Influenza vaccination accelerates recovery of ferrets from lymphopenia.

Ferrets are a useful animal model for human influenza virus infections, since they closely mimic the pathogenesis of influenza viruses observed in humans. However, a lack of reagents, especially for flow cytometry of immune cell subsets, has limited research in this model. Here we use a panel of primarily species cross-reactive antibodies to identify ferret T cells, cytotoxic T lymphocytes (CTL), B cells, and granulocytes in peripheral blood. Following infection with seasonal H3N2 or H1N1pdm09 influenza viruses, these cell types showed rapid and dramatic changes in frequency, even though clinically the infections were mild. The loss of B cells and CD4 and CD8 T cells, and the increase in neutrophils, were especially marked 1-2 days after infection, when about 90% of CD8+ T cells disappeared from the peripheral blood. The different virus strains led to different kinetics of leukocyte subset alterations. Vaccination with homologous vaccine reduced clinical symptoms slightly, but led to a much more rapid return to normal leukocyte parameters. Assessment of clinical symptoms may underestimate the effectiveness of influenza vaccine in restoring homeostasis.

Increased immunogenicity of avian influenza DNA vaccine delivered to the skin using a microneedle patch.

Effective public health responses to an influenza pandemic require an effective vaccine that can be manufactured and administered to large populations in the shortest possible time. In this study, we evaluated a method for vaccination against avian influenza virus that uses a DNA vaccine for rapid manufacturing and delivered by a microneedle skin patch for simplified administration and increased immunogenicity. We prepared patches containing 700-µm long microneedles coated with an avian H5 influenza hemagglutinin DNA vaccine from A/Viet Nam/1203/04 influenza virus. The coating DNA dose increased with DNA concentration in the coating solution and the number of dip-coating cycles. Coated DNA was released into the skin tissue by dissolution within minutes. Vaccination of mice using microneedles induced higher levels of antibody responses and hemagglutination inhibition titers, and improved protection against lethal infection with avian influenza as compared to conventional intramuscular delivery of the same dose of the DNA vaccine. Additional analysis showed that the microneedle coating solution containing carboxymethylcellulose and a surfactant may have negatively affected the immunogenicity of the DNA vaccine. Overall, this study shows that DNA vaccine delivery by microneedles can be a promising approach for improved vaccination to mitigate an influenza pandemic.

In vitro evolution of H5N1 avian influenza virus toward human-type receptor specificity.

Acquisition of α2-6 sialoside receptor specificity by α2-3 specific highly-pathogenic avian influenza viruses (H5N1) is thought to be a prerequisite for efficient transmission in humans. By in vitro selection for binding α2-6 sialosides, we identified four variant viruses with amino acid substitutions in the hemagglutinin (S227N, D187G, E190G, and Q196R) that revealed modestly increased α2-6 and minimally decreased α2-3 binding by glycan array analysis. However, a mutant virus combining Q196R with mutations from previous pandemic viruses (Q226L and G228S) revealed predominantly α2-6 binding. Unlike the wild type H5N1, this mutant virus was transmitted by direct contact in the ferret model although not by airborne respiratory droplets. However, a reassortant virus with the mutant hemagglutinin, a human N2 neuraminidase and internal genes from an H5N1 virus was partially transmitted via respiratory droplets. The complex changes required for airborne transmissibility in ferrets suggest that extensive evolution is needed for H5N1 transmissibility in humans.

Virus-like particle vaccine containing hemagglutinin confers protection against 2009 H1N1 pandemic influenza.

