Mucosal delivery of inactivated influenza vaccine induces B-cell-dependent heterosubtypic cross-protection against lethal influenza A H5N1 virus infection

Influenza vaccines that induce greater cross-reactive or heterosubtypic immunity (Het-I) may overcome limitations in vaccine efficacy imposed by the antigenic variability of influenza A viruses. We have compared mucosal versus traditional parenteral administration of inactivated influenza vaccine for the ability to induce Het-I in BALB/c mice and evaluated a modified Escherichia coli heat-labile enterotoxin adjuvant, LT(R192G), for augmentation of Het-I. Mice that received three intranasal (i.n.) immunizations of H3N2 vaccine in the presence of LT(R192G) were completely protected against lethal challenge with a highly pathogenic human H5N1 virus and had nasal and lung viral titers that were at least 2,500-fold lower than those of control mice receiving LT(R192G) alone. In contrast, mice that received three vaccinations of H3N2 vaccine subcutaneously in the presence or absence of LT(R192G) or incomplete Freund's adjuvant were not protected against lethal challenge and had no significant reductions in tissue virus titers observed on day 5 post-H5N1 virus challenge. Mice that were i.n. administered H3N2 vaccine alone, without LT(R192G), displayed partial protection against heterosubtypic challenge. The immune mediators of Het-I were investigated. The functional role of B and CD8+ T cells in Het-I were evaluated by using gene-targeted B-cell (IgH-6(-/-))- or beta2-microglobulin (beta2m(-/-))-deficient mice, respectively. beta2m(-/-) but not IgH-6(-/-) vaccinated mice were protected by Het-I and survived a lethal infection with H5N1, suggesting that B cells, but not CD8+ T cells, were vital for protection of mice against heterosubtypic challenge. Nevertheless, CD8+ T cells contributed to viral clearance in the lungs and brain tissues of heterotypically immune mice. Mucosal but not parenteral vaccination induced subtype cross-reactive lung immunoglobulin G (IgG), IgA, and serum IgG anti-hemagglutinin antibodies, suggesting the presence of a common cross-reactive epitope in the hemagglutinins of H3 and H5. These results suggest a strategy of mucosal vaccination that stimulates cross-protection against multiple influenza virus subtypes, including viruses with pandemic potential.     

Flavonoids from Houttuynia cordata attenuate H1N1-induced acute lung injury in mice via inhibition of influenza virus and Toll-like receptor signalling

BackgroundInfluenza virus is one of the most important human pathogens, causing substantial seasonal and pandemic morbidity and mortality. Houttuynia cordata is a traditionally used medicinal plant for the treatment of pneumonia. Flavonoids are one of the major bioactive constituents of Houttuynia cordata.PurposeThis study was designed to investigate the therapeutic effect and mechanism of flavonoid glycosides from H. cordata on influenza A virus (IAV)-induced acute lung injury (ALI) in mice.MethodsFlavonoids from H. cordata (HCF) were extracted from H. cordata and identified by high-performance liquid chromatography. Mice were infected intranasally with influenza virus H1N1 (A/FM/1/47). HCF (50, 100, or 200 mg/kg) or Ribavirin (100 mg/kg, the positive control) were administered intragastrically. Survival rates, life spans, weight losses, lung indexes, histological changes, inflammatory infiltration, and inflammatory markers in the lungs were measured. Lung virus titers and neuraminidase (NA) activities were detected. The expression of Toll-like receptors (TLRs) and levels of NF-κB p65 phosphorylation (NF-κB p65(p)) in the lungs were analysed. The effects of HCF on viral replication and TLR signalling were further evaluated in cells.ResultsHCF contained 78.5% flavonoid glycosides. The contents of rutin, hyperin, isoquercitrin, and quercitrin in HCF were 8.8%, 26.7%, 9.9% and 31.7%. HCF (50, 100 and 200 mg/kg) increased the survival rate and life span of mice infected with the lethal H1N1 virus. In H1N1-induced ALI, mice treated with HCF (50, 100 and 200 mg/kg) showed lesser weight loss and lower lung index than the model group. The lungs of HCF-treated ALI mice presented more intact lung microstructural morphology, milder inflammatory infiltration, and lower levels of monocyte chemotactic protein 1 (MCP-1), interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α) and malondialdehyde (MDA) than in the model group. Further investigation revealed that HCF exerted antiviral and TLR-inhibitory effects in vivo and in vitro. HCF (50, 100 and 200 mg/kg) reduced lung H1N1 virus titers and inhibited viral NA activity in mice. HCF (100 and 200 mg/kg) elevated the levels of interferon-β in lungs. HCF also decreased the expression of TLR3/4/7 and level of NF-κB p65(p) in lung tissues. In vitro experiments showed that HCF (50, 100 and 200 μg/ml) significantly inhibited viral proliferation and suppressed NA activity. In RAW 264.7 cells, TLR3, TLR4, and TLR7 agonist-stimulated cytokine secretion, NF-κB p65 phosphorylation, and nuclear translocation were constrained by HCF treatment. Furthermore, among the four major flavonoid glycosides in HCF, hyperin and quercitrin inhibited both viral replication and TLR signalling in cells.ConclusionHCF significantly alleviated H1N1-induced ALI in mice, which were associated with its dual antiviral and anti-inflammatory effects via inhibiting influenzal NA activity and TLR signalling. among the four major flavonoid glycosides in HCF, hyperin and quercitrin played key roles in the therapeutic effect of HCF.     

