HA Antigen Research Articles

Rac GTPase-activating Protein (Rac GAP) α1-Chimaerin Undergoes Proteasomal Degradation and Is Stabilized by Diacylglycerol Signaling in Neurons

α1-Chimaerin is a neuron-specific member of the Rho GTPase-activating protein family that selectively inactivates the small GTPase Rac. It is known to regulate the structure of dendrites and dendritic spines. We describe here that under basal conditions α1-chimaerin becomes polyubiquitinated and undergoes rapid proteasomal degradation. This degradation is partly dependent on the N-terminal region that is unique to this isoform. Mimicking diacylglycerol (DAG) signaling with a phorbol ester stabilizes endogenous α1-chimaerin against degradation and causes accumulation of the protein. The stabilization requires phorbol ester binding via the C1 domain of the protein and is independent of PKC activity. In addition, overexpression of a constitutively active Rac1 mutant is sufficient to cause an accumulation of α1-chimaerin through a phospholipase C-dependent mechanism, showing that endogenous DAG signaling can also stabilize the protein. These results suggest that signaling via DAG may regulate the abundance of α1-chimaerin under physiological conditions, providing a new model for understanding how its activity could be controlled.

Single dose of oil-adjuvanted inactivated vaccine protects chickens from lethal infections of highly pathogenic H5N1 influenza virus

The highly pathogenic H5N1 influenza viruses are endemic in poultry in many countries, but continuously infect humans and cause human mortality. H5N1 influenza viruses have been regarded as a pandemic candidate. In a pandemic event by this virus, the protection of poultry with an effective vaccine will help to greatly reduce the spread of this virus to humans since it easily infects poultry. Here we showed that immunization with one dose of oil-adjuvanted inactivated H5N1 vaccine could protect chickens from lethal infection by highly pathogenic H5N1 influenza virus until 12 weeks post-immunization. The complete protection of chickens depended on the amount of HA antigens in the vaccine. Complete homologous protection required over 1.25μg of HA antigens and complete heterologous protection required over 5.0μg of HA antigens. The bivalent H5N1 inactivated vaccine composed of 1.25μg of each antigen from clade 1 and clade 2.3.4 H5N1 influenza virus completely protected chickens from the lethal challenge of both viruses. When we determined the induction of antibody subtypes in tissues including nasal cavity, trachea, and lungs, the IgG subtype of antibody was induced more than the IgM or IgA subtype of antibody. Taken together, our results suggest that one dose of oil-adjuvanted inactivated H5N1 vaccine could provide chickens with sterile immunity against the homologous highly pathogenic H5N1 influenza virus.

Safety and immunogenicity of a recombinant hemagglutinin influenza–flagellin fusion vaccine (VAX125) in healthy young adults

Background The need for worldwide seasonal and pandemic vaccine production has increased interest in the development of innovative technologies for influenza vaccine production. We evaluated a novel influenza vaccine consisting of the globular head of the HA1 domain of the A/Solomon Islands/3/2006 (H1N1) influenza virus (VAX125) genetically fused to the TLR5 ligand, flagellin, and produced in E. coli. Methods 128 healthy adult subjects 18–49 years old were enrolled in a clinical trial conducted in three stages at a single center. Stage 1 was an open-label, dose escalation study in which the VAX125 vaccine was administered intramuscularly (im) at doses of 0.1 μg, 0.3 μg, 1 μg, 2 μg, 3 μg, 5 μg and 8 μg to groups of 8 subjects each. Stage 2 was a double-blind, placebo-controlled study in which subjects were randomized to receive 1.0 μg and 2.0 μg VAX125 vaccine doses or placebo, with 16 subjects per group. Finally, an additional 24 subjects received a 0.5 μg dose of VAX125 in stage 3, which was a non-randomized, open label study. In all parts subjects were followed for adverse events and sera was tested by hemagglutination-inhibition (HAI) and microneutralization (MN) against egg-grown virus on days 0, 7, 14, and 28. Serum C-reactive protein (CRP), cytokine levels, and anti-flagellin antibody were also assessed. Results Vaccine was generally well tolerated and there were no serious adverse events. Pain at the injection site was the most common local adverse event, and was mild or moderate in intensity. Systemic symptoms after vaccination include fatigue and headache, and two subjects, who received either 3 or 8 μg, had moderately severe systemic symptoms accompanied by substantial increases in serum CRP. Serum antibody responses against SI were seen by HAI and MN in most study subjects, with the geometric mean titer of post vaccination antibody increasing in a dose-dependent fashion. Overall, four-fold or greater serum HAI responses were seen in 61 of 96 (64%) subjects who received doses of 0.5 μg or greater, including in 46 of 72 subjects who received doses from 0.5 μg to 2 μg. Conclusions The globular head of the influenza HA expressed in a prokaryotic system was able to induce a functional antibody response against native virions. Vigorous responses were seen at relatively low doses of HA antigen suggesting that the addition of flagellin provided a substantial adjuvanting effect. The high levels of immune response at low doses of antigen and the relative ease of production associated with E. coli expression suggests that this approach may represent an effective strategy for enhancing the global influenza vaccine supply.

