Heterologous H5N1 Virus Research Articles

M2e nanovaccines supplemented with recombinant hemagglutinin protect chickens against heterologous HPAI H5N1 challenge

Current poultry vaccines against influenza A viruses target the globular head region of the hemagglutinin (HA1), providing limited protection against antigenically divergent strains. Experimental subunit vaccines based on the conserved ectodomain of the matrix protein 2 (M2e) induce cross-reactive antibody responses, but fail to fully prevent virus shedding after low pathogenic avian influenza (LPAI) virus challenge, and are ineffective against highly pathogenic avian influenza (HPAI) viruses. This study assessed the benefits of combining nanoparticles bearing three tandem M2e repeats (NR-3M2e nanorings or NF-3M2e nanofilaments) with an HA1 subunit vaccine in protecting chickens against a heterologous HPAI H5N1 virus challenge. Chickens vaccinated with the combined formulations developed M2e and HA1-specific antibodies, were fully protected from clinical disease and mortality, and showed no histopathological lesions or virus shedding, unlike those given only HA1, NR-3M2e, or NF-3M2e. Thus, the combined vaccine formulations provided complete cross-protection against HPAI H5N1 virus, and prevented environmental virus shedding, crucial for controlling avian influenza outbreaks.

A Chymotrypsin-Dependent Live-Attenuated Influenza Vaccine Provides Protective Immunity against Homologous and Heterologous Viruses.

Influenza virus is one of the main pathogens causing respiratory diseases in humans. Vaccines are the most effective ways to prevent viral diseases. However, the limited protective efficacy of current influenza vaccines highlights the importance of novel, safe, and effective universal influenza vaccines. With the progress of the COVID-19 pandemic, live-attenuated vaccines delivered through respiratory mucosa have shown robustly protective efficacy. How to obtain a safe and effective live-attenuated vaccine has become a major challenge. Herein, using the influenza virus as a model, we have established a strategy to quickly obtain a live-attenuated vaccine by mutating the cleavage site of the influenza virus. This mutated influenza virus can be specifically cleaved by chymotrypsin. It has similar biological characteristics to the original strain in vitro, but the safety is improved by at least 100 times in mice. It can effectively protect against lethal doses of both homologous H1N1 and heterologous H5N1 viruses post mucosal administration, confirming that the vaccine generated by this strategy has good safety and broad-spectrum protective activities. Therefore, this study can provide valuable insights for the development of attenuated vaccines for respiratory viruses or other viruses with cleavage sites.

Neuraminidase (NA) 370-Loop Mutations of the 2009 Pandemic H1N1 Viruses Affect NA Enzyme Activity, Hemagglutination Titer, Mouse Virulence, and Inactivated-Virus Immunogenicity.

Hemagglutinin (HA) and neuraminidase (NA) are the two major envelope proteins of influenza viruses. The spatial organization of HA and NA on the virus surface needs to be optimized to promote viral fitness, host specificity, transmissibility, infectivity, and virulence. We previously demonstrated that the recombinant NA protein of the 2009 pandemic H1N1 (pH1N1) with the I365T/S366N mutation in the NA 370-loop elicited higher NA-inhibition antibody titers against the homologous pH1N1 virus and three heterologous H5N1, H3N2, and H7N9 viruses in mice. In this study, we used PR8-based reverse genetics (RG) by replacing the HA and NA genes of A/Texas/05/2009 pH1N1 virus to obtain the wild-type pH1N1 and three NA 370-loop mutant viruses of pH1N1 (I365T/S366N), RG pH1N1 (I365E/S366D), and RG pH1N1 (I365T/S366A). Our results revealed that the viral NA enzyme activity increased for the RG pH1N1(I365T/S366N) and RG pH1N1 (I365E/S366D) viruses but reduced for the RG pH1N1 (I365T/S366A) virus. The increased or decreased NA enzyme activity was found to correlate with the increase or decrease in HA titers of these NA 370-loop mutant viruses. All of these three NA 370-loop mutant RG pH1N1 viruses were less virulent than the wild-type RG pH1N1 virus in mice. Immunizations with the inactivated viruses carrying the three NA 370-loop mutations and the wild-type RG pH1N1 virus were found to elicit approximately the same titers of NA-inhibition antibodies against H1N1 and H5N1 viruses. These results may provide information for developing NA-based influenza virus vaccines.