Human immune responses elicited by an intranasal inactivated H5 influenza vaccine

Intranasally administered influenza vaccines could be more effective than injected vaccines, because intranasal vaccination can induce virus-specific immunoglobulin A (IgA) antibodies in the upper respiratory tract, which is the initial site of infection. In this study, immune responses elicited by an intranasal inactivated vaccine of influenza A(H5N1) virus were evaluated in healthy individuals naive for influenza A(H5N1) virus. Three doses of intranasal inactivated whole-virion H5 influenza vaccine induced strong neutralizing nasal IgA and serum IgG antibodies. In addition, a mucoadhesive excipient, carboxy vinyl polymer, had a notable impact on the induction of nasal IgA antibody responses but not on serum IgG antibody responses. The nasal hemagglutinin (HA)-specific IgA antibody responses clearly correlated with mucosal neutralizing antibody responses, indicating that measurement of nasal HA-specific IgA titers could be used as a surrogate for the mucosal antibody response. Furthermore, increased numbers of plasma cells and vaccine antigen-specific Th cells in the peripheral blood were observed after vaccination, suggesting that peripheral blood biomarkers may also be used to evaluate the intranasal vaccine-induced immune response. However, peripheral blood immune cell responses correlated with neutralizing antibody titers in serum samples but not in nasal wash samples. Thus, analysis of the peripheral blood immune response could be a surrogate for the systemic immune response to intranasal vaccination but not for the mucosal immune response. The current study suggests the clinical potential of intranasal inactivated vaccines against influenza A(H5N1) viruses and highlights the need to develop novel means to evaluate intranasal vaccine-induced mucosal immune responses.     

An avian influenza H5N1 virus that binds to a human-type receptor

Avian influenza viruses preferentially recognize sialosugar chains terminating in sialic acid-alpha2,3-galactose (SAalpha2,3Gal), whereas human influenza viruses preferentially recognize SAalpha2,6Gal. A conversion to SAalpha2,6Gal specificity is believed to be one of the changes required for the introduction of new hemagglutinin (HA) subtypes to the human population, which can lead to pandemics. Avian influenza H5N1 virus is a major threat for the emergence of a pandemic virus. As of 12 June 2007, the virus has been reported in 45 countries, and 312 human cases with 190 deaths have been confirmed. We describe here substitutions at position 129 and 134 identified in a virus isolated from a fatal human case that could change the receptor-binding preference of HA of H5N1 virus from SAalpha2,3Gal to both SAalpha2,3Gal and SAalpha2,6Gal. Molecular modeling demonstrated that the mutation may stabilize SAalpha2,6Gal in its optimal cis conformation in the binding pocket. The mutation was found in approximately half of the viral sequences directly amplified from a respiratory specimen of the patient. Our data confirm the presence of H5N1 virus with the ability to bind to a human-type receptor in this patient and suggest the selection and expansion of the mutant with human-type receptor specificity in the human host environment.     

Subclinical brain injury caused by H5N1 influenza virus infection

Although H5N1 influenza A viruses can cause systemic infection, their neurotropism and long-term effects on the central nervous system (CNS) are not fully understood. We assessed H5N1viral invasion of the CNS and its long-term effects in a ferret model. An H5N1 virus caused nonsuppurative encephalitis, which lasted for 3 months without neurologic signs. Further, another H5N1 virus caused nonsuppurative vasculitis with brain hemorrhage. Three-dimensional analysis of viral distribution in the brain identified the olfactory system as a major route for brain invasion. The efficient growth of virus in the upper respiratory tract may thus facilitate viral brain invasion.     