Immunoprotection against influenza H5N1 virus by oral administration of enteric-coated recombinant Lactococcus lactis mini-capsules

Edible vaccines that can be made widely available and easily administered could bring great benefit to the worldwide battle against pandemic viral infections. They can be used not only for the vaccination of humans and domesticated animals, but also for wild herds and live stock which are otherwise difficult to vaccinate. In this study, we report the development of an edible mini-capsule form of live, non-persisting, recombinant Lactococcus lactis (L. lactis) vaccine against the highly virulent influenza H5N1 strain. Recombinant L. lactis-based H5N1 HA antigen expression constructs were made and shown to be able to induce higher levels of HA-specific serum IgG and fecal IgA antibody production after oral administration. The vectors were then formulated into a mini-capsule dosage form and fed to mouse. Four doses of oral administration rendered complete protection of the mouse against lethal challenges of H5N1 virus.

Genetic content of Influenza H3N2 vaccine seeds

Influenza vaccine seeds produced in chicken eggs are selected through HA and NA surface glycoproteins antigenicity, as well as through high replicative ability. Here we characterize the genetic content of recently used thirteen H3N2 influenza vaccine seeds. Interestingly, sequence analysis of the vaccine seeds shows reassortment events leading to PR8:H3N2 segment constellations, ranging from the 6:2 to 2:6 constellations. This study shows that the H3N2 PB1 is the most frequent internal segment incorporated in the tested vaccines seeds.

CD8α+ DC are not the sole subset cross‐presenting cell‐associated tumor antigens from a solid tumor

One of the clear paradoxes in tumor immunology is the fact that cross-presentation of cell-associated tumor antigens to CD8(+) T cells is efficient, yet CTL generation is weak, and tumors continue to grow. We examined, for the first time whether this may be due to alterations in the phenotype or function of cross-presenting DC using a solid tumor model expressing a membrane bound neo-antigen (hemagglutinin, HA). Tumor antigen was constitutively cross-presented in the tumor-draining LN throughout tumor progression by CD11c(+) DC. Further analysis revealed that both CD8alpha(+) and CD8alpha(-) DC subsets, but not plasmacytoid DC, were effective at cross-presenting HA tumor antigen. The proportions of DC subsets in the tumor-draining LN were equivalent to those seen in the LN of naïve mice; however, a significant increase in the expression of the potential inhibitory B7 molecule, B7-DC, was noted and appeared to be restricted to the CD8alpha(-) DC subset. Therefore LN resident CD8alpha(+) DC are not the sole DC subset capable of cross-presenting cell-associated tumor antigens. Migratory tumor DC subsets with altered co-stimulatory receptor expression may contribute to induction and regulation of tumor-specific responses.

Mucosal vaccination with a codon-optimized hemagglutinin gene expressed by attenuated Salmonella elicits a protective immune response in chickens against highly pathogenic avian influenza

The purpose of this study was to evaluate clinical protection from challenge conferred by two attenuated Salmonella enteria serovar typhimurium vaccine strains expressing the hemagglutinin (HA1) gene from a highly pathogenic avian influenza (HPAI) H5N1 (A/whooper swan/Mongolia/3/2005), under control of the anaerobically inducible nir15 promoter. Two-week-old White Leghorn chickens were immunized by oral gavage with one milliliter doses of >109 Salmonella colony-forming units once weekly for 4 weeks prior to challenge. Expression of recombinant protein was confirmed via Western blot. Serum and mucosal gavage samples were collected prior to, and following immunization and antibodies against avian influenza HA were confirmed by Western blot and hemagglutination-inhibition (HI) assay. Chickens were challenged with homologous (A/whooper swan/Mongolia/3/2005), or heterologous (A/Chicken/Queretaro/14588-19/95) HPAI virus strains. Chickens immunized with attenuated Salmonella strains containing plasmid expression vector (pTETnir15HA) demonstrated a statistically significant increase in survival compared to control groups. Results provide evidence of effectiveness of attenuated Salmonella strains for delivery of recombinant avian influenza HA antigens and induction of mucosal and systemic immune responses protective against lethal challenge with HPAI.

