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.     

The immune response of a recombinant fowlpox virus coexpressing the HA gene of the H5N1 highly pathogenic avian influenza virus and chicken interleukin 6 gene in ducks

Ducks have played an important role in the emergence of H5N1 subtype of highly pathogenic avian influenza (HPAI), and the development of an effective vaccine against HPAI in ducks is a top priority. It has been shown that a recombinant fowlpox virus (FPV)-vectored vaccine can provide protection against HPAI in ducks. In this study, a recombinant fowlpox virus (rFPV-AIH5AIL6) coexpressing the haemagglutinin (HA) gene of the H5N1 subtype of the avian influenza virus (AIV) and chicken interleukin 6 gene was constructed and tested in Gaoyou and cherry valley ducks to evaluate the immune response in ducks. These animal studies demonstrated that rFPV-AIH5AIL6 induced a higher anti-AIV HI antibody response, an enhanced lymphocyte proliferation response, an elevated immune protection, and a reduction in virus shedding compared to a recombinant fowlpox virus expressing the HA gene alone (rFPV-SYHA). These data indicate that rFPV-AIH5AIL6 may be a potential vaccine against the H5 subtype of avian influenza in ducks and chicken interleukin 6 may be an effective adjuvant for increasing the immunogenicity of FPV-vectored AIV vaccines in ducks.     

H9N2 avian influenza virus-like particle vaccine provides protective immunity and a strategy for the differentiation of infected from vaccinated animals

In the present study, virus-like particles (VLPs) were evaluated as a candidate poultry vaccine against avian influenza virus (AIV) subtype H9N2. Specific pathogen-free chickens received a single injection of the VLP vaccine expressing HA and M1 protein of AIV H9N2 (H9 HA VLP) at escalating doses in the presence or absence of ISA70 water-in-oil adjuvant. At 3 weeks post vaccination, we performed hemagglutination inhibition (HI) test and enzyme-linked immunosorbent assay (ELISA) to determine serological immune responses, and challenge studies using SPF chickens. A single dose of H9 HA VLP vaccine induced high levels of HI antibodies and lowered frequencies of virus isolation after the wild-type virus challenge. The addition of ISA70 adjuvant significantly increased the immunogenicity of H9 HA VLP vaccines. Furthermore, it allows differentiation of AIV-infected chickens from vaccinated chickens with an ELISA using nucleocapsid antigen, which offers a promising strategy to differentiate infected from vaccinated animals (DIVA). These results provide support for continued development of the VLP as an animal vaccine against influenza virus.     

Multi-antigen vaccines based on complex adenovirus vectors induce protective immune responses against H5N1 avian influenza viruses

There are legitimate concerns that the highly pathogenic H5N1 avian influenza virus could adapt for human-to-human transmission and cause a pandemic similar to the 1918 "Spanish flu" that killed 50 million people worldwide. We have developed pandemic influenza vaccines by incorporating multiple antigens from both avian and Spanish influenza viruses into complex recombinant adenovirus vectors. In vaccinated mice, these vaccines induced strong humoral and cellular immune responses against pandemic influenza virus antigens, and protected vaccinated mice against lethal H5N1 virus challenge. These results indicate that this multi-antigen, broadly protective vaccine may serve as a safer and more effective approach than traditional methods for development of a pandemic influenza vaccine.     

Preclinical evaluation of a modified vaccinia virus Ankara (MVA)-based vaccine against influenza A/H5N1 viruses

Highly pathogenic avian influenza viruses of the H5N1 subtype are responsible for an increasing number of infections in humans since 2003. More than 60% of the infections is lethal and new infections are reported frequently. In the light of the pandemic threat caused by these events the rapid availability of safe and effective vaccines is desirable. Modified vaccinia virus Ankara (MVA) expressing the HA gene of an influenza A/H5N1 virus is a promising candidate vaccine that induced protective immunity against infection with homologous and heterologous influenza A/H5N1 viruses in mice. We also evaluated the recombinant MVA vector expressing the HA of influenza A/H5N1 virus A/Vietnam/1194/04 (MVA-HA-VN/04) in non-human primates. Cynomolgus macaques were immunized twice and then challenged with influenza virus A/Vietnam/1194/04 (clade 1) or A/Indonesia/5/05 (clade 2.1) to assess the level of protective immunity. Immunization with MVA-HA-VN/04 induced (cross-reactive) antibodies and prevented virus replication in the upper and lower respiratory tract and the development of severe necrotizing bronchointerstitial pneumonia. Therefore MVA-HA-VN/04 is a promising vaccine candidate for the induction of protective immunity against highly pathogenic avian influenza A/H5N1 viruses.     