H5N1亚型禽流感病毒进化的研究进展

由H5N1流感病毒引起的高致病性禽流感,在禽类之间广泛传播。当人类接触这些禽类时,可能会被感染并产生严重的呼吸道症状,且死亡率高达60%。血凝素(hemagglutinin,HA)是H5N1病毒中和抗体的主要抗原,为了便于对病毒的HA突变进行研究,根据HA遗传基因的差异远近,所有的H5病毒株都被划分在20个分支内。对于H5N1病毒进化的研究在禽流感疫苗的研制、禽流感大流行的预防等方面均具有重要意义。现对禽流感、H5N1病毒特征、血凝素的结构功能、H5N1病毒的分支以及病毒进化的研究进行概述。     

Cold-adapted pandemic 2009 H1N1 influenza virus live vaccine elicits cross-reactive immune responses against seasonal and H5 influenza A viruses

The rapid transmission of the pandemic 2009 H1N1 influenza virus (pH1N1) among humans has raised the concern of a potential emergence of reassortment between pH1N1 and highly pathogenic influenza strains, especially the avian H5N1 influenza virus. Here, we report that the cold-adapted pH1N1 live attenuated vaccine (CApH1N1) elicits cross-reactive immunity to seasonal and H5 influenza A viruses in the mouse model. Immunization with CApH1N1 induced both systemic and mucosal antibodies with broad reactivity to seasonal and H5 strains, including HAPI H5N1 and the avian H5N2 virus, providing complete protection against heterologous and heterosubtypic lethal challenges. Our results not only accentuate the merit of using live attenuated influenza virus vaccines in view of cross-reactivity but also represent the potential of CApH1N1 live vaccine for mitigating the clinical severity of infections that arise from reassortments between pH1N1 and highly pathogenic H5 subtype viruses.     

Recombinant trimeric HA protein immunogenicity of H5N1 avian influenza viruses and their combined use with inactivated or adenovirus vaccines

Background

The highly pathogenic avian influenza (HPAI) H5N1 virus continues to cause disease in poultry and humans. The hemagglutinin (HA) envelope protein is the primary target for subunit vaccine development.

Methodology/Principal Findings

We used baculovirus-insect cell expression to obtain trimeric recombinant HA (rHA) proteins from two HPAI H5N1 viruses. We investigated trimeric rHA protein immunogenicity in mice via immunizations, and found that the highest levels of neutralizing antibodies resulted from coupling with a PELC/CpG adjuvant. We also found that the combined use of trimeric rHA proteins with (a) an inactivated H5N1 vaccine virus, or (b) a recombinant adenovirus encoding full-length HA sequences for prime-boost immunization, further improved antibody responses against homologous and heterologous H5N1 virus strains. Data from cross-clade prime-boost immunization regimens indicate that sequential immunization with different clade HA antigens increased antibody responses in terms of total IgG level and neutralizing antibody titers.

Conclusion/Significance

Our findings suggest that the use of trimeric rHA in prime-boost vaccine regimens represents an alternative strategy for recombinant H5N1 vaccine development.     

Source of high pathogenicity of an avian influenza virus H5N1: why H5 is better cleaved by furin

The origin of the high pathogenicity of an emerging avian influenza H5N1 due to the -RRRKK- insertion at the cleavage loop of the hemagglutinin H5, was studied using the molecular dynamics technique, in comparison with those of the noninserted H5 and H3 bound to the furin (FR) active site. The cleavage loop of the highly pathogenic H5 was found to bind strongly to the FR cavity, serving as a conformation suitable for the proteolytic reaction. With this configuration, the appropriate interatomic distances were found for all three reaction centers of the enzyme-substrate complex: the arrangement of the catalytic triad, attachment of the catalytic Ser³⁶⁸ to the reactive S1-Arg, and formation of the oxyanion hole. Experimentally, the -RRRKK- insertion was also found to increase in cleavage of hemagglutinin by FR. The simulated data provide a clear answer to the question of why inserted H5 is better cleaved by FR than the other subtypes, explaining the high pathogenicity of avian influenza H5N1.     