Estimation of the Effective Doses of Nasal-Inactivated Influenza Vaccine in Humans from Mouse-Model Experiments

Mouse models of influenza play an important role in developing effective human influenza vaccines. We have demonstrated that intranasal immunization with inactivated subvirion (SV) vaccines, in conjunction with a cholera toxin B subunit adjuvant (CTB*), provides more effective cross-protection than parenteral immunization in BALB/c mice. In addition, the minimal effective dose of nasal vaccine for providing complete protection against a lethal influenza virus infection is 0.1 microg SV vaccine (containing about 30 ng hemagglutinin [HA]) (with 0.1 microg CTB*) in BALB/c mice (20 g body weight) immunized twice intranasally 4 weeks apart. The effective dose in humans can be estimated to be roughly 100 microg SV vaccine (containing approximately 30 microg HA) (with 100 microg CTB*)/20 kg body weight by using the dose/body weight ratio from actual vaccination trials. This estimation can be rationalized by the hypothesis that the distribution of strains (or individuals) in a mouse (or human) population, in relation to their HA antigen responsiveness, follows a normal distribution, and by the fact that BALB/c mice are intermediate responders for anti-HA antibody responses, and correspond to human intermediate responders, which form the largest population. We also discuss the use of innate immune responses, as well as antibody responses, in BALB/c mice to assess the efficacy of unknown adjuvants and the development of other adjuvants for nasal vaccines that should be clinically safer than CTB*.

Particle-mediated DNA vaccines against seasonal and pandemic influenza viruses elicit strong mucosal antibody and T cell responses in the lung

The recent outbreaks of H5N1 avian and H1N1 swine-origin influenza viruses highlight the urgent need for vaccine strategies able to confer broad host immunity against both seasonal and pandemic strains of influenza. As the lung mucosa is the first barrier against pulmonary influenza infection, an effective influenza vaccine strategy should elicit anti-viral immune responses in the respiratory tract where they may afford better protection. As a safe alternative to live attenuated vaccines, we evaluated the immunogenicity of gene gun-based immunization using a tetravalent DNA vaccine encoding the HA genes from three seasonal flu strains (H1, H3, and B) and a H5N1 pandemic strain. Vaccinated mice were found to exhibit high titers of serum antibodies specific for each of the encoded HA antigens, as well as cross-reactivity to drifted strains of virus. Importantly, significant levels of HA-specific mucosal antibodies were also detected in the lung airways of vaccinated mice. Consistent with the belief that immune responses against conserved regions of the virus may provide cross-protection against divergent strains of influenza, we also evaluated the immunogenicity of particle-mediated DNA vaccines encoding the highly conserved viral nucleoprotein (NP). Mice immunized with a DNA vaccine encoding a consensus NP sequence derived from circulating strains of virus exhibited robust NP-specific CD8 T cell responses in both spleen and lung tissue. Together, these results demonstrate the ability of skindelivered particle-mediated DNA vaccines to elicit strong systemic and mucosal immune responses against both variable and conserved influenza antigens and support the concept that particle-mediated DNA vaccines may offer an effective strategy to improve protection against seasonal and pandemic influenza.

Construction and cellular immune response induction of HA-based alphavirus replicon vaccines against human-avian influenza (H5N1)

Several approaches are being taken worldwide to develop vaccines against H5N1 viruses; most of them, however, pose both practical and immunological challenges. One potential strategy for improving the immunogenicity of vaccines involves the use of alphavirus replicons and VP22, a herpes simplex type 1 (HSV-1) protein. In this study, we analysed the antigenic peptides and homogeneity of the HA sequences (human isolates of the H5N1 subtype, from 1997 to 2003) and explored a novel alphavirus replicon system of VP22 fused with HA, to assess whether the immunogenicity of an HA-based replicon vaccine could be induced and augmented via fusion with VP22. Further, replicon particles expressing VP22, and enhanced green fluorescent protein (EGFP) were individually used as controls. Cellular immune responses in mice immunised with replicons were evaluated by identifying specific intracellular cytokine production with flow cytometry (FCM). Animal-based experimentation indicated that both the IL-4 expression of CD4 + T cells and the IFN-γ expression of CD8 + T cells were significantly increased in mice immunised with VPR-HA and VPR-VP22/HA. A dose titration effect vis-à-vis both IL-4 expression and IFN-γ expression were observed in VPR-HA- and VPR-VP22/HA-vaccinated mice. Our results revealed that both VPR-VP22/HA and VPR-HA replicon particles presented a promising approach for developing vaccines against human-avian influenza, and VP22 could enhance the immunogenicity of the HA antigens to which it is fused.