Activation of cross-reactive mucosal T and B cell responses in human nasopharynx-associated lymphoid tissue in vitro by Modified Vaccinia Ankara-vectored influenza vaccines

Recent efforts have been focused on the development of vaccines that could induce broad immunity against influenza virus, either through T cell responses to conserved internal antigens or B cell response to cross-reactive haemagglutinin (HA). We studied the capacity of Modified Vaccinia Ankara (MVA)-vectored influenza vaccines to induce cross-reactive immunity to influenza virus in human nasopharynx-associated lymphoid tissue (NALT) in vitro. Adenotonsillar cells were isolated and stimulated with MVA vaccines expressing either conserved nucleoprotein (NP) and matrix protein 1 (M1) (MVA-NP-M1) or pandemic H1N1 HA (MVA-pdmH1HA). The MVA vaccine uptake and expression, and T and B cell responses were analyzed. MVA-vectored vaccines were highly efficient infecting NALT and vaccine antigens were highly expressed by B cells. MVA-NP-M1 elicited T cell response with greater numbers of IFNγ-producing CD4+ T cells and tissue-resident memory T cells than controls. MVA-pdmH1HA induced cross-reactive anti-HA antibodies to a number of influenza subtypes, in an age-dependent manner. The cross-reactive antibodies include anti-avian H5N1 and mainly target HA2 domain. Conclusion: MVA vaccines are efficient in infecting NALT and the vaccine antigen is highly expressed by B cells. MVA vaccines expressing conserved influenza antigens induce cross-reactive T and B cell responses in human NALT in vitro, suggesting the potential as mucosal vaccines for broader immunity against influenza.     

禽流感病毒研究进展及抗H7N9型病毒疫苗与抗体研究

禽流感病毒（avian influenza virus,AIV）是一种可引起急性呼吸道传染病的人畜共患病毒。自2013年我国出现了全球首例人感染H7N9型AIV病例以来,人们对该病毒产生了担忧与恐慌。AIV在全球广泛传播,人感染不同型别AIV事件也持续发生,造成了巨大的经济损失。目前尚无针对该病的特异性治疗措施与药物,疫苗成为最有可能预防控制病毒传播的手段。现有针对H7N9型AIV的兽用与人用疫苗种类繁多,其中,4类人用H7N9型AIV疫苗已经率先进入了临床试验阶段,主要包括了病毒样颗粒疫苗、减毒活疫苗、灭活疫苗及DNA疫苗,并显示出了良好的安全性和免疫原性。因为暂无上市的人用AIV疫苗,所以其真实效力不得而知。此外,现有的流感疫苗在人群中虽然具有良好的安全性和免疫原性,但对H7N9型AIV并无交叉抗体反应。本文回顾AIV的病原学、流行病学、职业暴露人群调查与防控策略、H7N9型AIV疫苗及H7N9型AIV全人源单克隆抗体研究进展,讨论尚存的问题和挑战以及未来的发展方向,为加深对疾病的了解以及控制AIV在全球的蔓延提供防控策略与方针。     

Vaccine protection of chickens against antigenically diverse H5 highly pathogenic avian influenza isolates with a live HVT vector vaccine expressing the influenza hemagglutinin gene derived from a clade 2.2 avian influenza virus