Impact of preexisting adenovirus vector immunity on immunogenicity and protection conferred with an adenovirus-based H5N1 influenza vaccine

The prevalence of preexisting immunity to adenoviruses in the majority of the human population might adversely impact the development of adaptive immune responses against adenovirus vector-based vaccines. To address this issue, we primed BALB/c mice either intranasally (i.n.) or intramuscularly (i.m.) with varying doses of wild type (WT) human adenovirus subtype 5 (HAd5). Following the development of immunity against HAd5, we immunized animals via the i.n. or i.m. route of inoculation with a HAd vector (HAd-HA-NP) expressing the hemagglutinin (HA) and nucleoprotein (NP) of A/Vietnam/1203/04 (H5N1) influenza virus. The immunogenicity and protection results suggest that low levels of vector immunity (<520 virus-neutralization titer) induced by priming mice with up to 10(7) plaque forming units (p.f.u.) of HAd-WT did not adversely impact the protective efficacy of the vaccine. Furthermore, high levels of vector immunity (approximately 1500 virus-neutralization titer) induced by priming mice with 10(8) p.f.u. of HAd-WT were overcome by either increasing the vaccine dose or using alternate routes of vaccination. A further increase in the priming dose to 10(9) p.f.u. allowed only partial protection. These results suggest possible strategies to overcome the variable levels of human immunity against adenoviruses, leading to better utilization of HAd vector-based vaccines.     

Proteomic characterization of influenza H5N1 virus-like particles and their protective immunogenicity

Recombinant virus-like particles (VLPs) have been shown to induce protective immunity. Despite their potential significance as promising vaccine candidates, the protein composition of VLPs produced in insect cells has not been well characterized. Here we report a proteomic analysis of influenza VLPs containing hemagglutinin (HA) and matrix M1 proteins from a human isolate of avian influenza H5N1 virus (H5 VLPs) produced in insect cells using the recombinant baculovirus expression system. Comprehensive proteomic analysis of purified H5 VLPs identified viral proteins and 37 additional host-derived proteins, many of which are known to be present in other enveloped viruses. Proteins involved in different cellular structures and functions were found to be present in H5 VLPs including those from the cytoskeleton, translation, chaperone, and metabolism. Immunization with purified H5 VLPs induced protective immunity, which was comparable to the inactivated whole virus containing all viral components. Unpurified H5 VLPs containing excess amounts of noninfluenza soluble proteins also conferred 100% protection against lethal challenge although lower immune responses were induced. These results provide important implications consistent with the idea that VLP production in insect cells may involve similar cellular machinery as other RNA enveloped viruses during synthesis, assembly, trafficking, and budding processes.     

Multiple-clade H5N1 influenza split vaccine elicits broad cross protection against lethal influenza virus challenge in mice by intranasal vaccination

Background

The increase in recent outbreaks and unpredictable changes of highly pathogenic avian influenza (HPAI) H5N1 in birds and humans highlights the urgent need to develop a cross-protective H5N1 vaccine. We here report our development of a multiple-clade H5N1 influenza vaccine tested for immunogenicity and efficacy to confer cross-protection in an animal model.

Methodology/Principal Findings

Mice received two doses of influenza split vaccine with oil-in-water emulsion adjuvant SP01 by intranasal administration separated by two weeks. Single vaccines (3 µg HA per dose) included rg-A/Vietnam/1203/2004(Clade 1), rg-A/Indonesia/05/2005(Clade 2.1), and rg-A/Anhui/1/2005(Clade 2.3.4). The trivalent vaccine contained 1 µg HA per dose of each single vaccine. Importantly, complete cross-protection was observed in mice immunized using trivalent vaccine with oil-in-water emulsion adjuvant SP01 that was subsequently challenged with the lethal A/OT/SZ/097/03 influenza strain (Clade 0), whereas only the survival rate was up to 60% in single A/Anhui/1/2005 vaccine group.

Conclusion/Significance

Our findings demonstrated that the multiple-clade H5N1 influenza vaccine was able to elicit a cross-protective immune response to heterologous HPAI H5N1 virus, thus giving rise to a broadly cross-reactive vaccine to potential prevention use ahead of the strain-specific pandemic influenza vaccine in the event of an HPAI H5N1 influenza outbreak. Also, the multiple-clade adjuvanted vaccine could be useful in allowing timely initiation of vaccination against unknown pandemic virus.     