High immune efficacy against different avian influenza H5N1 viruses due to oral administration of a Saccharomyces cerevisiae-based vaccine in chickens

A safe and effective vaccine is the best way to control large-scale highly pathogenic avian influenza virus (HPAI) A (H5N1) outbreaks. Saccharomyces cerevisiae (S. cerevisiae) is an ideal mucosal delivery vector for vaccine development, and we have previously shown that conventional administration of a S. cerevisiae-based vaccine (EBY100/pYD1-HA) via injection led to protection against the homologous H5N1 virus in a mouse model. Because the diameter of S. cerevisiae is approximately 10 μm, which results in a severe inflammation by injection route, therefore, oral administration is a more suitable approach for EBY100/pYD1-HA conferring protection in poultry. We extended our work by evaluating the immunogenicity and protective efficacy of oral vaccination with EBY100/pYD1-HA in the chicken model. Oral immunization with EBY100/pYD1-HA could induce robust serum IgG, mucosal IgA and cellular immune responses. Importantly, EBY100/pYD1-HA provided protection against challenges with a homologous and a heterologous H5N1 viruses. These findings suggest that EBY100/pYD1-HA, a promising H5N1 oral vaccine candidate, can avoid potential reassortment of other avian influenza viruses in oral administration of live virus vaccines and overcome the limitations of conventional injection routes. Importantly, this platform will be able to provide opportunities for broader applications in poultry during HPAI A (H5N1) outbreaks.

Longevity of adenovirus vector immunity in mice and its implications for vaccine efficacy

There is a high incidence of adenovirus (AdV) infection in humans due to the presence of more than 60 types of human adenoviruses (HAdVs). The majority of individuals are exposed to one or more HAdV types early in their lives, leading to the development of AdV type-specific neutralizing antibodies. Similarly, immunization or gene therapy with AdV vectors leads to immune responses to the AdV vector. This ‘vector immunity’ is a concern for AdV vector-based applications for vaccines or gene therapy, especially when the repeated administration of a vector is required. The objective of this investigation was to establish whether AdV neutralizing antibody titers decline sufficiently in a year to permit annual vaccination with the same AdV vector. Naïve or human adenoviral vector group C, type 5 (HAdV-C5)-primed mice were mock-inoculated (with PBS) or inoculated i.m. with 108 PFU of either HAd-GFP [HAdV-C5 vector expressing the green fluorescent protein (GFP)] to mimic the conditions for the first inoculation with an AdV vector-based vaccine. At 1, 3, 6, and 10 months post-HAd-GFP inoculation, naïve- or HAdV-primed animals were vaccinated i.m. with 108 PFU of HAd-H5HA [HAdV-C5 vector expressing hemagglutinin (HA) of H5N1 influenza virus]. There was a significant continual decrease in vector immunity titers with time, thereby leading to significant continual increases in the levels of HA-specific humoral and cell-mediated immune responses. In addition, significant improvement in protection efficacy against challenge with an antigenically heterologous H5N1 virus was observed in HAdV-primed animals at 6 months and onwards. These results indicate that the annual immunization with the same AdV vector may be effective due to a significant decline in vector immunity.

Inclusion of membrane-anchored LTB or flagellin protein in H5N1 virus-like particles enhances protective responses following intramuscular and oral immunization of mice

