Preparation of vaccines against H5N1 influenza

In response to the pandemic warning provided by the highly pathogenic H5N1 influenza virus infections in Hong Kong, there were world-wide attempts to develop vaccines. Three strategies were followed and although each was associated with some success, there were also some problems. Pre-clinical vaccine efficacy results are presented from one such strategy, that of using an apathogenic H5N3 avian strain for vaccine production.     

Intranasal administration of a live non-pathogenic avian H5N1 influenza virus from a virus library confers protective immunity against H5N1 highly pathogenic avian influenza virus infection in mice: Comparison of formulations and administration routes of vaccines

Outbreaks of highly pathogenic avian influenza viruses (HPAIVs) would cause disasters worldwide. Various strategies against HPAIVs are required to control damage. It is thought that the use of non-pathogenic avian influenza viruses as live vaccines will be effective in an emergency, even though there might be some adverse effects, because small amounts of live vaccines will confer immunity to protect against HPAIV infection. Therefore, live vaccines have the advantage of being able to be distributed worldwide soon after an outbreak. In the present study, we found that intranasal administration of a live H5N1 subtype non-pathogenic virus induced antibody and cytotoxic T lymphocyte responses and protected mice against H5N1 HPAIV infection. In addition, it was found that a small amount (100 PFU) of the live vaccine was as effective as 100 μg (approximately 10^10–11 PFU of virus particles) of the inactivated whole particle vaccine in mice. Consequently, the use of live virus vaccines might be one strategy for preventing pandemics of HPAIVs in an emergency.     

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,...     

Phylogenetic characterization of a reassortant H5N2 influenza A virus from a resident Mexican duck (Anas diazi)

Congregation of different migratory and resident bird species on aquatic ecosystems during winter migration increases contact rates and enhances influenza A virus (IAV) transmission. However, scarce research has been focused on the resident bird's contribution to the viral ecology at a local scale. The Mexican duck (Anas diazi) is an endemic endangered anatid from Mexico. This resident species shares aquatic habitats with migratory birds in the wetlands of Central Mexico. Therefore, here we describe the phylogenetic analysis of an IAV (A/Mexican duck/EstadodeMexico; Lerma/UIFMVZ377/2016(H5N2)) isolated in this species, during spatiotemporal concurrence with migratory anatids in the winter season. All eight gene sequences were obtained by nextgeneration sequencing. Maximum Likelihood trees were constructed using MEGA-X, with General Time Reversible + Invariant (GTR+I), Subtree Pruning and Regrafting (SPR) heuristic method, and 1000 bootstrap replicates. Similarities with six different IAV subtypes were observed through a BLAST search: H6N5, H7N7, H5N2, H4N6, H9N2, and H11N9, detected in wild ducks during 2015 in the Pacific, Central and Mississippi flyways stop sites across the United States of America and Canada. The molecular identification of this reassortant H5N2 IAV highlights the importance of resident species as a reservoir host and its potential participation in the maintenance and transmission of IAV in wetlands surrounded by rural areas.     

Structural and antigenic characterization of a computationally-optimized H5 hemagglutinin influenza vaccine

Influenza A virus is a leading cause of death worldwide. Viruses of the H5 subtype have the potential to induce high mortality, and no vaccines are currently available to protect against H5 influenza viruses in the event of an outbreak. Experimental vaccination with one clade 2 virus does not protect against other subclades. The computationally optimized broadly reactive (COBRA) methodology was previously used to generate a H5 hemagglutinin (HA) antigen (COBRA2) that elicited increased serological breadth against multiple clade 2 H5N1 influenza viruses. In this report, we structurally and antigenically characterized the COBRA2 HA antigen. We examined the biochemical characteristics of the COBRA2 protein and determined the protein is correctly cleaved, properly folded into a trimeric structure, and antigenically correct by probing with HA head- and stem-specific monoclonal antibodies (mAbs). We further probed the antigenicity by examining binding of a panel of H5 mouse mAbs to the COBRA2 antigen, as well as several other HA antigens. We determined the X-ray crystal structure of the COBRA2 HA antigen to 2.8 Å and the protein was observed to be in the expected trimeric form. The COBRA2 HA was structurally similar to the naturally occurring H5 HA antigens and suggests the protein folds similar to known HA structures. Overall, our data allow us to formulate a hypothesis on the mechanism of increased breadth due to vaccination with the COBRA2 HA antigen, which is that the protein incorporates antigenic sites from numerous HA antigens, and elicits mAbs with limited breadth, but with diversity in targeted antigenic sites.     