Phylodynamics of H5N1 avian influenza virus in Indonesia

Understanding how pathogens invade and become established in novel host populations is central to the ecology and evolution of infectious disease. Influenza viruses provide unique opportunities to study these processes in nature because of their rapid evolution, extensive surveillance, large data sets and propensity to jump species boundaries. H5N1 highly pathogenic avian influenza virus (HPAIV) is a major animal pathogen and public health threat. The virus is of particular importance in Indonesia, causing severe outbreaks among poultry and sporadic human infections since 2003. However, little is known about how H5N1 HPAIV emerged and established in Indonesia. To address these questions, we analysed Indonesian H5N1 HPAIV gene sequences isolated during 2003-2007. We find that the virus originated from a single introduction into East Java between November 2002 and October 2003. This invasion was characterized by an initially rapid burst of viral genetic diversity followed by a steady rate of lineage replacement and the maintenance of genetic diversity. Several antigenic sites in the haemagglutinin gene were subject to positive selection during the early phase, suggesting that host-immune-driven selection played a role in host adaptation and expansion. Phylogeographic analyses show that after the initial invasion of H5N1, genetic variants moved both eastwards and westwards across Java, possibly involving long-distance transportation by humans. The phylodynamics we uncover share similarities with other recently studied viral invasions, thereby shedding light on the ecological and evolutionary processes that determine disease emergence in a new geographical region.     

Impact of anti-rheumatic treatment on immunogenicity of pandemic H1N1 influenza vaccine in patients with arthritis

Introduction

An adjuvanted pandemic H1N1 influenza (pH1N1) vaccine (Pandemrix®) was reported as highly immunogenic resulting in seroconversion in 77 to 94% of adults after administration of a single dose. The aim of the study was to investigate the impact of different anti-rheumatic treatments on antibody response to pH1N1 vaccination in patients with rheumatoid arthritis (RA) and spondylarthropathy (SpA).

Methods

Patients with arthritis (n = 291; mean age 57 years, 64% women) participated. Hemagglutination inhibition (HI) assay was performed on blood samples drawn before and after a mean (SD) of 8.3 (4) months following vaccination. A positive immune response i.e. seroconversion was defined as negative prevaccination serum and postvaccination HI titer ≥40 or a ≥4-fold increase in HI titer. All patients were divided into predefined groups based on diagnosis (RA or SpA) and ongoing treatment: methotrexate (MTX), anti-tumor necrosis factor (anti-TNF) as monotherapy, MTX combined with anti-TNF, other biologics (abatacept, rituximab, tocilizumab) and non-steroidal anti-inflammatory drugs (NSAIDs)/analgesics. Predictors of positive immune response were studied using logistic regression analysis.

Results

The percentage of patients with positive immune response in the different treatment groups was: 1. RA on MTX 42%; 2. RA on anti-TNF monotherapy 53%; 3. RA on anti-TNF + MTX 43%; 4. RA on other biologics (abatacept 20%, rituximab 10% and tocilizumab 50%); 5. SpA on anti-TNF monotherapy 76%; 6. SpA on anti-TNF + MTX 47%; and 7. SpA on NSAIDs/analgesics 59%. RA patients on rituximab had significantly lower (P < 0.001) and SpA on anti-TNF monotherapy significantly better response rates compared to other treatment groups (P 0.001 to 0.033). Higher age (P < 0.001) predicted impaired immune response. Antibody titers 3 to 6 months after vaccination was generally lower compared to those within the first 3 months but no further decrease in titers were observed 6 to 22 months after vaccination.

Conclusions

Rituximab treatment severely reduced antibody response to pH1N1 influenza vaccine. The other treatment groups showed acceptable antibody responses. Protective antibody titers could be detected up to 22 months after vaccination in the current patient population, with the exception of rituximab treated patients.     

Production of inactivated influenza H5N1 vaccines from MDCK cells in serum-free medium

Background

Highly pathogenic influenza viruses pose a constant threat which could lead to a global pandemic. Vaccination remains the principal measure to reduce morbidity and mortality from such pandemics. The availability and surging demand for pandemic vaccines needs to be addressed in the preparedness plans. This study presents an improved high-yield manufacturing process for the inactivated influenza H5N1 vaccines using Madin-Darby canine kidney (MDCK) cells grown in a serum-free (SF) medium microcarrier cell culture system.