We previously demonstrated that intramuscular immunization with virus-like particles (VLPs) composed of the haemagglutinin (HA), neuraminidase (NA), and matrix (M1) proteins of A/meerkat/Shanghai/SH-1/2012 (clade 2.3.2.1) protected mice from lethal challenge with viruses from other H5 HPAI clades. The inclusion of additional proteins that can serve as immunological adjuvants in VLPs may enhance adaptive immune responses following vaccination, and oral vaccines may represent the safest choice. Here, we report the generation of H5N1 VLPs composed of the viral HA, NA, and M1 proteins and membrane-anchored forms of the Escherichia coli heat-labile enterotoxin B subunit protein (LTB) or the Toll-like receptor 5 ligand flagellin (Flic). Mice intramuscularly or orally immunized with VLPs containing LTB or Flic generated greater humoural and cellular immune responses than those administered H5N1 VLPs without LTB or Flic. Intramuscular immunization with VLPs protected mice from lethal challenge with homologous or heterologous H5N1 viruses irrespective of whether the VLPs additionally included LTB or Flic. In contrast, oral immunization of mice with LTB- or Flic-VLPs conferred substantial protection against lethal challenge with both homologous and heterologous H5N1 influenza viruses, whereas mice immunized orally with VLPs lacking LTB and Flic universally succumbed to infection. Mice immunized orally with LTB- or Flic-VLPs showed 10-fold higher virus-specific IgG titres than mice immunized with H5N1-VLPs lacking LTB or Flic. Collectively, these results indicate that the inclusion of immunostimulatory proteins, such as LTB and Flic, in VLP-based vaccines may represent a promising new approach for the control of current H5N1 HPAI outbreaks by eliciting higher humoural and cellular immune responses and conferring improved cross-clade protection.

A Bovine Adenoviral Vector-Based H5N1 Influenza -Vaccine Provides Enhanced Immunogenicity and Protection at a Significantly Low Dose

Several human and nonhuman adenovirus (AdV) vectors including bovine AdV type 3 (BAdV-3) were developed as gene delivery vectors to supplement and/or elude human AdV (HAdV)-specific neutralizing antibodies (vector immunity). Here we evaluated the vaccine immunogenicity and efficacy of BAdV-3 vector (BAd-H5HA) expressing hemagglutinin (HA) of a H5N1 influenza virus in a dose escalation study in mice with the intranasal (IN) or intramuscular (IM) route of inoculation in comparison with the HAdV type C5 (HAdV-C5) vector (HAd-H5HA) expressing HA of a H5N1 influenza virus. Dose-related increases in the immune responses were clearly noticeable. A single IM inoculation with BAd-H5HA resulted in enhanced cellular immune responses compared with that of HAd-H5HA and conferred complete protection following challenge with a heterologous H5N1 virus at the dose of 3 × 107 plaque-forming units (PFUs), whereas a significant amount of influenza virus was detected in the lungs of mice immunized with 1 × 108 PFUs of HAd-H5HA. Similarly, compared with that of HAd-H5HA, a single IN inoculation with BAd-H5HA produced significantly enhanced humoral (HA-specific immunoglobulin [IgG] and its subclasses, as well as HA-specific IgA) and cellular immune responses, and conferred complete protection following challenge with a heterologous H5N1 virus. Complete protection with BAd-H5HA was observed with the lowest vaccine dose (1 × 106 PFUs), but similar protection with HAd-H5HA was observed at the highest vaccine dose (1 × 108 PFUs). These results suggest that at least 30-fold dose sparing can be achieved with BAd-H5HA vector compared with HAd-H5HA vaccine vector.

Coimmunization with recombinant epitope-expressing baculovirus enhances protective effects of inactivated H5N1 vaccine against heterologous virus

H5N1, a highly pathogenic avian influenza virus (HPAIV), poses a significant threat to poultry and human health. However, currently available inactivated influenza vaccines are less efficacious against viruses that display antigenic drift. In this study, we constructed a recombinant baculovirus (BV-HMNN) expressing four conserved antigen epitopes: H5N1 hemagglutinin stem area amino acids 76–130 (HA2 76–130); three tandem repeats from the ectodomain of the conserved influenza matrix protein M2 (3M2e); nucleoprotein amino acids 55–69 (NP55–69); and nucleoprotein amino acids 380–393 (NP380–393). We evaluated the immunogenicity and protective efficacy of coimmunization with an inactivated avian influenza virus vaccine (Re6) and the recombinant baculovirus (BV-HMNN) against heterologous viral infection in specific-pathogen-free chickens. The chickens immunized with both vaccines (Re6+BV-HMNN) achieved complete protection, was significantly greater than that of chickens vaccinated with Re6 alone. BV-HMNN-supplemented vaccination also reduced viral shedding more effectively than nonsupplemented vaccination. We conclude that coimmunization with both vaccines was superior to immunization with the inactivated vaccine alone in inducing cross-protection against heterologous H5N1 virus.