Cross-clade protective immunity of H5N1 influenza vaccines in a mouse model

H5N1 highly pathogenic avian influenza viruses evolved into several clades, leading to appreciably distinct antigenicities of their hemagglutinins. As such, candidate H5N1 pre-pandemic vaccines for human use should be sought. Here, to evaluate fundamental immunogenic variations between H5N1 vaccines, we prepared four inactivated H5N1 test vaccines from different phylogenetic clades (clade 1, 2.1, 2.2, and 2.3.4) in accordance with the WHO recommendation, and tested their cross-clade immunity in a mouse model by vaccination followed by challenge with heterologous virulent viruses. All H5N1 vaccines tested provided full or partial cross-clade protective immunity, except one clade 2.2-based vaccine, which did not protect mice from clade 2.3.4 virus challenge. Among the test vaccines, a clade 2.1-based vaccine possessed the broadest-spectrum cross-immunity. These results suggest that currently stockpiled pre-pandemic vaccines, especially clade 2.1-based vaccines, will likely be useful as backup vaccines in a pandemic situation, even one involving antigenic-drifted viruses.     

ISCOM vaccine induced protection against a lethal challenge with a human H5N1 influenza virus

Recently avian influenza A viruses of the H5N1 subtype were shown to infect humans in the Hong Kong area, resulting in the death of six people. Although these viruses did not efficiently spread amongst humans, these events illustrated that influenza viruses of subtypes not previously detected in humans could be at the basis of a new pandemic. In the light of this pandemic threat we evaluated and compared the efficacy of a classical non-adjuvanted subunit vaccine and a vaccine based on immune stimulating complexes (ISCOM) prepared with the membrane glycoproteins of the human influenza virus A/Hong Kong 156/97 (H5N1) to protect roosters against a lethal challenge with this virus. The ISCOM vaccine induced protective immunity against the challenge infection whereas the non-adjuvanted subunit vaccine proved to be poorly immunogenic and failed to induce protection in this model.     

The current state of H5N1 vaccines and the use of the ferret model for influenza therapeutic and prophylactic development

Highly pathogenic avian influenza H5N1 is a threat to global public health as a natural pandemic causing agent but has recently been considered a bioterrorism concern. The evolving view of the H5N1 virus necessitates the re-evaluation of the current status of H5N1 therapeutics and prophylactics, in particular the preparation of viable H5N1 vaccination strategies as well as the use of ferrets in influenza research. Here the highly pathogenic H5N1 virus dilemma is discussed in context with the current H5N1 vaccine status and the use of the ferret model. Previously, the development of various H5N1 vaccine platforms have been attempted, many of them tested in the ferret model, including vector vaccines, adjuvant vaccines, DNA vaccines, and reverse engineered vaccines. Moreover, as ferrets are a superlative animal model for influenza investigation and vaccine testing, it is imperative that this model is recognized for its uses in prophylactic development and not only as an agent for creating transmissible influenza viruses. Elucidating the ferret immune response and creating ferret immune reagents remain important goals in conjunction with the development and manufacture of H5N1 vaccines. In summary, an efficacious H5N1 vaccine is urgently needed and the ferret model remains an appropriate model for its development.     

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.     

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.     

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.     

Extrapolating theoretical efficacy of inactivated influenza A/H5N1 virus vaccine from human immunogenicity studies

Influenza A virus subtype H5N1 has been a public health concern for almost 20 years due to its potential ability to become transmissible among humans. Phase I and II clinical trials have assessed safety, reactogenicity and immunogenicity of inactivated influenza A/H5N1 virus vaccines. A shortage of vaccine is likely to occur during the first months of a pandemic. Hence, determining whether to give one dose to more people or two doses to fewer people to best protect the population is essential. We use hemagglutination–inhibition antibody titers as an immune correlate for avian influenza vaccines. Using an established relationship to obtain a theoretical vaccine efficacy from immunogenicity data from thirteen arms of six phase I and phase II clinical trials of inactivated influenza A/H5N1 virus vaccines, we assessed: (1) the proportion of theoretical vaccine efficacy achieved after a single dose (defined as primary response level), and (2) whether theoretical efficacy increases after a second dose, with and without adjuvant. Participants receiving vaccine with AS03 adjuvant had higher primary response levels (range: 0.48–0.57) compared to participants receiving vaccine with MF59 adjuvant (range: 0.32–0.47), with no observed trends in primary response levels by antigen dosage. After the first and second doses, vaccine with AS03 at dosage levels 3.75, 7.5 and 15 mcg had the highest estimated theoretical vaccine efficacy: Dose (1) 45% (95% CI: 36–57%), 53% (95% CI: 42–63%) and 55% (95% CI: 44–64%), respectively and Dose (2) 93% (95% CI: 89–96%), 97% (95% CI: 95–98%) and 97% (95% CI: 96–100%), respectively. On average, the estimated theoretical vaccine efficacy of lower dose adjuvanted vaccines (AS03 and MF59) was 17% higher than that of higher dose unadjuvanted vaccines, suggesting that including an adjuvant is dose-sparing. These data indicate adjuvanted inactivated influenza A/H5N1 virus vaccine produces high theoretical efficacy after two doses to protect individuals against a potential avian influenza pandemic.     