Vaccination is an important tool in the protection of poultry against avian influenza (AI). For field use, the overwhelming majority of AI vaccines produced are inactivated whole virus formulated into an oil emulsion. However, recombinant vectored vaccines are gaining use for their ability to induce protection against heterologous isolates and ability to overcome maternal antibody interference. In these studies, we compared protection of chickens provided by a turkey herpesvirus (HVT) vector vaccine expressing the hemagglutinin (HA) gene from a clade 2.2 H5N1 strain (A/swan/Hungary/4999/2006) against homologous H5N1 as well as heterologous H5N1 and H5N2 highly pathogenic (HP) AI challenge. The results demonstrated all vaccinated birds were protected from clinical signs of disease and mortality following homologous challenge. In addition, oral and cloacal swabs taken from challenged birds demonstrated that vaccinated birds had lower incidence and titers of viral shedding compared to sham-vaccinated birds. Following heterologous H5N1 or H5N2 HPAI challenge, 80–95% of birds receiving the HVT vector AI vaccine at day of age survived challenge with fewer birds shedding virus after challenge than sham vaccinated birds. In vitro cytotoxicity analysis demonstrated that splenic T lymphocytes from HVT-vector-AI vaccinated chickens recognized MHC-matched target cells infected with H5, as well as H6, H7, or H9 AI virus. Taken together, these studies provide support for the use of HVT vector vaccines expressing HA to protect poultry against multiple lineages of HPAI, and that both humoral and cellular immunity induced by live vaccines likely contributes to protection.     

New DIVA vaccine for the protection of poultry against H5 highly pathogenic avian influenza viruses irrespective of the N-subtype

Most human cases of highly pathogenic H5N1 avian influenza virus (HPAIV) infection are the result of direct contact with infected poultry. Therefore, infection of poultry should be prevented to avoid human exposure. One method to combat HPAIV outbreaks relies on depopulation. An alternative or supplementary method is the use of DIVA (discriminating infected from vaccinated animals) vaccines to prevent infection of animals on holdings surrounding an outbreak. Discrimination between infected and vaccinated animals is often based on the ‘heterologous neuraminidase’ strategy. This implies that a suitable vaccine can only be selected when the N-subtype of the outbreak strain is known. Thus, at least two vaccines with different N-subtypes must be available, allowing a switch of vaccine in the event that one of them matches the outbreak strain. However, such vaccines cannot be used preventively in situations in which the N-subtype of the outbreak strain is unknown. In order to circumvent these drawbacks we generated a recombinant influenza virus containing the HA gene of a contemporary H5N1 HPAIV strain in combination with the NA gene of a human type B influenza virus. An inactivated vaccine based on this virus protected chickens against clinical disease, and completely prevented virus shedding after H5N1 HPAIV challenge infection. Serological analyses confirmed that the vaccine complied with the DIVA principle. Since NA of type B does not occur in avian influenza strains, this vaccine is suitable as a DIVA vaccine against any H5 HPAIV, and may be used preventively without compromising the DIVA principle.     

Protective efficacy in farmed ducks of a duck enteritis virus-vectored vaccine against H5N1, H5N6, and H5N8 avian influenza viruses

Ducks play a key role in the maintenance and spread of avian influenza viruses (AIVs) in nature, and control of AIVs in ducks has important implications for AIV eradication from poultry. We previously constructed a recombinant duck enteritis virus (DEV), rDEVus78HA, that expresses the HA gene of an H5N1 AIV and showed that rDEVus78HA immunization provides complete protection against both DEV and H5N1 AIV challenge in specific-pathogen-free ducks. In this study, we performed a 60-week clinical trial and found that this rDEVus78HA vaccine can function as a bivalent vaccine in farmed ducks against lethal challenge with DEV and H5N1 virus. Moreover, we found that rDEVus78HA-vaccinated ducks were efficiently protected against challenges with recently isolated heterologous H5N6 and H5N8 viruses. Our results demonstrate that rDEVus78HA could be extremely valuable for the control of DEV and H5 AIVs in ducks.     

Heterosubtypic protection against avian influenza virus by live attenuated and chimeric norovirus P-particle-M2e vaccines in chickens