Benefits and Limits of Egg Yolkvs. Serum Samples for Avian Influenza Virus Serosurveillance

Serologic tests are a valuable tool for retrospective surveillance of avian influenza viruses (AIV) and monitoring of postvaccination host immune response. Yet collection of serum samples, particularly in adult breeder chickens, is laborious, intrusive to birds, and may pose a serious risk to the biosecurity of a flock. In this study we compared the level of AIV-specific antibody titers in eggs and serum samples obtained from broiler breeder chickens vaccinated at 6, 12, and 18 wk of age with H5N2-inactivated vaccine. Nucleocapsid protein-specific ELISA and hemagglutination inhibition test (HI) against homologous as well as heterologous antigens were used. The eggs and sera were collected at 22, 30, 45, and 50 wk of age (i.e., 4, 12, 27, and 32 wk after the third and final immunization, respectively). Using ELISA, the number of positive egg yolk samples decreased over time after vaccination, from 97% to 47%, while the seropositivity rate of serum samples was 97%-100% during the whole investigation period. No antibody titers were detected in egg white. By HI, antibody titers in serum samples were higher than in egg yolk samples. Compared to the homologous H5N2 antigen, significantly lower HI titers were obtained by using a heterologous H5N1 virus of clade 2.2.1.2. In addition, no HI titers were detected in egg yolk and/or serum samples tested against the antigen of an Egyptian H5N1 antigenic drift variant of clade 2.2.1.1. This study indicates that egg yolk may be used to monitor the postvaccination immune status of broiler breeder chickens and retrospective serosurveillance-by HI when a matching antigen is available as well as by ELISA-particularly for up to 12 wk postvaccination.

A cationic liposome–DNA complexes adjuvant (JVRS-100) enhances the immunogenicity and cross-protective efficacy of pre-pandemic influenza A (H5N1) vaccine in ferrets

Influenza A (H5N1) viruses continue to pose a public health threat. As inactivated H5N1 vaccines are poorly immunogenic, adjuvants are needed to improve the immunogenicity of H5N1 vaccine in humans. Here, we investigated the immunogenicity and cross-protective efficacy in ferrets of a clade 2.2-derived vaccine with addition of JVRS-100, an adjuvant consisting of cationic liposome–DNA complexes (CLDC). After the first vaccination, significantly higher levels of hemagglutination-inhibition (HAI) and neutralizing antibody titers were detected in ferrets immunized with adjuvanted vaccine compared to unadjuvanted vaccine. Following a second dose of adjuvanted vaccine, HAI antibody titers of≥40 were detected against viruses from multiple H5N1 clades. HAI antibodies against newly isolated H5N2 and H5N8 viruses were also augmented by JVRS-100. Ferrets were challenged with a heterologous H5N1 virus. All ferrets that received two doses of adjuvanted vaccine exhibited mild illness, significantly reduced nasal wash virus titers and protection from lethal challenge. In contrast, ferrets that received unadjuvanted vaccine showed greater weight loss, high viral titers and 3 of 6 animals succumbed to the lethal challenge. Our results indicate that the addition of JVRS-100 to H5N1 vaccine enhanced immunogenicity and cross-protection against lethal H5N1 virus disease in ferrets. JVRS-100 warrants further investigation as a potential adjuvant for influenza vaccines.

Yeast Surface-Displayed H5N1 Avian Influenza Vaccines.

Highly pathogenic H5N1 avian influenza viruses pose a pandemic threat to human health. A rapid vaccine production against fast outbreak is desired. We report, herein, a paradigm-shift influenza vaccine technology by presenting H5N1 hemagglutinin (HA) to the surface of yeast. We demonstrated, for the first time, that the HA surface-presented yeast can be used as influenza vaccines to elicit both humoral and cell-mediated immunity in mice. The HI titer of antisera reached up to 128 in vaccinated mice. A high level of H5N1 HA-specific IgG1 and IgG2a antibody production was detected after boost immunization. Furthermore, we demonstrated that the yeast surface-displayed HA preserves its antigenic sites. It preferentially binds to both avian- and human-type receptors. In addition, the vaccine exhibited high cross-reactivity to both homologous and heterologous H5N1 viruses. A high level production of anti-HA antibodies was detected in the mice five months after vaccination. Finally, our animal experimental results indicated that the yeast vaccine offered complete protection of mice from lethal H5N1 virus challenge. No severe side effect of yeast vaccines was noted in animal studies. This new technology allows for rapid and large-scale production of influenza vaccines for prepandemic preparation.