Immunization of the world population before an influenza pandemic such as the 2009 H1N1 virus spreads globally is not possible with current vaccine production platforms. New influenza vaccine technologies, such as virus-like-particles (VLPs), offer a promising alternative. Here, we tested the immunogenicity and protective efficacy of a VLP vaccine containing hemagglutinin (HA) and M1 from the 2009 pandemic H1N1 influenza virus (H1N1pdm) in ferrets and compared intramuscular (i.m.) and intranasal (i.n.) routes of immunization. Vaccination of ferrets with VLPs containing the M1 and HA proteins from A/California/04/2009 (H1N1pdm) induced high antibody titers and conferred significant protection against virus challenge. VLP-vaccinated animals lost less weight, shed less virus in nasal washes, and had markedly lower virus titers in all organs tested than naïve controls. A single dose of VLPs, either i.m. or i.n., induced higher levels of antibody than did two doses of commercial split vaccine. Ferrets vaccinated with split vaccine were incompletely protected against challenge; these animals had lower virus titers in olfactory bulbs, tonsils, and intestines, but lost weight and shed virus in nasal washes to a similar extent as naïve controls. Challenge with heterologous A/Brisbane/59/07 (H1N1) virus revealed that the VLPs conferred minimal cross-protection to heterologous infection, as revealed by the lack of reduction in nasal wash and lung virus titers and slightly higher weight loss relative to controls. In summary, these experiments demonstrate the strong immunogenicity and protective efficacy of VLPs compared to the split vaccine and show that i.n. vaccination with VLPs has the potential for highly efficacious vaccination against influenza.

The virulence of 1997 H5N1 influenza viruses in the mouse model is increased by correcting a defect in their NS1 proteins.

The NS1 protein of human influenza A viruses binds the 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30), a protein required for 3' end processing of cellular pre-mRNAs, thereby inhibiting production of beta interferon (IFN-ß) mRNA. The NS1 proteins of pathogenic 1997 H5N1 viruses contain the CPSF30-binding site but lack the consensus amino acids at positions 103 and 106, F and M, respectively, that are required for the stabilization of CPSF30 binding, resulting in nonoptimal CPSF30 binding in infected cells. Here we have demonstrated that strengthening CPSF30 binding, by changing positions 103 and 106 in the 1997 H5N1 NS1 protein to the consensus amino acids, results in a remarkable 300-fold increase in the lethality of the virus in mice. Unexpectedly, this increase in virulence is not associated with increased lung pathology but rather is characterized by faster systemic spread of the virus, particularly to the brain, where increased replication and severe pathology occur. This increased spread is associated with increased cytokine and chemokine levels in extrapulmonary tissues. We conclude that strengthening CPSF30 binding by the NS1 protein of 1997 H5N1 viruses enhances virulence in mice by increasing the systemic spread of the virus from the lungs, particularly to the brain.

The development of vaccine viruses against pandemic A(H1N1) influenza.

Wild type human influenza viruses do not usually grow well in embryonated hens' eggs, the substrate of choice for the production of inactivated influenza vaccine, and vaccine viruses need to be developed specifically for this purpose. In the event of a pandemic of influenza, vaccine viruses need to be created with utmost speed. At the onset of the current A(H1N1) pandemic in April 2009, a network of laboratories began a race against time to develop suitable candidate vaccine viruses. Two approaches were followed, the classical reassortment approach and the more recent reverse genetics approach. This report describes the development and the characteristics of current pandemic H1N1 candidate vaccine viruses.

Flexible label-free quantitative assay for antibodies to influenza virus hemagglutinins.

During the initial pandemic influenza H1N1 virus outbreak, assays such as hemagglutination inhibition and microneutralization provided important information on the relative protection afforded by the population's cross-reactivity from prior infections and immunizations with seasonal vaccines. However, these assays continue to be limited in that they are difficult to automate for high throughput, such as in pandemic situations, as well as to standardize between labs. Thus, new technologies are being sought to improve standardization, reliability, and throughput by using chemically defined reagents rather than whole cells and virions. We now report the use of a cell-free and label-free flu antibody biosensor assay (f-AbBA) for influenza research and diagnostics that utilizes recombinant hemagglutinin (HA) in conjunction with label-free biolayer interferometry technology to measure biomolecular interactions between the HA and specific anti-HA antibodies or sialylated ligands. We evaluated f-AbBA to determine anti-HA antibody binding activity in serum or plasma to assess vaccine-induced humoral responses. This assay can reveal the impact of antigenic difference on antibody binding to HA and also measure binding to different subtypes of HA. We also show that the biosensor assay can measure the ability of HA to bind a model sialylated receptor-like ligand. f-AbBA could be used in global surveillance laboratories since preliminary tests on desiccated HA probes showed no loss of activity after >2 months in storage at room temperature, indicating that the same reagent lots could be used in different laboratories to minimize interlaboratory assay fluctuation. Future development of such reagents and similar technologies may offer a robust platform for future influenza surveillance activities.

