Hemagglutinin Vaccine Research Articles

Induction of cross‐protective immunity against influenza A virus H5N1 by an intranasal vaccine with extracts of mushroom mycelia

The identification of a safe and effective adjuvant that is able to enhance mucosal immune responses is necessary for the development of an efficient inactivated intranasal influenza vaccine. The present study demonstrated the effectiveness of extracts of mycelia derived from edible mushrooms as adjuvants for intranasal influenza vaccine. The adjuvant effect of extracts of mycelia was examined by intranasal co-administration of the extracts and inactivated A/PR8 (H1N1) influenza virus hemagglutinin (HA) vaccine in BALB/c mice. The inactivated vaccine in combination with mycelial extracts induced a high anti-A/PR8 HA-specific IgA and IgG response in nasal washings and serum, respectively. Virus-specific cytotoxic T-lymphocyte responses were also induced by administration of the vaccine with extract of mycelia, resulting in protection against lethal lung infection with influenza virus A/PR8. In addition, intranasal administration of NIBRG14 vaccine derived from the influenza A/Vietnam/1194/2004 (H5N1) virus strain administered in conjunction with mycelial extracts from Phellinus linteus conferred cross-protection against heterologous influenza A/Indonesia/6/2005 virus challenge in the nasal infection model. In addition, mycelial extracts induced proinflammatory cytokines and CD40 expression in bone marrow-derived dendritic cells. These results suggest that mycelial extract-adjuvanted vaccines can confer cross-protection against variant H5N1 influenza viruses. The use of extracts of mycelia derived from edible mushrooms is proposed as a new safe and effective mucosal adjuvant for use for nasal vaccination against influenza virus infection.

Comparative immunogenicity of recombinant influenza hemagglutinin (rHA) and trivalent inactivated vaccine (TIV) among persons ≥65 years old

Alternative substrates for influenza vaccine production are needed to ensure adequate supplies. We evaluated the relative safety and immunogenicity of recombinant hemagglutinin (rHA) or trivalent inactivated vaccine (TIV) among 869 ≥65-year-old subjects in a randomized clinical trial. Virologic surveillance for influenza-like illness (ILI) was conducted during the 2006–2007 epidemic. Vaccines were well tolerated. Seroconversion rates vs. influenza A/H1N1 and H3N2 antigens were superior in the rHA group, but were inferior vs. influenza B; however, results for influenza B are confounded since the vaccine antigens were different. ILI frequencies were low and similar in both groups. Studies assessing relative immunogenicity of vaccines using identical B Ags are warranted.

Rapid Development of an Efficacious Swine Vaccine for Novel H1N1

Recombinant hemagglutinin (HA) from a novel H1N1 influenza strain was produced using an alphavirus replicon expression system. The recombinant HA vaccine was produced more rapidly than traditional vaccines, and was evaluated as a swine vaccine candidate at different doses in a challenge model utilizing the homologous influenza A/California/04/2009 (H1N1) strain. Vaccinated animals showed significantly higher specific antibody response, reduced lung lesions and viral shedding, and higher average daily gain when compared to non-vaccinated control animals. These data demonstrate that the swine vaccine candidate was efficacious at all of the evaluated doses.

Preparation, characterization, and immunogenicity in mice of a recombinant influenza H5 hemagglutinin vaccine against the avian H5N1 A/Vietnam/1203/2004 influenza virus

Production of influenza vaccines requires a minimum of 6 months after the circulating strain is isolated and the use of infectious viruses. The hemagglutinin (protective antigen) of circulating influenza viruses mutates rapidly requiring reformulation of the vaccines. Our goal is to eliminate the risk of working with infectious virus and reduce significantly the production time. A cDNA fragment encoding the influenza virus A/Vietnam/1203/2004 (H5N1) HA gene was prepared using RT-PCR with viral RNA as a template. Recombinant HA (rHA) protein was produced in Escherichia coli and purified from isolated inclusion bodies by urea solubilization and Ni +-ion column chromatography. Vaccine candidates were prepared by treating the rHA with formalin, adsorption onto alum or with both. Mice were injected subcutaneously with candidate vaccines two or three times 2 weeks apart. Sera were collected 1 week after the last injection and antibody measured by ELISA and hemagglutination inhibition (HI). The highest antibody response (GM 449 EU) was elicited by three injections of 15 μg alum-adsorbed rHA. Dosages of 5 μg of rHA formulated with formalin and alum, and 5 μg alum-adsorbed rHA elicited IgG anti-HA of GM 212 and 177 EU, respectively. HI titers, ≥40 were obtained in ≥80% of mice with three doses of all formulations. We developed a method to produce rHA in a time-frame suitable for annual and pandemic influenza vaccination. Using this method, rHA vaccine can be produced in 3–4 weeks and when formulated with alum, induces HA antibody levels in young outbred mice consistent with the FDA guidelines for vaccines against epidemic and pandemic influenza.