Tumor antigen cross-presentation by tumor dendritic cells is blocked at a post-processing stage but can be reversed by selected pro-apoptotic agents (88.23)

Abstract Cross presentation defines the unique capacity of an antigen presenting cell (APC) to present exogenous antigen via MHC class I molecules to CD8+ T cells. This process serves a critical role in host anti-tumor immunity. Dendritic cells (DC), specifically the CD8α+ subset, are specialised cross-presenting cells; however there is little understanding of how different DC subsets contribute to anti-tumour immunity. In this study we have examined the cross-presentation of a marker tumour antigen (transfected HA) expressed by the murine AB1-HA cell line. We have found that tumor antigen is constitutively cross-presented in the draining lymph node throughout disease progression and that both CD8α+ and CD8α - DC subsets are responsible for the observed cross-presentation of HA antigen to CD8+ T cells. Interestingly, tumor-infiltrating DCs fail to cross present, though they are not deficient in tumor antigen uptake or processing. Importantly, a tumor apoptosis-inducing false nucleotide agent, gemcitabine, which primes for anti-tumor immunity, also reverses the defect in antigen cross presentation of tumor DCs. These data suggest that lymph node resident CD8α+ DC are not the sole DC subset capable of cross-priming against tumor antigens and that local cross-presentation within tumors ie. TCR restimulation of tumor-infiltrating CD8 T-cells may be essential for effective anti-tumor immunity. These data have important implications for tumor immunotherapy particularly the use of immunotherapy in conjunction with tumor apoptosis-inducing therapy.

A recombinant vesicular stomatitis virus replicon vaccine protects chickens from highly pathogenic avian influenza virus (H7N1)

Highly pathogenic avian influenza viruses (HPAIV) of subtypes H5 and H7 cause fatal disease in poultry (fowl plague) but also have zoonotic potential. Currently commercially available vaccines often do not provide sufficient protection and do not allow easy discrimination between vaccinated and infected birds. Therefore, vaccination of domestic poultry against H5 and H7 HPAIV is not allowed in many countries, or is only possible after special permission has been provided. We generated a recombinant marker vaccine based on non-transmissible vesicular stomatitis virus (VSV) expressing the HA antigen of HPAIV A/FPV/Rostock/34 (H7N1) in place of the VSV G gene. This virus, VSV*ΔG(HA), was propagated on a helper cell line providing VSV G in trans. Since no progeny virus was produced after infection of non-complementing cells, the vector was classified as biosafety level 1 organism (“safe”). Chickens were immunized via the intramuscular route. Following booster vaccination with the same replicons high titers of serum antibodies were induced, which neutralized avian influenza viruses of subtypes H7N1 and H7N7 but not H5N2. Vaccinated chickens were protected against a lethal dose of heterologous HPAIV A/chicken/Italy/445/99 (H7N1). Secretion of challenge virus was short-term and significantly reduced. Finally, it was possible to discriminate vaccinated chickens from infected ones by a simple ELISA assay. We propose that VSV replicons have the potential to be developed to high-quality vaccines for protection of poultry against different subtypes of avian influenza viruses.

Recombinant Influenza B virus HA and NA antigens administered in equivalent amounts are immunogenically equivalent and induce equivalent homotypic and broader heterovariant protection in mice than conventional and live influenza vaccines

Influenza B virus is an important cause of acute upper respiratory disease in humans. Vaccination is the primary method of control of influenza related disease, yet vaccine methodology and production technology have not changed in over 40 years. In this study, we compare the efficacy of recombinant baculovirus produced protein based neuraminidase containing influenza B vaccines with conventional inactivated influenza vaccine (CIV) and live-attenuated influenza vaccine (LAIV) in a murine model. All HA containing vaccines stimulated antibody and protected against an infectious challenge with homotypic virus (B/Harbin/7/94), only recombinant protein based (rHA+rNA and rNA) vaccines containing immunogenic amounts of influenza neuraminidase (NA) protected against challenge with a significantly antigenically different heterovariant virus (B/Beijing/243/1997), as measured by a reduction in mean pulmonary virus titers. This report demonstrates with influenza B virus, in a side-by-side comparison with CIV and LAIV in a murine model system the superiority of vaccines containing immunogenic NA over currently approved CIV and LAIV vaccines.