Broadly protective immunity against divergent influenza viruses by oral co-administration of Lactococcus lactis expressing nucleoprotein adjuvanted with cholera toxin B subunit in mice.

BackgroundCurrent influenza vaccines need to be annually reformulated to well match the predicated circulating strains. Thus, it is critical for developing a novel universal influenza vaccine that would be able to confer cross-protection against constantly emerging divergent influenza virus strains. Influenza virus A is a genus of the Orthomyxoviridae family of viruses. Influenza virus nucleoprotein (NP) is a structural protein which encapsidates the negative strand viral RNA, and anti-NP antibodies play role in cross-protective immunity. Lactococcus lactis (L. lactis) is an ideal vaccine delivery vehicle via oral administration route. However, L. lactis vectored vaccine exhibits poor immunogenicity without the use of mucosal adjuvant. To enhance the immunogenicity of L. lactis vectored vaccine, cholera toxin B (CTB) subunit, one of mucosal adjuvants, is a safe adjuvant for oral route, when combined with L. lactis vectored vaccine. In this study, we hypothesized that pNZ8008, a L. lactis expression plasmid, encoding NP antigen, would be able to elicit cross-protection with the use of CTB via oral administration route.ResultsTo construct L. lactis vectored vaccine, nucleoprotein (NP) gene of A/California/04/2009(H1N1) was sub-cloned into a L. lactis expression plasmid, pNZ8008. The expression of recombinant L. lactis/pNZ8008-NP was confirmed by Western blot, immunofluorescence assay and flow cytometric analysis. Further, immunogenicity of L. lactis/pNZ8008-NP alone or adjuvanted with cholera toxin B (CTB) subunit was evaluated in a mouse model via oral administration route. Antibodies responses were detected by ELISA. The result indicated that oral administration of L. lactis/pNZ8008-NP adjuvanted with CTB could elicit significant humoral and mucosal immune responses, as well as cellular immune response, compared with L. lactis/pNZ8008-NP alone. To further assess the cross-protective immunity of L. lactis/pNZ8008-NP adjuvanted with CTB, we used L. lactis/pNZ8110-pgsA-HA1 alone or adjuvanted with CTB as controls. Mice that received L. lactis/pNZ8008-NP adjuvanted with CTB were completely protected from homologous H1N1 virus and showed 80% protection against heterologous H3N2 or H5N1 virus, respectively. By contrast, L. lactis/pNZ8110-pgsA-HA1 adjuvanted with CTB also conferred 100% protection against H5N1 virus infection, but indicated no cross-protection against H1N1 or H5N1 virus challenge. As controls, mice vaccinated orally with L. lactis/pNZ8008-NP alone or L. lactis/pNZ8110-pgsA-HA1 alone could not survive.ConclusionThis study is the first to report the construction of recombinant L. lactis/pNZ8008-NP and investigate its immunogenicity with the use of CTB. Compared with L. lactis/pNZ8110-pgsA-HA1 adjuvanted with CTB, our data support 5 × 1011 CFU of L. lactis/pNZ8008-NP adjuvanted with 1 µg of CTB is a better combination for universal influenza vaccines development that would provide cross-protective immunity against divergent influenza A viruses.

Expression of H5 hemagglutinin vaccine antigen in common duckweed (Lemna minor) protects against H5N1 high pathogenicity avian influenza virus challenge in immunized chickens

