Heterologous prime-boost immunization with live SPY1 and DnaJ protein of <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis> induces strong Th1 and Th17 cellular immune responses in mice

Streptococcus pneumoniae is a leading cause of infectious diseases in children under 5-year-old. Vaccine has been used as an indispensable strategy to prevent S. pneumoniae infection for more than 30 years. Our previous studies confirmed that mucosal immunization with live attenuated strain SPY1 can protect mice against nasopharyngeal colonization of S. pneumoniae and lethal pneumococcal infection, and the protective effects are comparable with those induced by commercially available 23-valent polysaccharide vaccine. However, live attenuated vaccine SPY1 needs four inoculations to get satisfactory protective effect, which may increase the risk of virulence recovery. It is reported that heterologous primeboost approach is more effective than homologous primeboost approach. In the present study, to decrease the doses of live SPY1 and improve the safety of SPY1 vaccine, we immunized mice with SPY1 and DnaJ protein alternately. Our results showed that heterologous prime-boost immunization with SPY1 and DnaJ protein could significantly reduce the colonization of S. pneumoniae in the respiratory tract of mice, and induce stronger Th1 and Th17 cellular immune responses than SPY1 alone. These results indicate heterologous prime-boost immunization method not only elicits better protective effect than SPY1 alone, but also reduces the doses of live SPY1 and decreases the risk of SPY1 vaccine. This work is the first time to study the protective efficiency with two different forms of S. pneumoniae candidate vaccine, and provides a new strategy for the development of S. pneumoniae vaccine.     

Toxoplasma gondii: Protective effect of an intranasal SAG1 and MIC4 DNA vaccine in mice

Infections by the intracellular protozoan parasite Toxoplasma gondii are widely prevalent in humans and other animals which can cause severe or lethal toxoplasmosis. So the development of a more effective vaccine is needed urgently. A multiantigenic vaccine against toxoplasmosis was constructed in the present study, which contains two T. gondii antigens, SAG1 and MIC4 on the basis of previous immunological and immunization studies. The eukaryotic plasmid pcDNA3.1-SAG1-MIC4, pcDNA3.1-SAG1, pcDNA3.1-MIC4 were constructed first, which can express surface protein SAG1 and microneme protein MIC4 from different stages of T. gondii life cycle, and the expression ability of these DNA vaccine in HeLa cells were examined by Western blot. The efficacy of these plasmids with or without co-administration of a plasmid encoding cholera toxin A2/B as a genetic adjuvant by mucosal way to protect BALB/c mice against toxoplasmosis was evaluated. We found these vaccines were able to elicit a significant humoral and cellular immune response in vaccinated mice and they can increase survival rate and prolong the life of mice that were infected by T. gondii especially in the pcDNA3.1-SAG1-MIC4 group. Co-delivery of cholera toxin A2/B further enhanced the potency of multiantigenic DNA vaccine by intranasal route. These results encourage further research towards achieving vaccinal protection against the T. gondii in animals and humans.     

DNA vaccines based on genetically detoxified derivatives of pneumolysin fail to protect mice against challenge with Streptococcus pneumoniae

The 7-valent polysaccharide conjugate vaccine currently administered against Streptococcus pneumoniae has been shown to be highly effective in high risk-groups, but its use in developing countries will probably not be possible due to high costs. The use of conserved protein antigens using the genetic vaccination strategy is an interesting alternative for the development of a cost-effective vaccine. We have analyzed the potential of DNA vaccines expressing genetically detoxified derivatives of pneumolysin (pneumolysoids) against pneumococcal infections, and compared this with immunization using recombinant protein. The purified recombinant pneumolysoid with the highest residual cytolytic activity was able to confer partial protection against a lethal intraperitoneal challenge, with the induction of high antibody levels. Immunization with DNA vaccines expressing pneumolysoids, on the other hand, induced a significantly lower antibody response and no protection was observed.     