Microneedle delivery of H5N1 influenza virus-like particles to the skin induces long-lasting B- and T-cell responses in mice.

A simple method suitable for self-administration of vaccine would improve mass immunization, particularly during a pandemic outbreak. Influenza virus-like particles (VLPs) have been suggested as promising vaccine candidates against potentially pandemic influenza viruses, as they confer long-lasting immunity but are not infectious. We investigated the immunogenicity and protective efficacy of influenza H5 VLPs containing the hemagglutinin (HA) of A/Vietnam/1203/04 (H5N1) virus delivered into the skin of mice using metal microneedle patches and also studied the response of Langerhans cells in a human skin model. Prime-boost microneedle vaccinations with H5 VLPs elicited higher levels of virus-specific IgG1 and IgG2a antibodies, virus-specific antibody-secreting cells, and cytokine-producing cells up to 8 months after vaccination compared to the same antigen delivered intramuscularly. Both prime-boost microneedle and intramuscular vaccinations with H5 VLPs induced similar hemagglutination inhibition titers and conferred 100% protection against lethal challenge with the wild-type A/Vietnam/1203/04 virus 16 weeks after vaccination. Microneedle delivery of influenza VLPs to viable human skin using microneedles induced the movement of CD207(+) Langerhans cells toward the basement membrane. Microneedle vaccination in the skin with H5 VLPs represents a promising approach for a self-administered vaccine against viruses with pandemic potential.

Protective immunity against H5N1 influenza virus by a single dose vaccination with virus-like particles.

We generated influenza virus-like particles (VLPs) containing the wild type (WT) H5 hemagglutinin (HA) from A/Viet Nam/1203/04 virus or a mutant H5 HA with a deletion of the multibasic cleavage motif. VLPs containing mutant H5 HA were found to be as immunogenic as VLPs containing WT HA. A single intramuscular vaccination with either type of H5 VLPs provided complete protection against lethal challenge. In contrast, the recombinant H5 HA vaccine was less immunogenic and vaccination even with a 5 fold higher dose did not induce protective immunity. VLP vaccines were superior to the recombinant HA in inducing T helper type 1 immune responses, hemagglutination inhibition titers, and antibody secreting cells, which significantly contribute to inducing protective immunity after a single dose vaccination. This study provides insights into the potential mechanisms of improved immunogenicity by H5 VLP vaccines as an approach to improve the protective efficacy against potential pandemic viruses.

Induction of long-term protective immune responses by influenza H5N1 virus-like particles.

BACKGROUND: Recurrent outbreaks of highly pathogenic H5N1 avian influenza virus pose a threat of eventually causing a pandemic. Early vaccination of the population would be the single most effective measure for the control of an emerging influenza pandemic. METHODOLOGY/PRINCIPAL FINDINGS: Influenza virus-like particles (VLPs) produced in insect cell-culture substrates do not depend on the availability of fertile eggs for vaccine manufacturing. We produced VLPs containing influenza A/Viet Nam1203/04 (H5N1) hemagglutinin, neuraminidase, and matrix proteins, and investigated their preclinical immunogenicity and protective efficacy. Mice immunized intranasally with H5N1 VLPs developed high levels of H5N1 specific antibodies and were 100% protected against a high dose of homologous H5N1 virus infection at 30 weeks after immunization. Protection is likely to be correlated with humoral and cellular immunologic memory at systemic and mucosal sites as evidenced by rapid anamnestic responses to re-stimulation with viral antigen in vivo and in vitro. CONCLUSIONS/SIGNIFICANCE: These results provide support for clinical evaluation of H5N1 VLP vaccination as a public health intervention to mitigate a possible pandemic of H5N1 influenza.

Pathogenesis of H5N1 influenza virus infections in mice and ferret models differs according to respiratory tract or digestive system exposure.