Poly(γ-glutamic acid) nano-particles combined with mucosal influenza virus hemagglutinin vaccine protects against influenza virus infection in mice

Adding poly(γ-glutamic acid) nano-particles (γ-PGA-NPs), a safe, natural material, to subcutaneous immunization with influenza virus hemagglutinin (HA) vaccine increases the protective immune responses against influenza virus in mice. Here, we examined whether intranasal administration of the HA vaccine with γ-PGA-NPs would induce protection from influenza virus challenge in mice. Intranasal immunization with the mixture of γ-PGA-NPs and HA vaccine from an influenza virus strain A/PR/8/34 (H1N1) or A/New Caledonia/20/99 (H1N1) enhanced protection of mice from A/PR/8/34 infection. Intranasal immunization with A/New Caledonia/20/99 HA vaccine and γ-PGA-NPs induced cell-mediated immune responses and neutralizing antibody production for both A/New Caledonia/20/99 and A/PR/8/34. These data suggest that γ-PGA-NPs may have potential for clinical applications as a mucosal adjuvant.

Immune effects against influenza A virus and a novel DNA vaccine with co-expression of haemagglutinin- and neuraminidase-encoding genes

The high variability of influenza virus causes difficulties in the control and prevention of influenza, thus seeking a promising approach for dealing with these problems is a hot topic. Haemagglutinin (HA) and neuraminidase (NA) are major surface antigens of the influenza virus, and provide effective protection against lethal challenges with this virus. We constructed a DNA vaccine (pHA-IRES2-NA) that co-expressed both HA and NA, and compared its protective efficacy and immunogenic ability with that of singly expressed HA or NA, or a mixture of the two singly expressed proteins. Our findings showed that both HA and NA proteins expressed by pHA-IRES2-NA could be detected in vivo and in vitro. The protection of DNA vaccines was evaluated by serum antibody titres, residual lung virus titres and survival rates of the mice. In the murine model, immunization of pHA-IRES2-NA generated significant anti-HA and anti-NA antibody, increased the percentage of CD8(+) cells and gamma interferon-producing CD8(+) cells and the ratio of Th1/Th2 (T helper) cells, which was comparable to the effects of immunization with HA or NA DNA alone or with a mixture of HA and NA DNA. All the mice inoculated by pHA-IRES2-NA resisted the lethal challenge by homologous influenza virus and survived with low lung virus titre. In addition, previous studies reported that co-expression allowed higher-frequency transduction compared to co-transduction of separated vector systems encoding different genes. The novel HA and NA co-expression DNA vaccine is a successful alternative to using a mixture of purified HA and NA proteins or HA and NA DNA.

Evaluation of Vaccines for H5N1 Influenza Virus in Ferrets Reveals the Potential for Protective Single-Shot Immunization

As part of influenza pandemic preparedness, policy decisions need to be made about how best to utilize vaccines once they are manufactured. Since H5N1 avian influenza virus has the potential to initiate the next human pandemic, isolates of this subtype have been used for the production and testing of prepandemic vaccines. Clinical trials of such vaccines indicate that two injections of preparations containing adjuvant will be required to induce protective immunity. However, this is a working assumption based on classical serological measures only. Examined here are the dose of viral hemagglutinin (HA) and the number of inoculations required for two different H5N1 vaccines to achieve protection in ferrets after lethal H5N1 challenge. Ferrets inoculated twice with 30 microg of A/Vietnam/1194/2004 HA vaccine with AlPO4, or with doses as low as 3.8 microg of HA with Iscomatrix (ISCOMATRIX, referred to as Iscomatrix herein, is a registered trademark of CSL Limited) adjuvant, were completely protected against death and disease after H5N1 challenge, and the protection lasted at least 15 months. Cross-clade protection was also observed with both vaccines. Significantly, complete protection against death could be achieved with only a single inoculation of H5N1 vaccine containing as little as 15 microg of HA with AlPO4 or 3.8 microg of HA with Iscomatrix adjuvant. Ferrets vaccinated with the single-injection Iscomatrix vaccines showed fewer clinical manifestations of infection than those given AlPO4 vaccines and remained highly active. Our data provide the first indication that in the event of a future influenza pandemic, effective mass vaccination may be achievable with a low-dose "single-shot" vaccine and provide not only increased survival but also significant reduction in disease severity.