A synthetic hemagglutinin (HA) gene from the highly pathogenic avian influenza (HPAI) virus A/chicken/Indonesia/7/2003 (H5N1) (Indo/03) was expressed in aquatic plant Lemna minor (rLemna-HA). In Experiment 1, efficacy of rLemna-HA was tested on birds immunized with 0.2μg or 2.3μg HA and challenged with 106 mean chicken embryo infectious doses (EID50) of homologous virus strain. Both dosages of rLemna-HA conferred clinical protection and dramatically reduced viral shedding. Almost all the birds immunized with either dosage of rLemna-HA elicited HA antibody titers against Indo/03 antigen, suggesting an association between levels of anti-Indo/03 antibodies and protection. In Experiment 2, efficacy of rLemna-HA was tested on birds immunized with 0.9μg or 2.2μg HA and challenged with 106 EID50 of heterologous H5N1 virus strains A/chicken/Vietnam/NCVD-421/2010 (VN/10) or A/chicken/West Java/PWT-WIJ/2006 (PWT/06). Birds challenged with VN/10 exhibited 100% survival regardless of immunization dosage, while birds challenged with PWT/06 had 50% and 30% mortality at 0.9μg HA and 2.2μg HA, respectively. For each challenge virus, viral shedding titers from 2.2μg HA vaccinated birds were significantly lower than those from 0.9μg HA vaccinated birds, and titers from both immunized groups were in turn significantly lower than those from sham vaccinated birds. Even if immunized birds elicited HA titers against the vaccine antigen Indo/03, only the groups challenged with VN/10 developed humoral immunity against the challenge antigen. None (rLemna-HA 0.9μg HA) and 40% (rLemna-HA 2.2μg HA) of the immunized birds challenged with PWT/06 elicited pre-challenge antibody titers, respectively. In conclusion, Lemna-expressed HA demonstrated complete protective immunity against homologous challenge and suboptimal protection against heterologous challenge, the latter being similar to results from inactivated whole virus vaccines. Transgenic duckweed-derived HA could be a good alternative for producing high quality antigen for an injectable vaccine against H5N1 HPAI viruses.

Sialic acid-binding protein Sp2CBMTD protects mice against lethal challenge with emerging influenza A (H7N9) virus.

Compounds that target the cellular factors essential for influenza virus replication represent an innovative approach to antiviral therapy. Sp2CBMTD is a genetically engineered multivalent protein that masks sialic acid-containing cellular receptors on the respiratory epithelium, which are recognized by influenza viruses. Here, we evaluated the antiviral potential of Sp2CBMTD against lethal infection in mice with an emerging A/Anhui/1/2013 (H7N9) influenza virus and addressed the mechanistic basis of its activity in vivo. Sp2CBMTD was administered to mice intranasally as a single or repeated dose (0.1, 1, 10, or 100 μg) before (day -7, -3, and/or -1) or after (6 or 24 h) H7N9 virus inoculation. A single Sp2CBMTD dose (10 or 100 μg) protected 80% to 100% of the mice when administered 7 days before the H7N9 lethal challenge. Repeated Sp2CBMTD administration conferred the highest protection, resulting in 100% survival of the mice even at the lowest dose tested (0.1 μg). When treatment began 24 h after exposure to the H7N9 virus, a single administration of 100 μg of Sp2CBMTD protected 40% of the mice from death. The administration of Sp2CBMTD induced the pulmonary expression of proinflammatory mediators (interleukin-6 [IL-6], IL-1β, RANTES, monocyte chemotactic protein-1 [MCP-1], macrophage inflammatory protein-1α [MIP-1α], and inducible protein [IP-10]) and recruited neutrophils to the respiratory tract before H7N9 virus infection, which resulted in less pronounced inflammation and rapid virus clearance from mouse lungs. Sp2CBMTD administration did not affect the virus-specific adaptive immune response, which was sufficient to protect against reinfection with a higher dose of homologous H7N9 virus or heterologous H5N1 virus. Thus, Sp2CBMTD was effective in preventing H7N9 infections in a lethal mouse model and holds promise as a prophylaxis option against zoonotic influenza viruses.

Point-of-Use Mixing of Influenza H5N1 Vaccine and MF59 Adjuvant for Pandemic Vaccination Preparedness: Antibody Responses and Safety. A Phase 1 Clinical Trial.