BACKGROUND: Epidemiologic, clinical, and laboratory data suggest that H5N1 influenza viruses are transmitted through and predominantly affect the respiratory system of mammals. Some data suggest digestive system involvement. However, direct evidence of alimentary transmission and infection in mammals is lacking. METHODS: Infection with and pathogenesis of 4 H5N1 viruses were assessed in mice and ferrets inoculated intranasally or intragastrically with virus in liquid. In addition, ferrets were fed infected raw chicken meat or minced meat administered into the stomach by gavage with a tube. RESULTS: Only one virus, A/Whooper swan/Mongolia/244/05, was able to infect mice after intragastric inoculation in liquid, whereas no evidence of infection was observed in ferrets after intragastric inoculation. Consumption of infected meat by ferrets resulted in respiratory system infection only (due to A/Muscovy duck/Vietnam/209/05 and A/Whooper swan/Mongolia/244/05 viruses) or in both severe respiratory and systemic infection with predominant involvement of the liver, pancreas, and large and small intestine (due to A/Vietnam/1203/04 virus). Direct intragastric exposure to infected meat (A/Vietnam/1203/04 virus) resulted in lethal systemic disease mainly affecting the intestine, liver, and pancreas but not involving the lungs. CONCLUSIONS: Our results demonstrated that exposure of the digestive system to H5N1 influenza viruses could initiate infection either through the tonsil, with spread to respiratory tissues, or through intestinal infection, with spread to the liver and pancreas.

Changes in H5N1 influenza virus hemagglutinin receptor binding domain affect systemic spread.

The HA of influenza virus is a receptor-binding and fusion protein that is required to initiate infection. The HA receptor-binding domain determines the species of sialyl receptors recognized by influenza viruses. Here, we demonstrate that changes in the HA receptor-binding domain alter the ability of the H5N1 virus to spread systemically in mice. The A/Vietnam/1203/04 (VN1203) and A/Hong Kong/213/03 (HK213) viruses are consistently lethal to domestic chickens but differ in their pathogenicity to mammals. Insertion of the VN1203 HA and neuraminidase (NA) genes into recombinant HK213 virus expanded its tissue tropism and increased its lethality in mice; conversely, insertion of HK213 HA and NA genes into recombinant VN1203 virus decreased its systemic spread and lethality. The VN1203 and HK213 HAs differ by 10 aa, and HK213 HA has shown greater binding affinity for synthetic alpha2,6-linked sialyl receptor. Introduction of an S227N change and removal of N-linked glycosylation at residue 158 increased the alpha2,6-binding affinity of VN1203 HA. Recombinant VN1203 virus carrying the S227N change alone or with the residue-158 glycosylation site removed showed reduced lethality and systemic spread in mice but not in domestic chickens. Wild-type VN1203 virus exhibited the greatest efficiency in systemic spread after intramuscular inoculation and in infection of mouse bone marrow-derived dendritic cells and conventional pulmonary dendritic cells. These results show that VN1203 HA glycoprotein confers pathogenicity by facilitating systemic spread in mice; they also suggest that a minor change in receptor binding domain may modulate the virulence of H5N1 viruses.

Domestic pigs have low susceptibility to H5N1 highly pathogenic avian influenza viruses.

Genetic reassortment of H5N1 highly pathogenic avian influenza viruses (HPAI) with currently circulating human influenza A strains is one possibility that could lead to efficient human-to-human transmissibility. Domestic pigs which are susceptible to infection with both human and avian influenza A viruses are one of the natural hosts where such reassortment events could occur. Virological, histological and serological features of H5N1 virus infection in pigs were characterized in this study. Two- to three-week-old domestic piglets were intranasally inoculated with 10(6) EID(50) of A/Vietnam/1203/04 (VN/04), A/chicken/Indonesia/7/03 (Ck/Indo/03), A/Whooper swan/Mongolia/244/05 (WS/Mong/05), and A/Muscovy duck/Vietnam/ 209/05 (MDk/VN/05) viruses. Swine H3N2 and H1N1 viruses were studied as a positive control for swine influenza virus infection. The pathogenicity of the H5N1 HPAI viruses was also characterized in mouse and ferret animal models. Intranasal inoculation of pigs with H5N1 viruses or consumption of infected chicken meat did not result in severe disease. Mild weight loss was seen in pigs inoculated with WS/Mong/05, Ck/Indo/03 H5N1 and H1N1 swine influenza viruses. WS/Mong/05, Ck/Indo/03 and VN/04 viruses were detected in nasal swabs of inoculated pigs mainly on days 1 and 3. Titers of H5N1 viruses in nasal swabs were remarkably lower compared with those of swine influenza viruses. Replication of all four H5N1 viruses in pigs was restricted to the respiratory tract, mainly to the lungs. Titers of H5N1 viruses in the lungs were lower than those of swine viruses. WS/Mong/05 virus was isolated from trachea and tonsils, and MDk/VN/05 virus was isolated from nasal turbinate of infected pigs. Histological examination revealed mild to moderate bronchiolitis and multifocal alveolitis in the lungs of pigs infected with H5N1 viruses, while infection with swine influenza viruses resulted in severe tracheobronchitis and bronchointerstitial pneumonia. Pigs had low susceptibility to infection with H5N1 HPAI viruses. Inoculation of pigs with H5N1 viruses resulted in asymptomatic to mild symptomatic infection restricted to the respiratory tract and tonsils in contrast to mouse and ferrets animal models, where some of the viruses studied were highly pathogenic and replicated systemically.