Evaluation of conserved and variable influenza antigens for immunization against different isolates of H5N1 viruses

The combination of rapid evolution and high mortality in human cases of infections has raised concerns that the H5N1 avian influenza virus may become a new, possibly severe, pandemic virus. Vaccination is likely to be the most efficient strategy to mitigate the impact of the next influenza pandemic. The present study evaluates B and T cell immune responses generated by the H5N1 viral antigens, hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), or the M2 ion channel in parallel, expressed from a DNA vaccine vehicle. Protection studies of immunized mice challenged with 100 LD50 of homologous or heterologous H5N1 viruses indicate that HA afforded better protection than the NA, NP or M2 DNA vaccines. The antibody response was also higher in HA-vaccinated mice as determined by hemagglutination inhibition (HI) and neutralizing antibodies (NAB) assays. Interestingly, the T cell response was higher against HA than against NA, NP or M2 and was detectable at low doses of the DNA–HA vaccine capable of inducing complete protection, despite the absence of a detectable B cell response. This study emphasizes the need to evaluate the relationship between both arms of the adaptive immune responses in regards to protective efficacy against influenza virus.

FluBlok, a next generation influenza vaccine manufactured in insect cells

FluBlok, a recombinant trivalent hemagglutinin (rHA) vaccine produced in insect cell culture using the baculovirus expression system, provides an attractive alternative to the current egg-based trivalent inactivated influenza vaccine (TIV). Its manufacturing process presents the possibility for safe and expeditious vaccine production. FluBlok contains three times more HA than TIV and does not contain egg-protein or preservatives. The high purity of the antigen enables administration at higher doses without a significant increase in side-effects in human subjects. The insect cell–baculovirus production technology is particularly suitable for influenza where annual adjustment of the vaccine is required. The baculovirus–insect expression system is generally considered a safe production system, with limited growth potential for adventitious agents. Still regulators question and challenge the safety of this novel cell substrate as FluBlok continues to advance toward product approval. This review provides an overview of cell substrate characterization for expresSF cell line used for the manufacturing of FluBlok. In addition, this review includes an update on the clinical development of FluBlok. The highly purified protein vaccine, administered at three times higher antigen content than TIV, is well tolerated and results in stronger immunogenicity, a long lasting immune response and provides cross-protection against drift influenza viruses.

FluBlok, a recombinant hemagglutinin influenza vaccine

FluBlok, a recombinant trivalent hemagglutinin (HA) vaccine produced in insect cell culture using the baculovirus expression system, provides an attractive alternative to the current egg-based trivalent inactivated influenza vaccine (TIV) manufacturing process. FluBlok contains three times more HA than TIV and does not contain egg-protein or preservatives. This review discusses the four main clinical studies that were used to support licensure of FluBlok under the 'Accelerated Approval' mechanism in the United States.

Protective immunity against influenza H5N1 virus challenge in mice by intranasal co-administration of baculovirus surface-displayed HA and recombinant CTB as an adjuvant

The increasing number of recent outbreaks of HPAI H5N1 in birds and humans brings out an urgent need to develop potent H5N1 vaccine regimens. Here we present a study on the intranasal vaccination of recombinant baculovirus surface-displayed hemagglutinin (BacHA) or inactivated whole H5N1 viral (IWV) vaccine with a recombinant cholera toxin B subunit (rCTB) as a mucosal adjuvant in a BALB/c mouse model. Two groups of mice were vaccinated with different doses (HA titer of log 2 4 or log 2 8) of either HA surface-displayed baculovirus or inactivated whole viral vaccine virus adjuvanted with different doses (2 μg or 10 μg) of rCTB. The vaccinations were repeated after 28 days. HA specific serum IgG and mucosal IgA antibodies were quantified by indirect ELISA, and serum neutralizing antibody titer were estimated by hemagglutination inhibition (HI) assay and virus neutralization titer assay. Functional protective efficacy of the vaccine was assessed by host challenge against HPAI H5N1 strains. The results revealed that mice co-administered with log 2 8 HA titer of BacHA vaccine and adjuvanted with 10 μg of rCTB had a significantly enhanced serum IgG and mucosal IgA immune response and serum microneutralization titer compared with mice administered with unadjuvanted log 2 4 or log 2 8 HA titer of BacHA alone. Also vaccination with 10 μg of rCTB and log 2 8 HA titer of BacHA elicited higher HA specific serum and mucosal antibody levels and serum HI titer than vaccination with log 2 8 HA titer of inactivated H5N1 virus adjuvanted with the same dose of rCTB. The host challenge study also showed that 10 μg rCTB combined with log 2 8 HA titer of BacHA provided 100% protection against 10MLD 50 of homologous and heterologous H5N1 strains. The study shows that the combination of rH5 HA expressed on baculovirus surface and rCTB mucosal adjuvant form an effective mucosal vaccine against H5N1 infection. This baculovirus surface-displayed vaccine is more efficacious than inactivated H5N1 influenza vaccine when administered by intranasal route and has no biosafety concerns associated with isolation, purification and production of the latter vaccine.