Background Avian influenza A/H5N1 has threatened human health for nearly 2 decades. Avian influenza A vaccine without adjuvant is poorly immunogenic. A flexible rapid tactic for mass vaccination will be needed if a pandemic occurs.Methods A multicenter, randomized, blinded phase 1 clinical trial evaluated safety and antibody responses after point-of-use mixing of influenza A/Indonesia/05/2005 (H5N1) vaccine with MF59 adjuvant. Field-site pharmacies mixed 3.75, 7.5, or 15 mcg of antigen with or without MF59 adjuvant just prior to intramuscular administration on days 0 and 21 of healthy adults aged 18–49 years.Results Two hundred and seventy subjects were enrolled. After vaccination, titers of hemagglutination inhibition antibody ≥1:40 were achieved in 80% of subjects receiving 3.75 mcg + MF59 vs only 14% receiving 15 mcg without adjuvant (P < .0001). Peak hemagglutination inhibition antibody geometric mean titers for vaccine + MF59 were ∼65 regardless of antigen dose, and neutralizing titers were 2- to 3-fold higher. Vaccine + MF59 produced cross-reactive antibody responses against 4 heterologous H5N1 viruses. Excellent safety and tolerability were demonstrated.Conclusions Point-of-use mixing of H5N1 antigen and MF59 adjuvant achieved target antibody titers in a high percentage of subjects and was safe. The feasibility of the point-of-use mixing should be studied further.

Protective efficacy of a single dose of baculovirus hemagglutinin-based vaccine in chickens and ducks against homologous and heterologous H5N1 virus infections.

Outbreaks of the highly pathogenic H5N1 virus in poultry and humans are ongoing. Vaccination is an efficient method for prevention of H5N1 infection. Using chickens and ducks, we assessed the efficacy of a vaccine comprising H5N1 hemagglutinin (HA) protein produced in a baculovirus expression system. The immunized chickens and ducks were protected against lethal infection by H5N1 in an antigen dose-dependent manner. Complete protection against homologous challenge and partial protection against heterologous challenge were achieved in chickens immunized with 5 μg HA protein and in ducks immunized with 10 μg HA protein. The IgG antibody subtype was mainly detected in the sera and tissues, including the lungs. The neuraminidase (NA) inhibition assay was negative in immunized chickens and ducks. Our results indicated that the expressed HA protein by baculovirus was immunogenic to both chickens and ducks, and the immunized chickens and ducks were protected from the lethal infections of highly pathogenic H5N1 influenza virus, though ducks required more HA protein than chickens to be protected. Also, baculovirus HA-vaccinated poultry can be differentiated from infected poultry by NA inhibition assay.

A cationic lipid-DNA complex adjuvant (JVRS-100) enhances the immunogenicity and cross-protective efficacy of influenza pre-pandemic H5N1 vaccine in ferrets (VAC2P.931)

Abstract Highly pathogenic avian influenza A H5N1 viruses continue to pose a public health threat. Adjuvants are needed to improve the immunogenicity of H5N1 vaccine in humans. JVRS-100 is a cationic lipid-DNA complex adjuvant. We investigated the immunogenicity and cross-protective efficacy of JVRS-100-adjuvanted H5N1 (clade 2.2) vaccine in ferrets. Four weeks after the first vaccination, significantly higher levels of serum hemagglutination-inhibition (HI), neutralizing and H5 IgG antibody were detected in ferrets immunized with adjuvanted vaccine compared to the unadjuvanted vaccine group. Following a second dose of JVRS-100 adjuvanted vaccine, cross-reactive HI antibody titers of ≥40 were detected against viruses from multiple H5N1 clades. Ferrets were challenged with a high lethal dose of heterologous H5N1 virus (clade 2.1.3.2) 2 months after the second vaccination. Ferrets that received JVRS-100 adjuvanted vaccine had significantly reduced virus titers in the nasal washes and were completely protected from severe disease and death. In contrast, ferrets that received unadjuvanted vaccine had high viral titers through day 6 and 3 of 6 animals succumbed to the lethal infection. Our results indicate that the addition of JVRS-100 to H5N1 vaccine enhanced immunogenicity and cross-protection against lethal H5N1 virus disease in ferrets. JVRS-100 warrants further investigation as a potential adjuvant for use in human vaccination against avian influenza viruses with pandemic potential.