Influenza (H5N1) viruses in poultry, Russian Federation, 2005-2006.

We studied 7 influenza (H5N1) viruses isolated from poultry in western Siberia and the European part of the Russian Federation during July 2005-February 2006. Full genome sequences showed high homology to Qinghai-like influenza (H5N1) viruses. Phylogenetic analysis not only showed a close genetic relationship between the H5N1 strains isolated from poultry and wild migratory waterfowls but also suggested genetic reassortment among the analyzed isolates. Analysis of deduced amino acid sequences of the M2 and neuraminidase proteins showed that all isolates are potentially sensitive to currently available antiviral drugs. Pathogenicity testing showed that all studied viruses were highly pathogenic in chickens; for 3 isolates tested in mice and 2 tested in ferrets, pathogenicity was heterogeneous. Pathogenicity in mammalian models was generally correlated with Lys at residue 627 of polymerase basic protein 2.

Inefficient transmission of H5N1 influenza viruses in a ferret contact model.

The abilities to infect and transmit efficiently among humans are essential for a novel influenza A virus to cause a pandemic. To evaluate the pandemic potential of widely disseminated H5N1 influenza viruses, a ferret contact model using experimental groups comprised of one inoculated ferret and two contact ferrets was used to study the transmissibility of four human H5N1 viruses isolated from 2003 to 2006. The effects of viral pathogenicity and receptor binding specificity (affinity to synthetic sialosaccharides with alpha2,3 or alpha2,6 linkages) on transmissibility were assessed. A/Vietnam/1203/04 and A/Vietnam/JP36-2/05 viruses, which possess "avian-like" alpha2,3-linked sialic acid (SA) receptor specificity, caused neurological symptoms and death in ferrets inoculated with 10(3) 50% tissue culture infectious doses. A/Hong Kong/213/03 and A/Turkey/65-596/06 viruses, which show binding affinity for "human-like" alpha2,6-linked SA receptors in addition to their affinity for alpha2,3-linked SA receptors, caused mild clinical symptoms and were not lethal to the ferrets. No transmission of A/Vietnam/1203/04 or A/Turkey/65-596/06 virus was detected. One contact ferret developed neutralizing antibodies to A/Hong Kong/213/03 but did not exhibit any clinical signs or detectable virus shedding. In two groups, one of two naïve contact ferrets had detectable virus after 6 to 8 days when housed together with the A/Vietnam/JP36-2/05 virus-inoculated ferrets. Infected contact ferrets showed severe clinical signs, although little or no virus was detected in nasal washes. This limited virus shedding explained the absence of secondary transmission from the infected contact ferret to the other naïve ferret that were housed together. Our results suggest that despite their receptor binding affinity, circulating H5N1 viruses retain molecular determinants that restrict their spread among mammalian species.

Cross-protectiveness and immunogenicity of influenza A/Duck/Singapore/3/97(H5) vaccines against infection with A/Vietnam/1203/04(H5N1) virus in ferrets.

Ferrets were immunized with two 7- mu g doses of hemagglutinin from inactivated whole-virus vaccines containing the hemagglutinin gene of A/Duck/Singapore/3/97(H5N3) then inoculated with a lethal dose of A/Vietnam/1203/04(H5N1) (Viet/1203/04). Serum samples did not react with Viet/1203/04 in hemagglutination-inhibition (HI) or virus-neutralization (VN) tests. All vaccinated ferrets survived the challenge, whereas all mock-immunized ferrets died. Immunized ferrets had significantly lower virus titers in the upper respiratory tract and less-severe disease. Vaccine generated from antigenically different H5 virus protects against infection by a highly pathogenic H5 strain. Neither HI nor VN testing provides correlates of cross-protection in ferrets.