Multivalent HA DNA Vaccination Protects against Highly Pathogenic H5N1 Avian Influenza Infection in Chickens and Mice

BackgroundSustained outbreaks of highly pathogenic avian influenza (HPAI) H5N1 in avian species increase the risk of reassortment and adaptation to humans. The ability to contain its spread in chickens would reduce this threat and help maintain the capacity for egg-based vaccine production. While vaccines offer the potential to control avian disease, a major concern of current vaccines is their potency and inability to protect against evolving avian influenza viruses.Methodology / Principal FindingsThe ability of DNA vaccines encoding hemagglutinin (HA) proteins from different HPAI H5N1 serotypes was evaluated for its ability to elicit neutralizing antibodies and to protect against homologous and heterologous HPAI H5N1 strain challenge in mice and chickens after DNA immunization by needle and syringe or with a pressure injection device. These vaccines elicited antibodies that neutralized multiple strains of HPAI H5N1 when given in combinations containing up to 10 HAs. The response was dose-dependent, and breadth was determined by the choice of the influenza virus HA in the vaccine. Monovalent and trivalent HA vaccines were tested first in mice and conferred protection against lethal H5N1 A/Vietnam/1203/2004 challenge 68 weeks after vaccination. In chickens, protection was observed against heterologous strains of HPAI H5N1 after vaccination with a trivalent H5 serotype DNA vaccine with doses as low as 5 µg DNA given twice either by intramuscular needle injection or with a needle-free device.Conclusions/SignificanceDNA vaccines offer a generic approach to influenza virus immunization applicable to multiple animal species. In addition, the ability to substitute plasmids encoding different strains enables rapid adaptation of the vaccine to newly evolving field isolates.

Phase I and II randomised trials of the safety and immunogenicity of a prototype adjuvanted inactivated split-virus influenza A (H5N1) vaccine in healthy adults

Objective The primary objective was to evaluate the safety and immunogenicity of a prototype inactivated, split-virus H5N1 (avian influenza A) vaccine. A secondary objective was to assess the cross-reactivity of immune responses to two variant clade 2 H5N1 strains. Methods In two randomised, dose comparison, parallel assignment, multicentre trials conducted in Australia, healthy adult volunteers received two doses of 7.5 μg or 15 μg H5 haemagglutinin (HA) vaccine ± AlPO 4 adjuvant (phase I trial; N = 400) or two doses of 30 μg or 45 μg H5 HA with AlPO 4 adjuvant (phase II trial; N = 400). Revaccination with a booster dose was offered 6 months after dose 2 (phase I trial only). Main outcome measures were the change in immunogenicity at each follow-up visit from baseline, measured using HA inhibition (HI) and virus microneutralisation (MN) assays, and the frequency and nature of adverse events (AEs). Computer generated tables were used to randomly allocate treatments; participants and investigators were blinded to treatment allocation. Findings All formulations were well-tolerated; no unexpected serious adverse events were reported. Two doses of 30 μg or 45 μg H5 HA adjuvanted formulations elicited the highest immune responses, with considerable MN antibody (≥1:20) persistence up to 6 months post-vaccination. The 7.5 and 15 μg formulations (±adjuvant) were less immunogenic than the higher dose formulations; HI and MN antibody titres decreased to near pre-vaccination levels at 6 months but were restored to post-dose 2 levels after the booster dose. Immune responses in the phase I trial demonstrated modest levels of cross-protective MN antibodies against two currently circulating, distinct clade 2 H5N1 strains. Interpretation Two doses of prototype 30 μg or 45 μg aluminium-adjuvanted, clade 1 H5N1 vaccines were immunogenic and well-tolerated with considerable 6-month antibody persistence. The prototype H5N1 vaccine also elicited modest levels of cross-protective MN antibodies against variant clade 2 H5N1 strains [ClinicalTrials.gov identifiers: NCT00136331, NCT00320346; Funding: CSL Limited, Australia].