Increased sensitivity for detecting avian influenza-specific antibodies by a modified hemagglutination inhibition assay using horse erythrocytes

The hemagglutination inhibition (HI) assay is a widely used serological method to measure the levels of protective antibody responses against influenza viruses. However, the traditional HI assay which uses chicken erythrocytes is not sufficiently sensitive for detecting HI antibodies specific to avian influenza viruses. Previously, it was demonstrated that employing an assay using horse erythrocytes was able to increase the sensitivity of HI assay. The current report describes further optimization of this modified HI assay. It was shown that this method was able to increase detection of HI activities in rabbit sera immunized with H5 HA antigens, and proved that this increased sensitivity is useful in dissecting the strain specificity of HI antibody responses. In addition, the modified HI assay using horse erythrocytes increased the sensitivity of detecting HI antibodies specific for three major serotypes of avian influenza viruses, H5, H7 and H9, in people who may have asymptomatic infection with avian influenza viruses. Based on these results, the optimized use of horse erythrocytes should be standard practice for detecting HI activities against avian influenza viruses.

Generation of Influenza HA-Specific Lymphocytes from Autologous Umbilical Cord Blood-Derived Dendritic Cells.

INTRODUCTION: Umbilical cord blood transplantation (CBT) is being performed with increasing frequency for patients lacking HLA-matched donors. CBT is associated with delayed immune reconstitution and high rates of viral infections. Particularly problematic is the reactivation of latent viral infections such as CMV, EBV, and VZV, as is overt infection from common agents like adenovirus and influenza. To address these issues, we proposed to generate influenza-specific CB T-cells ex vivo for potential cell therapy applications, allowing for effective therapeutic interventions until such time that immune reconstitution has occurred.METHODS: Dendritic cells (DC) were generated from both the adherent and the CD14+ CB fractions cultured in AIM-V medium supplemented with human serum, GM-CSF, and IL-4. Antigen loading was performed by incubation with recombinant, purified A/New Caledonia HA antigen. Maturation was performed with the ITIP inflammatory cocktail (IL-1β, TNF-α, IL-6, and PGE2). Autologous CB lymphocytes were co-cultured with mature DC at a ratio of 10:1 for 4 days in IL-12 after which they were expanded in a cytokine cocktail of IL-2, IL-7, IL-12, and IL-15. Lymphocytes were restimulated similarly every 7-10 days. Antigen specificity was demonstrated by IFN-γ ELISpot.RESULTS: Mature DC expressed the appropriate markers including CD80, CD83, CD86, CD209, and HLA-DR. Some DC expressed CD14, others CD11c, an indication of a mixed interstitial/Langerhans myeloid phenotype. We demonstrated a 4–10 fold enhancement of IFN-γ ELISpots when primed lymphocytes were incubated with HA-loaded DC in comparison to autologous unloaded DC (p<0.003) or loaded allogeneic DC (p<0.0001). The CD8 content of the HA-specific lines was typically greater than 50% after two stimulations. At the time of phenotyping CD3+ cells were primarily CD62LposCD197(CCR7)neg and CD127(IL-7Rα)neg, an effector phenotype; however, 6–22% of cells expressed CD127 (effector or central memory), and up to 6% of cells were CD62LposCD197pos (central memory).CONCLUSIONS: The results demonstrate that, despite the generally naïve nature of CB lymphocytes, influenza-specific responses can be generated ex vivo, and could potentially be used clinically in CBT patients who develop infectious complications prior to immune reconstitution.

Myoferlin Regulates Vascular Endothelial Growth Factor Receptor-2 Stability and Function

Myoferlin and dysferlin are members of the ferlin family of membrane proteins. Recent studies have shown that mutation or genetic disruption of myoferlin or dysferlin promotes muscular dystrophy-related phenotypes in mice, which are the result of impaired plasma membrane integrity. However, no biological functions have been ascribed to myoferlin in non-muscle tissues. Herein, using a proteomic analysis of endothelial cell (EC) caveolae/lipid raft microdomains we identified myoferlin in these domains and show that myoferlin is highly expressed in ECs and vascular tissues. The loss of myoferlin results in lack of proliferation, migration, and nitric oxide (NO) release in response to vascular endothelial growth factor (VEGF). Western blotting and surface biotinylation experiments show that loss of myoferlin reduces the expression level and autophosphorylation of VEGF receptor-2 (VEGFR-2) in native ECs. In a reconstituted cell system, transfection of myoferlin increases VEGFR-2 membrane expression and autophosphorylation in response to VEGF. In vivo, VEGFR-2 levels and VEGF-induced permeability are impaired in myoferlin-deficient mice. Mechanistically, myoferlin forms a complex with dynamin-2 and VEGFR-2, which prevents CBL-dependent VEGFR-2 polyubiquitination and proteasomal degradation. These data are the first to report novel biological activities for myoferlin and reveal the role of membrane integrity to VEGF signaling.