Avian influenza in poultry continues to be a great concern worldwide, and the currently licensed inactivated influenza vaccines are not effective against the novel strains of influenza virus that continue to emerge in the field. This warrants the development of more broadly protective influenza vaccines or vaccination regimens. Live attenuated influenza vaccines (LAIVs) and subunit vaccines derived from viral peptides, such as the highly conserved ectodomain of influenza virus matrix protein 2 (M2e), can offer a more broadly reactive immune response. In chickens, we previously showed that a chimeric norovirus P particle containing M2e (M2eP) could provide partial but broad immunity, when administered as a standalone vaccine, and also enhanced the protective efficacy of inactivated vaccine when used in a combination regimen. We also demonstrated that a naturally-selected NS1-truncated H7N3 LAIV (pc4-LAIV) was highly efficacious against antigenically distant heterologous H7N2 low pathogenicity avian influenza virus challenge, especially when used as the priming vaccine in a prime-boost vaccination regimen. In this study, we investigated the cross-subtype protective efficacy of pc4-LAIV in conjunction with M2eP using single vaccination, combined treatment, and prime-boost approaches. Chickens vaccinated with pc4-LAIV showed significant reduction of tracheal shedding of a low pathogenicity H5N2 challenge virus. This cross-subtype protective efficacy was further enhanced, during the initial stages of challenge virus replication, in chickens that received a vaccination regimen consisting of priming with pc4-LAIV at 1?day of age and boosting with M2eP. Further, H5N2-specific serum IgG and pc4-LAIV-specific hemagglutination-inhibition antibody titers were enhanced in LAIV-primed and M2eP boost-vaccinated chickens. Taken together, our data point to the need of further investigation into the benefits of combined and prime-boost vaccination schemes utilizing LAIV and epitope-based vaccines, to develop more broadly protective vaccination regimens.     

Safety,immunogenicity and efficacy of poxvirus-based vector vaccines expressing the haemagglutinin gene of a highly pathogenic H5N1 avian influenza virus in pigs

This study investigates the safety, immunogenicity and efficacy of different pox-vector vaccines expressing the haemagglutinin of a highly pathogenic (HP) H5N1 avian influenza virus (AIV) (A/chicken/Indonesia/7/03) in pigs. Pigs were vaccinated twice, with a 4-week interval, with a fowlpox (TROVAC^®), a canarypox (ALVAC^®), or a vaccinia (NYVAC) vector vaccine combined with an oil-in-water adjuvant, with the unadjuvanted NYVAC, or left unvaccinated. Six weeks after the second vaccination, all pigs were challenged intra-tracheally with low pathogenic (LP) H5N2 AIV A/chicken/Belgium/150/99. Sera were examined in haemagglutination inhibition (HI) tests against the H5N1 AIV from which the vaccine haemagglutinin derived, the challenge virus and the human A/Vietnam/1194/04 HPAIV. After challenge pigs were compared for H5N2 virus replication in the trachea and 4 lung lobes at 24 or 72 h post-challenge. Vaccination was well tolerated by all animals. Antibody titres peaked 2 weeks after the second vaccination and were 2- to 4-fold higher against the vaccine virus than heterologous H5 viruses. The NYVAC and ALVAC adjuvanted vaccines consistently induced higher antibody titres than TROVAC or NYVAC without adjuvant. Following challenge, the H5N2 challenge virus was isolated from all unvaccinated pigs, while 19 out of 21 vaccinates showed complete virological protection. Pox-vector vaccines were safe, immunogenic and efficacious against challenge with a heterologous H5 AIV, offering an alternative to classical inactivated vaccines. It remains to be seen whether they would protect against a swine-adapted H5 virus, which may replicate 100–1000 times better than our challenge virus.     

Highly pathogenic avian influenza virus H5N1 from Egypt escapes vaccine-induced immunity but confers clinical protection against a heterologous clade 2.2.1 Egyptian isolate

The poultry populations of Egypt are endemically infected by highly pathogenic avian influenza viruses (HPAIV) of subtype H5N1. Vaccination was chosen as an auxiliary tool to control HPAIV in poultry. Potency of commercial vaccines regarding emerging variants is under discussion. In the current study efficacy of four different inactivated whole H5 virus vaccines representing different sublineages of HPAIV H5N1 were tested in chickens against challenge viruses currently co-circulating in Egypt and representing two antigenically widely distinct HPAIV H5N1 lineages, i.e., “variant” (clade 2.2.1var) and “proper” (clade 2.2.1pro) viruses. All vaccines induced clinical protection against challenge with 2.2.1pro Egyptian strains. In contrast, when challenged with a variant strain, only chickens vaccinated with the homologous Egyptian clade 2.2.1var virus or an inactivated re-assorted H5N1 strain (Re-5, clade 2.3) were protected. However, only the homologous virus induced sterile immunity whereas chickens clinically protected after Re-5 vaccination shed virus at day two after infection indistinguishable to H5N2 vaccines. In conclusion, monitoring vaccine-driven evolution of HPAIV H5N1 by surveillance, antigenic characterization, and challenge studies is essential to assess efficacy of AIV vaccination campaigns.     