Baculovirus-Derived Hemagglutinin Vaccine Protects Chickens from Lethal Homologous Virus H5N1 Challenge

Since outbreaks of highly pathogenic avian influenza (HPAI) in both human and poultry from 2003, it is critical to have effective vaccines. A cDNA fragment coding the entire hemagglutinin (HA) gene derived from an H5N1 strain (A/duck/China/E319-2/03) was cloned and expressed using the baculovirus system. Two weeks after receiving two doses of recombinant HA (rHA) vaccines, chickens develop high antibody response for hemagglutination inhibition (HI) at titer 7.2 log(2). Challenge studies revealed that vaccinated chickens with HI titers greater than 3 log(2) could have immunoprotection against the same HPAI H5N1 strain virus challenge through intranasal route. Additionally, HI titer of 5 log(2) determined whether the live viruses could not be detected from oropharyngeal, cloacal discharge or in tissues. This result suggests that the rHA expressed from baculovirus system could be a candidate for the development of a safe and efficient subunit vaccine for HPAI (H5N1).

Influenza hemagglutinin vaccine with poly(γ-glutamic acid) nanoparticles enhances the protection against influenza virus infection through both humoral and cell-mediated immunity

Subcutaneous immunization with an influenza hemagglutinin (HA) vaccine can induce the production of virus-neutralizing antibodies, but not a cell-mediated immune response. Here we tested whether amphiphilic poly(gamma-glutamic acid)-graft- l-phenylalanine copolymers (gamma-PGA-NPs), which are derived from a bacterial capsular exopolymer produced by certain Bacillus natto strains, were an effective adjuvant for systemic influenza HA vaccination. Subcutaneous immunization with a mixture of HA vaccine and gamma-PGA-NPs induced higher mononuclear cell proliferation and the production of gamma-interferon (IFN-gamma), interleukin (IL)-4, and IL-6 upon HA restimulation, and enhanced not only anti-HA neutralizing antibody production but also the influenza virus-specific cell-mediated immune response, including CTL activity, compared with immunization with HA alone or a mixture of HA and aluminum adjuvant. HA vaccine with gamma-PGA-NPs protected mice against challenges with lethal doses of homologous influenza virus. The results indicate that adding gamma-PGA-NPs to the HA vaccine promotes effective protection and identifies gamma-PGA-NPs as a new, effective, and potent candidate adjuvant for a subcutaneous influenza virus vaccine.

New Approaches to Confronting an Imminent Influenza Pandemic

Scientists and health officials are concerned that an H5N1 influenza pandemic could be both imminent and catastrophic. Managing it will be difficult. Supplies of antiviral agents will be limited and expensive. Clinical development of adjuvant-combined, antigen-sparing, inactivated vaccines has been slow; the vaccines will take several months to produce and the global capacity to produce them will remain limited for several years. People who live in countries without vaccine companies - more than 85% of humankind - will have little prospect for being immunized. Thus, new approaches are needed to confront an imminent pandemic. The interventions must be scientifically promising and already licensed or near licensure. Moreover, the global industrial capacity to produce them must be large and already in place. Three interventions meet these criteria. Within a few months, several billion doses of live-attenuated H5N1 vaccines could be produced in existing egg-based or cell culture production facilities and several billion doses of an H5 recombinant hemagglutinin (rHA) vaccine could be produced in existing pharmaceutical bioreactors. In addition, generic medications such as statins might be able to moderate the aberrant innate immune response that characterizes human cases of H5N1 influenza. Statins would be affordable and available worldwide on the first day of the pandemic. Given the limitations of current efforts to develop and produce antivirals and conventional vaccines, urgent attention must be given these promising new approaches to pandemic control.