Efficacy of inactivated swine influenza virus vaccines against the 2009 A/H1N1 influenza virus in pigs

The gene constellation of the 2009 pandemic A/H1N1 virus is a unique combination from swine influenza A viruses (SIV) of North American and Eurasian lineages, but prior to April 2009 had never before been identified in swine or other species. Although its hemagglutinin gene is related to North American H1 SIV, it is unknown if vaccines currently used in U.S. swine would cross-protect against infection with the pandemic A/H1N1. The objective of this study was to evaluate the efficacy of inactivated vaccines prepared with North American swine influenza viruses as well as an experimental homologous A/H1N1 vaccine to prevent infection and disease from 2009 pandemic A/H1N1. All vaccines tested provided partial protection ranging from reduction of pneumonia lesions to significant reduction in virus replication in the lung and nose. The multivalent vaccines demonstrated partial protection; however, none was able to prevent all nasal shedding or clinical disease. An experimental homologous 2009 A/H1N1 monovalent vaccine provided optimal protection with no virus detected from nose or lung at any time point in addition to amelioration of clinical disease. Based on cross-protection demonstrated with the vaccines evaluated in this study, the U.S. swine herd likely has significant immunity to the 2009 A/H1N1 from prior vaccination or natural exposure. However, consideration should be given for development of monovalent homologous vaccines to best protect the swine population thus limiting shedding and the potential transmission of 2009 A/H1N1 from pigs to people.     

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).     

The efficacy,effectiveness and cost-effectiveness of inactivated influenza virus vaccines

Influenza is a major cause of morbidity and mortality worldwide. Currently available inactivated influenza virus vaccines are safe and effective in preventing influenza. Substantial health benefits are seen across all age and risk groups. Studies assessing the economic benefits of vaccination suggest that vaccination is highly cost effective and in many cases cost saving among the elderly. Influenza vaccination has also been associated with significant economic benefits in younger adults and children. Additional health economic studies from developing countries and from tropical/subtropical regions will be vitally important for better understanding of the global burden of influenza and potential benefits of vaccination.     

A subunit vaccine candidate derived from a classic H5N1 avian influenza virus in China protects fowls and BALB/c mice from lethal challenge

In recent years, numerous human infections with avian influenza viruses in Asia have raised the concern that the next influenza pandemic is imminent. The most effective way to combat human avian influenza is through vaccination of the public. In this study, we developed an influenza A recombinant protein (rH5HA) directed against the hemagglutinin (HA) of a classic H5N1 high pathogenic avian influenza virus isolated in South China in 1996. Following purification of the recombinant protein expressed from a baculovirus expression system, we evaluated the efficiency of rH5HA on specific pathogen free (SPF) chicken, commercial chicken, and in BALB/c mice in an infection-protection model. The results demonstrated that rH5HA induced antibody responses and provided full protection in both SPF chickens and commercial chickens. Protective immunity was generated within 2 weeks in chickens as young as 7-day post-hatch using a minimum amount of rH5HA protein (2 μg/bird/vaccination). The serum antibody generated from rH5HA immunization was protective and lasted more than 6 months. Our data also demonstrated that rH5HA immunization protected BALB/c mice from a lethal challenge with pathogenic avian influenza virus. These results suggested that vaccination with rH5HA could be a vaccine candidate for the control of H5N1 avian influenza in poultry, in mice, and potentially in other mammals including human.     

Avian influenza a (H5N1) virus antibodies in poultry cullers, South Korea, 2003-2004

Transmission of influenza (H5N1) virus from birds to humans is a serious public health threat. In South Korea, serologic investigation among 2,512 poultry workers exposed during December 2003-March 2004 to poultry with confirmed or suspected influenza (H5N1) virus infection found antibodies in 9. Frequency of bird-to-human transmission was low.