Delivery of an inactivated avian influenza virus vaccine adjuvanted with poly(D,L-lactic-co-glycolic acid) encapsulated CpG ODN induces protective immune responses in chickens

In poultry, systemic administration of commercial vaccines consisting of inactivated avian influenza virus (AIV) requires the simultaneous delivery of an adjuvant (water-in-oil emulsion). These vaccines are often limited in their ability to induce quantitatively better local (mucosal) antibody responses capable of curtailing virus shedding. Therefore, more efficacious adjuvants with the ability to provide enhanced immunogenicity and protective anti-AIV immunity in chickens are needed. While the Toll-like receptor (TLR) 21 agonist, CpG oligodeoxynucleotides (ODNs) has been recognized as a potential vaccine adjuvant in chickens, poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles, successfully tested as vaccine delivery systems in other species, have not been extensively explored. The present study, therefore, assessed both systemic and mucosal antibody-mediated responses following intramuscular vaccination (administered at 7 and 21 days post-hatch) of chickens with PLGA encapsulated H9N2 AIV plus encapsulated CpG ODN 2007 (CpG 2007), and nonencapsulated AIV plus PLGA encapsulated CpG 2007 vaccine formulations. Virus challenge was performed at 2 weeks post-secondary vaccination using the oculo-nasal route. Our results showed that chickens vaccinated with the nonencapsulated AIV vaccine plus PLGA encapsulated CpG 2007 developed significantly higher systemic IgY and local (mucosal) IgY antibodies as well as haemagglutination inhibition antibody titres compared to PLGA encapsulated AIV plus encapsulated CpG 2007 vaccinated chickens. Furthermore, chickens that received CpG 2007 as an adjuvant in the vaccine formulation had antibodies exhibiting higher avidity indicating that the TLR21-mediated pathway may enhance antibody affinity maturation qualitatively. Collectively, our data indicate that vaccination of chickens with nonencapsulated AIV plus PLGA encapsulated CpG 2007 results in qualitatively and quantitatively augmented antibody responses leading to a reduction in virus shedding compared to the encapsulated AIV plus PLGA encapsulated CpG 2007 formulation.     

Recombinant herpesvirus of turkeys as a vector-based vaccine against highly pathogenic H7N1 avian influenza and Marek's disease

A major challenge for poultry vaccination is the design of vaccines that protect against multiple pathogens via a single protective dose delivered through mass vaccination methods. In this investigation, we examined herpesvirus of turkeys (HVT) as a vaccine vector for delivery of haemagglutinin (HA) antigen of highly pathogenic H7N1 avian influenza virus that can act as a dual vaccine against avian influenza and Marek's disease. The HVT vector was developed using reverse genetics based on an infectious bacterial artificial chromosome (BAC) clone of HVT. The BAC carrying the HVT genome was genetically modified to express the HA gene of a highly pathogenic H7N1 virus. The resultant recombinant BAC construct containing the modified HVT sequence was transfected into chicken embryo fibroblast (CEF) cells, and HVT recombinants (rHVT-H7HA) harbouring the H7N1 HA were recovered. Analysis of cultured CEF cells infected with the rHVT-H7HA showed that HA was expressed and that the rescued rHVT-H7HA stocks were stable during several in vitro passages with no difference in growth kinetics compared with the parent HVT. Immunisation of one-day-old chicks with rHVT-H7HA induced H7-specific antibodies and protected chickens challenged with homologous H7N1 virus against virus shedding, clinical disease and death. This vaccine supports differentiation between infected and vaccinated animals (DIVA) vaccination strategies because no nucleoprotein-(NP) specific antibodies were detected in the rHVT-H7HA vaccinated birds. The rHVT-H7HA not only provided protection against a lethal challenge with highly pathogenic H7N1 virus but also against highly virulent Marek's disease virus and can be used as a DIVA vaccine.     

