 strain. Finally, whole particle vaccines of H5N1 protected mice against infection by an H5N1 highly pathogenic avian influenza virus more effectively than did ether-split vaccines. These results suggest that formalin-inactivated virus particles of apathogenic strains are effective for induction of both cytotoxic T-lymphocyte and antibody responses against highly pathogenic avian influenza viruses in vivo, resulting in protection from infection by a highly pathogenic H5N1 virus.     

禽流感重组禽痘病毒rFPV-HA-NA活载体疫苗的研究

目的：确定重组禽痘病毒rFPV-HA-NA疫苗株的最佳免疫剂量、免疫日龄、免疫产生时间及免疫持续期。方法：用重组禽痘病毒rFPV-HA-NA疫苗免疫SPF鸡，免疫后用HPAIVH5N1和H7N1AIV进行致死性攻击，观察疫苗免疫后的保护效果。结果：大约含100个PFU的重组病毒即能使机体获得对强毒攻击的100％保护；对1日龄SPF鸡进行免疫接种，4周后能100％抵抗HPAIV的致死性攻击；2周和3周龄的免疫鸡对免疫周1后的强毒攻击具有100％的保护力；重组病毒在免疫后10个月时，其诱导产生的血凝抑制（Haemagglutinin inhibition，HI）抗体仍保持在2-3log2水平，并可以提供100％抵抗病毒的致死性感染。结论：重组禽痘病毒rFPV-HA-NA疫苗是一种安全、高效的基因工程疫苗，它有望在不久的将来替代全病毒灭活疫苗用于高致病力禽流感的预防。     

Assembly and immunological properties of a bivalent virus-like particle (VLP) for avian influenza and Newcastle disease

Avian influenza virus (AIV) and Newcastle disease virus (NDV) are both important pathogens in poultry worldwide. The protection of poultry from avian influenza and Newcastle disease can be achieved through vaccination. We embarked on the development of a bivalent vaccine that would allow for a single immunization against both avian influenza and Newcastle disease. We constructed a chimeric virus-like particle (VLP) that is composed of the M1 protein and HA protein of avian influenza virus and a chimeric protein containing the cytoplasmic and transmembrane domains of AIV neuraminidase protein (NA) and the ectodomain of the NDV hemagglutinin-neuraminidase (HN) protein (NA/HN). The single immunization of chickens with the chimeric VLP vaccine induced both AIV H5- and NDV-specific antibodies. The HI titers and specific antibodies elicited by the chimeric VLPs were statistically similar to those elicited in animals vaccinated with the corresponding commercial monovalent vaccines. Chickens vaccinated with chimeric VLP vaccine and then challenged with the Newcastle disease F48E9 virus displayed complete protection. Overall, the chimeric VLP vaccine elicits strong immunity and can protect against Newcastle disease virus challenge.     

Vaccination of gallinaceous poultry for H5N1 highly pathogenic avian influenza: Current questions and new technology

Vaccination of poultry for avian influenza virus (AIV) is a complex topic as there are numerous technical, logistic and regulatory aspects which must be considered. Historically, control of high pathogenicity (HP) AIV infection in poultry has been accomplished by eradication and stamping out when outbreaks occur locally. Since the H5N1 HPAIV from Asia has spread and become enzootic, vaccination has been used on a long-term basis by some countries to control the virus, other countries have used it temporarily to aid eradication efforts, while others have not used it at all. Currently, H5N1 HPAIV is considered enzootic in China, Egypt, Viet Nam, India, Bangladesh and Indonesia. All but Bangladesh and India have instituted vaccination programs for poultry. Importantly, the specifics of these programs differ to accommodate different situations, resources, and industry structure in each country. The current vaccines most commonly used are inactivated whole virus vaccines, but vectored vaccine use is increasing. Numerous technical improvements to these platforms and novel vaccine platforms for H5N1 vaccines have been reported, but most are not ready to be implemented in the field.     