Principal Finding

The current study has evaluated the performance of cell adaptation switched from serum-containing (SC) medium to several commercial SF media. The selected SF medium was further evaluated in various bioreactor culture systems for process scale-up evaluation. No significant difference was found in the cell growth in different sizes of bioreactors studied. In the 7.5 L bioreactor runs, the cell concentration reached to 2.3×10⁶ cells/mL after 5 days. The maximum virus titers of 1024 Hemagglutinin (HA) units/50 µL and 7.1±0.3×10⁸ pfu/mL were obtained after 3 days infection. The concentration of HA antigen as determined by SRID was found to be 14.1 µg/mL which was higher than those obtained from the SC medium. A mouse immunogenicity study showed that the formalin-inactivated purified SF vaccine candidate formulated with alum adjuvant could induce protective level of virus neutralization titers similar to those obtained from the SC medium. In addition, the H5N1 viruses produced from either SC or SF media showed the same antigenic reactivity with the NIBRG14 standard antisera.

Conclusions

The advantages of this SF cell-based manufacturing process could reduce the animal serum contamination, the cost and lot-to-lot variation of SC medium production. This study provides useful information to manufacturers that are planning to use SF medium for cell-based influenza vaccine production.     

Potent inhibition of human influenza H5N1 virus by oligonucleotides derived by SELEX

New therapeutics are urgently needed for the treatment of pandemic influenza caused by H5N1 influenza virus mutants. Aptamer was a promising candidate for treatment and prophylaxis of influenza virus infections. In this study, systemic evolution of ligands through exponential enrichment (SELEX) was used to screen DNA aptamers targeted to recombinant HA1 proteins of the H5N1 influenza virus. After 11 rounds of selection, DNA aptamers that bind to the HA1 protein were isolated and shown to have different binding capacities. Among them, aptamer 10 had the strongest binding to the HA1 protein, and had an inhibitory effect on H5N1 influenza virus, as shown by the hemagglutinin and MTT assays. These results should aid the development of new drugs for the prevention and control of influenza virus infections.     

Intranasal administration of adjuvant-combined recombinant influenza virus HA vaccine protects mice from the lethal H5N1 virus infection

Attenuated recombinant H5N1 influenza virus was constructed to develop a safe H5N1 influenza vaccine. The immunogenicity and protective effect of the vaccine prepared from haemagglutinin-modified recombinant H5N1 influenza virus was evaluated in mice intranasally co-administered with cholera toxin B subunit containing a trace amount of holotoxin (CTB*), synthetic double-stranded RNA, poly (I:C) or chitin microparticles (CMP) as adjuvants. Intranasal administration of recombinant H5 HA split vaccine with CTB* or poly(I:C) and/or CMP elicited an immunological response with both anti-H5 HA IgA in the nasal wash and anti-H5 HA IgG antibody in the serum, and showed a protective against lethal H5N1 A/Hong Kong/483/97 (HK483) infection. We also demonstrated that intranasal co-administration of antigen with both poly (I:C) and CMP enhanced the expression of Toll-like receptor (TLR) 3, TLR7 in the spleen. These results indicate that poly (I:C) and CMP are highly effective as mucosal adjuvants for use with the nasal H5N1 vaccine.     

Adaptation of high-growth influenza H5N1 vaccine virus in Vero cells: implications for pandemic preparedness

Current egg-based influenza vaccine production technology can't promptly meet the global demand during an influenza pandemic as shown in the 2009 H1N1 pandemic. Moreover, its manufacturing capacity would be vulnerable during pandemics caused by highly pathogenic avian influenza viruses. Therefore, vaccine production using mammalian cell technology is becoming attractive. Current influenza H5N1 vaccine strain (NIBRG-14), a reassortant virus between A/Vietnam/1194/2004 (H5N1) virus and egg-adapted high-growth A/PR/8/1934 virus, could grow efficiently in eggs and MDCK cells but not Vero cells which is the most popular cell line for manufacturing human vaccines. After serial passages and plaque purifications of the NIBRG-14 vaccine virus in Vero cells, one high-growth virus strain (Vero-15) was generated and can grow over 10(8) TCID(50)/ml. In conclusion, one high-growth H5N1 vaccine virus was generated in Vero cells, which can be used to manufacture influenza H5N1 vaccines and prepare reassortant vaccine viruses for other influenza A subtypes.     

An overview of the highly pathogenic H5N1 influenza virus

Since the first human case of H5N1 avian influenza virus infection was reported in 1997, this highly pathogenic virus has infected hundreds of people around the world and resulted in many deaths. The ability of H5N1 to cross species boundaries, and the presence of polymorphisms that enhance virulence, present challenges to developing clear strategies to prevent the pandemic spread of this highly pathogenic avian influenza (HPAI) virus. This review summarizes the current understanding of, and recent research on, the avian influenza H5N1 virus, including transmission, virulence, pathogenesis, clinical characteristics, treatment and prevention.