Protective efficacy of several vaccines against highly pathogenic H5N1 avian influenza virus under experimental conditions

Although several vaccines have been developed to protect against highly pathogenic avian influenza of subtype H5N1 'Asia' their efficiency has primarily been assessed individually. Thus, a direct comparison of their performance is still lacking. The following study was conducted to compare the protective efficacy of three commercially available inactivated vaccines based on influenza virus strains of subtypes H5N2 (vaccine A), H5N9 (vaccine B), and H5N3 (vaccine C), as well as two hemagglutinin expressing experimental vector vaccines (modified vaccinia virus Ankara-H5 and Newcastle disease virus-H5) against a lethal dose of highly pathogenic H5N1 avian influenza virus in chickens. To assess their potential as emergency vaccines, a single immunisation was performed for all vaccines, despite the recommendation of a double-vaccination schedule for commercial vaccines B and C. Overall, all vaccines induced clinical protection against challenge infection 3 weeks after immunisation. No mortality was observed in chickens immunised with vaccine A and viral shedding could not be detected. Immunisation with NDV-H5, vaccine C and MVA-H5 conferred also protection against lethal challenge. However, viral RNA was detected by real-time RT-PCR in swabs of 10%, 20% and 50% of animals, and 0%, 10% and 30% of animals, respectively, shed infectious virus. Immunisation with vaccine B was less protective since 50% of the vaccinated animals shed infectious virus after challenge and 20% of the chickens succumbed to disease. These results indicate that the NDV-H5 vectored vaccine is similarly effective as the best inactivated vaccine. Considering the advantage of live NDV which can be administered via spray or drinking water as well as the potential use of this H5 expressing vector vaccine for an easy DIVA (differentiating infected from vaccinated animals) strategy, NDV-H5 could represent an alternative for extensive vaccination against avian influenza in chickens.     

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.     

H7 avian influenza virus vaccines protect chickens against challenge with antigenically diverse isolates

Vaccination has been a critical tool in the control of some avian influenza viruses (AIV) and has been used routinely in Pakistan to help control sporadic outbreaks of highly pathogenic (HP) H7 AIV since 1995. During that time, several AIV isolates were utilized as inactivated vaccines with varying degrees of success. In order to evaluate which H7 AIV strains may serve as optimal vaccines for diverse H7 AIVs from Pakistan we conducted vaccination-challenge studies with five H7 vaccines against challenge with two HPAIVs: A/chicken/Murree/NARC-1/1995 H7N3 and A/chicken/Karachi/SPVC-4/2004 H7N3. To further characterize the isolates antigenic cartography was used to visually demonstrate the antigenic relationships among the isolates. All vaccines provided similar protection against mortality, morbidity and shedding of challenge virus from the respiratory tract. However, some minor (not statistically significant) differences were observed and correlated with antibody levels induced by the vaccine prior to challenge.     

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.     

Priming with a DNA vaccine delivered by attenuated Salmonella typhimurium and boosting with a killed vaccine confers protection of chickens against infection with the H9 subtype of avian influenza virus

Control of the circulation of H9 low-pathogenic avian influenza virus (LPAIV) is a major concern for both animal and public health. To improve vaccine efficacy against H9 LPAIV, we have utilized a novel prime–boost vaccination strategy. Specific-pathogen free (SPF) chickens were first orally immunized with a hemagglutinin (HA) DNA vaccine delivered by attenuated Salmonella typhimurium, followed by boosting with a killed avian influenza (AI) vaccine. Chickens in this combined vaccination group were completely protected against both oropharyngeal and cloacal virus shedding after intranasal challenge with H9N2 AIV, while viruses were detected from these sites in other vaccination groups. Prior to challenge, chickens in the prime–boost group also had higher (P < 0.05) serum hemagglutination inhibition (HI) titers and intestinal mucosal IgA ELISA titers against AIV, and higher lymphoproliferation stimulation indices than those from other groups. Thus, we have demonstrated the efficacy of a novel prime–boost vaccination strategy against H9N2 avian influenza virus, which could be also applied for the development of vaccines against other mucosally infectious pathogens.