Incorporation of the TLR5-ligand flagellin in a recombinant Influenza A hemagglutinin vaccine provides enhanced immunogenicity and efficacy in vivo (47.2)

Abstract The linkage between innate and adaptive immunity is well established. The recognition of specific pathogen associated molecular patterns (PAMPs) by members of the Toll-like receptor (TLR) family plays a prominent role in the activation of adaptive immune responses. Previously, we have demonstrated that the linkage of specific PAMPs with defined antigens provides more potent and protective antigen-specific responses in the absence of adjuvants or complex formulations. Here we utilized this approach to develop a recombinant protein based influenza vaccine. The TLR5 ligand flagellin was linked to hemagglutinin (HA) to generate a single component vaccine. The recombinant E. coli expressed protein is recognized by influenza-convalescent antisera, indicating that the HA portion of the fusion protein is properly folded and presents conformational epitopes. In mice, the recombinant vaccine induces strong humoral and cellular responses that are quantitatively and qualitatively superior to those observed when the antigen is delivered with alum or co-delivered with flagellin unlinked. Analysis of serum antibody responses reveals strong reactivity with cells infected with influenza A and the ability to inhibit viral entry in vitro. Moreover, immunization with the fusion protein fully protects BALB/c mice from a lethal intranasal challenge with influenza A. These data demonstrate that linkage of the TLR5-ligand flagellin with HA provides significant promise as a non-egg based approach in the development of novel vaccines to combat seasonal and pandemic influenza.

Production of a novel influenza vaccine using insect cells: protection against drifted strains.

A recombinant trivalent hemagglutinin (HA) vaccine produced in cell culture using the baculovirus expression system provides an attractive alternative to the current egg-based influenza vaccine (Trivalent Inactivated Influenza Vaccine [TIV]) manufacturing process. The HA genes from the annual World Health Organization-recommended strains are cloned, expressed, and purified using a general purification process. Here, we provide an overview of the expression technology used to make the annual adjustment of the vaccine and the clinical studies completed to date with recombinant HA. The highly purified protein vaccine, administered at three times higher antigen content than TIV, results in stronger immunogenicity, a long-lasting immune response and provides cross-protection against drift variant influenza viruses. Furthermore, the vaccine does not contain egg proteins, adjuvants, preservatives, endotoxins, or antibiotics and can therefore be used in a broader population.

Dose‐Related Safety and Immunogenicity of a Trivalent Baculovirus‐Expressed Influenza‐Virus Hemagglutinin Vaccine in Elderly Adults

Influenza-virus hemagglutinin (HA) protein expressed in insect cells by recombinant baculovirus is a candidate influenza vaccine. In a randomized, double-blind trial conducted in 399 adults > or = 65 years of age, the efficacy of trivalent inactivated influenza vaccine (TIV) licensed for intramuscular injection was compared with that of trivalent baculovirus-expressed HA vaccine administered at doses of 15 microg, 45 microg, or 135 microg of each HA. Compared with TIV, baculovirus-expressed HA vaccine was safe and induced better serum antibody responses to the H3 component when administered at doses of 45 microg or 135 microg of each HA. Baculovirus-expressed HA is a safe and immunogenic influenza vaccine in elderly adults.

Modified Pulmonary Surfactant Is a Potent Adjuvant That Stimulates the Mucosal IgA Production in Response to the Influenza Virus Antigen

The intranasal administration of influenza hemagglutinin (HA) vaccine with Surfacten, a modified pulmonary surfactant free of antigenic c-type lectins, as a mucosal adjuvant induced the highest protective mucosal immunity in the airway. The intranasal immunization of mice with HA vaccine (0.2 microg)-Surfacten (0.2 microg) selectively induced the neutralizing anti-HA IgA, but not IgG, and conferred nearly maximal protection in the airway, without inducing a systemic response. In contrast, intranasal inoculation of vaccine with 0.2 microg of the potent mucosal adjuvant cholera toxin B* (CT-B*), prepared by adding 0.2% native CT to the B subunit of CT, induced both anti-HA IgA and IgG in the airway and in the serum. The intranasal administration of HA vaccine alone induced a limited amount of mucosal IgA against influenza virus. Although the s.c. administration of HA vaccine prominently induced serum IgG and IgA, Surfacten and CT-B* did not enhance their induction, and the concentrations of Abs leaking into the airways were insufficient to prevent viral multiplication. The intranasal administration of HA-Surfacten stimulated the expression of MHC class II, CD40, and CD86 molecules in the CD11c-positive cells isolated from the nasal mucosa, but not the expression of cells from the lungs or spleens. Lymphocytes isolated from the airway mucosa after intranasal HA-Surfacten immunization prominently induced TGF-beta1 which, compared with inoculation without Surfacten, promoted an Ag-specific mucosal IgA response. Surfacten alone, however, did not induce TGF-beta1. Our observations suggest that Surfacten, by mimicking the natural surfactant, is an effective mucosal adjuvant in the process of airway immunization.