Protection afforded by avian influenza vaccination programmes consisting of a novel RNA particle and an inactivated avian influenza vaccine against a highly pathogenic avian influenza virus challenge in layer chickens up to 18 weeks post-vaccination

The efficacies of an oil adjuvanted-inactivated reverse genetics-derived H5 avian influenza virus (AIV) vaccine and an alphavirus replicon RNA particle (RP) AIV vaccine were evaluated in commercial Leghorn chickens. Challenge utilized A/turkey/MN/12582/2015, an isolate representing the U.S. H5N2 Clade 2.3.4.4 responsible for the 2015 highly pathogenic avian influenza (HPAI) epornitic in commercial poultry the United States. As part of a long-term, 36-week study, chickens were challenged at seven weeks of age after receiving a single vaccination, at 18 weeks of age following a vaccine prime-single boost, and at 36 weeks of age after a prime- double-boost. All vaccine programmes reduced virus oropharyngeal and cloacal shedding and mortality compared to the non-vaccinated control birds; however, chickens receiving at least one administration of the RP vaccine generally had diminished viral shedding especially from the cloacal swabbings. A detectable serum antibody response and protection were observed through 18 weeks post-vaccination. Our data suggest that, in conjunction with a comprehensive eradication, enhanced biosecurity and controlled marketing plan, vaccination programmes of commercial layer chickens with novel RP vaccines may represent an important tool for preventing HPAI-related mortalities and decreasing viral load during a catastrophic influenza outbreak.

RESEARCH HIGHLIGHTS

• Immunization of poultry following a vaccination schedule consisting of inactivated and RNA particle vaccines offered significant protection against lethal disease following HPAIV challenge.

• Virus shedding was significantly (P?<?0.05) reduced in chickens vaccinated with either inactivated and/or recombinant vaccines.

• Serum antibody titres were not a reliable indicator of protection.

• An inactivated vaccine containing 384 HAU of the homologous antigen was unable to induce complete protection.

     

New Strategies for the Development of H5N1 Subtype Influenza Vaccines

The emergence and spread of highly pathogenic avian influenza (H5N1) viruses among poultry in Asia, the Middle East, and Africa have fueled concerns of a possible human pandemic, and spurred efforts towards developing vaccines against H5N1 influenza viruses, as well as improving vaccine production methods. In recent years, promising experimental reverse genetics-derived H5N1 live attenuated vaccines have been generated and characterized, including vaccines that are attenuated through temperature-sensitive mutation, modulation of the interferon antagonist protein, or disruption of the M2 protein. Live attenuated influenza virus vaccines based on each of these modalities have conferred protection against homologous and heterologous challenge in animal models of influenza virus infection. Alternative vaccine strategies that do not require the use of live virus, such as virus-like particle (VLP) and DNA-based vaccines, have also been vigorously pursued in recent years. Studies have demonstrated that influenza VLP vaccination can confer homologous and heterologous protection from lethal challenge in a mouse model of infection. There have also been improvements in the formulation and production of vaccines following concerns over the threat of H5N1 influenza viruses. The use of novel substrates for the growth of vaccine virus stocks has been intensively researched in recent years, and several candidate cell culture-based systems for vaccine amplification have emerged, including production systems based on Madin-Darby canine kidney, Vero, and PerC6 cell lines. Such systems promise increased scalability of product, and reduced reliance on embryonated chicken eggs as a growth substrate. Studies into the use of adjuvants have shown that oil-in-water-based adjuvants can improve the immunogenicity of inactivated influenza vaccines and conserve antigen in such formulations. Finally, efforts to develop more broadly cross-protective immunization strategies through the inclusion of conserved influenza virus antigens in vaccines have led to experimental vaccines based on the influenza hemagglutinin (HA) stem domain. Such vaccines have been shown to confer protection from lethal challenge in mouse models of influenza virus infection. Through further development, vaccines based on the HA stem have the potential to protect vaccinated individuals against unanticipated pandemic and epidemic influenza virus strains. Overall, recent advances in experimental vaccines and in vaccine production processes provide the potential to lower mortality and morbidity resulting from influenza infection.