Highly immunogenic prime–boost DNA vaccination protects chickens against challenge with homologous and heterologous H5N1 virus

Highly pathogenic avian influenza viruses (HPAIVs) cause huge economic losses in the poultry industry because of high mortality rate in infected flocks and trade restrictions. Protective antibodies, directed mainly against hemagglutinin (HA), are the primary means of protection against influenza outbreaks. A recombinant DNA vaccine based on the sequence of H5 HA from the H5N1/A/swan/Poland/305-135V08/2006 strain of HPAIV was prepared. Sequence manipulation included deletion of the proteolytic cleavage site to improve protein stability, codon usage optimization to improve translation and stability of RNA in host cells, and cloning into a commercially available vector to enable expression in animal cells. Naked plasmid DNA was complexed with a liposomal carrier and the immunization followed the prime–boost strategy. The immunogenic potential of the DNA vaccine was first proved in broilers in near-to-field conditions resembling a commercial farm. Next, the protective activity of the vaccine was confirmed in SPF layer-type chickens. Experimental infections (challenge experiments) indicated that 100% of vaccinated chickens were protected against H5N1 of the same clade and that 70% of them were protected against H5N1 influenza virus of a different clade. Moreover, the DNA vaccine significantly limited (or even eliminated) transmission of the virus to contact control chickens. Two intramuscular doses of DNA vaccine encoding H5 HA induced a strong protective response in immunized chicken. The effective protection lasted for a minimum 8weeks after the second dose of the vaccine and was not limited to the homologous H5N1 virus. In addition, the vaccine reduced shedding of the virus.

Effects of route and coadministration of recombinant raccoon poxviruses on immune responses and protection against highly pathogenic avian influenza in mice

We previously demonstrated that recombinant raccoonpox (RCN) virus could serve as a vector for an influenza vaccine. RCN constructs expressing the hemagglutinin (HA) from H5N1 viruses were immunogenic in chickens. In the current study, we generated several recombinant RCN constructs expressing influenza (H5N1) antigens and a molecular adjuvant (Heat-Labile enterotoxin B from E. coli: RCN-LTB), demonstrated their expression in vitro, and evaluated their ability to protect mice against H5N1 virus challenge. RCN-HA provided strong protection when administered intradermally (ID), but not intranasally (IN). Conversely, the RCN-neuraminidase (NA) construct was highly efficacious by the IN route and elicited high titers of neutralizing antibodies in mice. Vaccination by combined ID (RCN-HA) and IN (RCN-NA) routes offered mice the best protection against an IN challenge with heterologous H5N1 virus. However, protection was reduced when the different RCN constructs were pre-mixed, perhaps due to reduced expression of antigen.

Evaluation of the efficacy of a pre-pandemic H5N1 vaccine (MG1109) in mouse and ferret models

The threat of a highly pathogenic avian influenza (HPAI) H5N1 virus causing the next pandemic remains a major concern. In this study, we evaluated the immunogenicity and efficacy of an inactivated whole-virus H5N1 pre-pandemic vaccine (MG1109) formulated by Green Cross Co., Ltd containing the hemagglutinin (HA) and neuraminidase (NA) genes of the clade 1 A/Vietnam/1194/04 virus in the backbone of A/Puerto Rico/8/34 (RgVietNam/04xPR8/34). Administration of the MG1109 vaccine (2-doses) in mice and ferrets elicited high HI and SN titers in a dose-dependent manner against the homologous (RgVietNam/04xPR8/34) and various heterologous H5N1 strains, (RgKor/W149/06xPR8/34, RgCambodia/04xPR8/34, RgGuangxi/05xPR8/34), including a heterosubtypic H5N2 (A/Aquatic bird/orea/W81/05) virus. However, efficient cross-reactivity was not observed against heterosubtypic H9N2 (A/Ck/Korea/H0802/08) and H1N1 (PR/8/34) viruses. Mice immunized with 1.9 μg HA/dose of MG1109 were completely protected from lethal challenge with heterologous wild-type HPAI H5N1 A/EM/Korea/W149/06 (clade 2.2) and mouse-adapted H5N2 viruses. Furthermore, ferrets administered at least 3.8 μg HA/dose efficiently suppressed virus growth in the upper respiratory tract and lungs. Vaccinated mice and ferrets also demonstrated attenuation of clinical disease signs and limited virus spread to other organs. Thus, this vaccine provided immunogenic responses in mouse and ferret models even against challenge with heterologous HPAI H5N1 and H5N2 viruses. Since the specific strain of HPAI H5N1 virus that would potentially cause the next outbreak is unknown, pre-pandemic vaccine preparation that could provide cross-protection against various H5 strains could be a useful approach in the selection of promising candidate vaccines in the future.