Development of an Immunochromatographic Kit for Rapid Diagnosis of H5 Avian Influenza Virus Infection

Highly pathogenic avian influenza (HPAI) caused by the H5N1 subtype has given rise to serious damage in poultry industries in Asia. The virus has expanded its geographical range to Europe and Africa, posing a great risk to human health as well. For the control of avian influenza, a rapid diagnosis by detecting the causative virus and identifying its subtype is essential. In the present study, a rapid diagnosis kit combining immunochromatography with enzyme immunoassay which detects the H5 HA antigen of influenza A virus was developed using newly established anti-H5 HA monoclonal antibodies. The present kit specifically detected all of the H5 influenza viruses tested, and did not react with the other HA subtypes. H5 HA antigens were detected from swabs and tissue homogenates of chickens infected with HPAI virus strain A/chicken/Yamaguchi/7/04 (H5N1) from 2 days post inoculation. The kit showed enough sensitivity and specificity for the rapid diagnosis of HPAI.

LTC Experts Take Panic Out of Pandemic

LTC Experts Take Panic Out of Pandemic

Toll-Like Receptor Adaptor Molecules Enhance DNA-Raised Adaptive Immune Responses against Influenza and Tumors through Activation of Innate Immunity

Toll-like receptors (TLRs) recognize microbial components and trigger the signaling cascade that activates the innate and adaptive immunity. TLR adaptor molecules play a central role in this cascade; thus, we hypothesized that overexpression of TLR adaptor molecules could mimic infection without any microbial components. Dual-promoter plasmids that carry an antigen and a TLR adaptor molecule such as the Toll-interleukin-1 receptor domain-containing adaptor-inducing beta interferon (TRIF) or myeloid differentiation factor 88 (MyD88) were constructed and administered to mice to determine if these molecules can act as an adjuvant. A DNA vaccine incorporated with the MyD88 genetic adjuvant enhanced antigen-specific humoral immune responses, whereas that with the TRIF genetic adjuvant enhanced cellular immune responses. Incorporating the TRIF genetic adjuvant in a DNA vaccine targeting the influenza HA antigen or the tumor-associated antigen E7 conferred superior protection. These results indicate that TLR adaptor molecules can bridge innate and adaptive immunity and potentiate the effects of DNA vaccines against virus infection and tumors.

Safety and immunogenicity of a Proteosome™-trivalent inactivated influenza vaccine, given nasally to healthy adults

We studied the safety and immunogenicity of a nasally administered vaccine comprising three monovalent inactivated influenza antigens (A/New Caledonia/20/99 (H1N1), A/Panama/2007/99 (H3N2), and B/Guangdong/120/2000) non-covalently associated with outer membrane proteins of Neisseria meningitidis (Proteosome™) in normal, healthy adults. In a randomized, double-blind trial participants ( n = 78) were allocated to placebo or a single nasal dose of vaccine containing 15, 30, or 45 μg of each of the three HA antigens, or two nasal doses containing 30 μg of each HA, separated by 2 weeks. The vaccine was generally well tolerated in all doses tested, and in a one or two-dose schedule. A shallow vaccine reactogenicity dose-response was seen. The most common local reaction was nasal congestion, which occurred in up to 48.3% of vaccine recipients in days 0–6 after vaccine but was mild and self-limiting; this reaction was not significantly more common among active vaccine recipients than placebo recipients. Mild to moderate headache was the most commonly reported systemic reactogenicity complaint in all treatment groups, and was the only solicited complaint to increase significantly in frequency after a second active dose. No severe systemic reactions occurred. A positive and statistically significant antibody response was observed, in serum and in nasal secretions, to increasing dose for all three antigens. Serum HAI titre responses and nasal secretory IgA immune responses were elicited against all three antigens. Further testing of this nasal influenza vaccine is warranted to determine its safety and immunogenicity in these populations and its efficacy in the prevention of clinical illness.