Role of specific hemagglutinin amino acids in the immunogenicity and protection of H5N1 influenza virus vaccines.

If H5N1 influenza viruses become transmissible among humans, vaccination will offer the most effective option to limit their spread. Two human vaccine candidates recently generated by reverse genetics are based on antigenically different hemagglutinin (HA) glycoproteins derived from the A/HK/213/03 (H5N1) and A/Vietnam/1203/04 (H5N1) viruses. Their HA1 amino acid sequences differ at 10 positions, one of which (N154) introduces a potential glycosylation site in A/Vietnam/1203/04 (H5N1). To assess the impact of five amino acids in the putative antigenic sites on immunogenicity and immune protection, we generated a series of whole-virus vaccines that differed only in one or two HA amino acids. Sera from ferrets vaccinated with these inactivated preparations had high virus neutralization titers, but their hemagglutination inhibition (HI) titers were usually low. Interestingly, a recombinant virus in which the HA amino acid S223 (characteristic of 2004 viruses) was converted to N223 (as in A/HK/213/03) resulted in higher HI titers. This observation indicates that specific HA residues, such as N223, increase the sensitivity of the HI assay by altering receptor specificity and/or antibody-antigen binding. Ferrets vaccinated with mutant vaccine viruses were protected against lethal challenge with wild-type A/Vietnam/1203/04 virus. Our results suggest that inclusion of the N223 residue in the HA glycoproteins of diagnostic reference viruses may facilitate the evaluation of vaccine efficacy in humans.

Efficacy of H5 influenza vaccines produced by reverse genetics in a lethal mouse model.

We studied the efficacy, in mice, of 2 H5 influenza vaccine viruses produced by reverse genetics. Mice were immunized with inactivated viruses and then inoculated with a human H5N1 1997 or 2003 virus or an avian H5N1 2001 virus. Vaccine viruses that we tested raised high levels of hemagglutination-inhibiting (1:160-1:1280) and virus-neutralizing (1:900-1:1900) antibodies on day 21 after a single dose of vaccine and decreased or prevented virus replication in mouse lungs; 54.5%-100% of immunized mice survived, whereas all control mice died. Protection was achieved despite antigenic differences and incomplete matching of the vaccine strain and the challenge virus. Therefore, high levels of cross-protection are predicted in the mouse model.

Pathogenesis of Hong Kong H5N1 influenza virus NS gene reassortants in mice: the role of cytokines and B- and T-cell responses.

The severity of disease caused in humans by H5N1 influenza viruses remains unexplained. The NS gene of Hong Kong H5N1/97 viruses was shown to contribute to high pathogenicity of reassortants in a pig model. However, the molecular pathogenesis and host immune response underlying this phenomenon remain unclear. Here, in a mouse model, H1N1 A/Puerto Rico/8/34 (PR/8) reassortants that contained the H5N1/97 NS gene, the H5N1/01 NS gene, or an altered H5N1/97 NS gene encoding a Glu92-->Asp substitution in NS1 was studied. The pathogenicity of reassortant viruses, the induction of cytokines and chemokine CXCL1 (KC) in the lungs and specific B- and T-cell responses was characterized. In mice infected with reassortant virus containing the H5N1/97 NS gene, the mouse lethal dose (50%) and lung virus titres were similar to those of PR/8, which is highly pathogenic to mice. This reassortant virus required two more days than PR/8 to be cleared from the lungs of infected mice. Reassortants containing the altered H5N1/97 NS gene or the H5N1/01 NS gene demonstrated attenuated pathogenicity and lower lung titres in mice. Specific B- and T-cell responses were consistent with viral pathogenicity and did not explain the delayed clearance of the H5N1/97 NS reassortant. The reassortant induced elevated pulmonary concentrations of the inflammatory cytokines IL1alpha, IL1beta, IL6, IFN-gamma and chemokine KC, and decreased concentrations of the anti-inflammatory cytokine IL10. This cytokine imbalance is reminiscent of the clinical findings in two humans who died of H5N1/97 infection and may explain the unusual severity of the disease.