Gamma-irradiation of <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis> for the use as an immunogenic whole cell vaccine

Streptococcus pneumoniae is a major respiratory pathogen that causes millions of deaths worldwide. Although subunit vaccines formulated with the capsular polysaccharides or their protein conjugates are currently-available, low-cost vaccines with wide serotype coverage still remain to be developed, especially for developing countries. Recently, gamma- irradiation has been considered as an effective inactivation method to prepare S. pneumoniae vaccine candidate. In this study, we investigated the immunogenicity and protective immunity of gamma-irradiated S. pneumoniae (r-SP), by comparing with heat-inactivated S. pneumoniae (h-SP) and formalin-inactivated S. pneumoniae (f-SP), both of which were made by traditional inactivation methods. Intranasal immunization of C57BL/6 mice with r-SP in combination with cholera toxin as an adjuvant enhanced S. pneumoniaespecific antibodies on the airway mucosal surface and in sera more potently than that with h-SP or f-SP under the same conditions. In addition, sera from mice immunized with r-SP potently induced opsonophagocytic killing activity more effectively than those of h-SP or f-SP, implying that r-SP could induce protective antibodies. Above all, immunization with r-SP effectively protected mice against S. pneumoniae infection. Collectively, these results suggest that gamma- irradiation is an effective method for the development of a killed whole cell pneumococcal vaccine that elicits robust mucosal and systemic immune responses.     

Tamarin model of pneumococcal bacteremia

Tamarins (Saguinus labiatus) were utilized to study host defenses against pneumococcal bacteremia. Tamarins had a poor antibody response to immunization with varying doses of pneumococcal capsular polysaccharide (PCP) vaccine (2 of 15 positive) or to infection with serotype 7F Streptococcus pneumoniae (2 of 14 positive). Tamarins were protected against challenge with a lethal dose of serotype 7F S. pneumoniae if the bacteria were preopsonized with human immune globulin intravenous or if the tamarins were injected with the immune globulin 30 min before challenge. There was minimal protection utilizing a mouse monoclonal anti-type 7F PCP antibody.     

Immunization with a MOMP-Based Vaccine Protects Mice against a Pulmonary Chlamydia Challenge and Identifies a Disconnection between Infection and Pathology

Chlamydia pneumoniae is responsible for up to 20% of community acquired pneumonia and can exacerbate chronic inflammatory diseases. As the majority of infections are either mild or asymptomatic, a vaccine is recognized to have the greatest potential to reduce infection and disease prevalence. Using the C. muridarum mouse model of infection, we immunized animals via the intranasal (IN), sublingual (SL) or transcutaneous (TC) routes, with recombinant chlamydial major outer membrane protein (MOMP) combined with adjuvants CTA1-DD or a combination of cholera toxin/CpG-oligodeoxynucleotide (CT/CpG). Vaccinated animals were challenged IN with C. muridarum and protection against infection and pathology was assessed. SL and TC immunization with MOMP and CT/CpG was the most protective, significantly reducing chlamydial burden in the lungs and preventing weight loss, which was similar to the protection induced by a previous live infection. Unlike a previous infection however, these vaccinations also provided almost complete protection against fibrotic scarring in the lungs. Protection against infection was associated with antigen-specific production of IFNγ, TNFα and IL-17 by splenocytes, however, protection against both infection and pathology required the induction of a similar pro-inflammatory response in the respiratory tract draining lymph nodes. Interestingly, we also identified two contrasting vaccinations capable of preventing infection or pathology individually. Animals IN immunized with MOMP and either adjuvant were protected from infection, but not the pathology. Conversely, animals TC immunized with MOMP and CTA1-DD were protected from pathology, even though the chlamydial burden in this group was equivalent to the unimmunized controls. This suggests that the development of pathology following an IN infection of vaccinated animals was independent of bacterial load and may have been driven instead by the adaptive immune response generated following immunization. This identifies a disconnection between the control of infection and the development of pathology, which may influence the design of future vaccines.     

Identification of glycosylated regions in pneumococcal PspA conjugated to serotype 6B capsular polysaccharide

Conjugate vaccines are being widely used since their introduction. Nowadays the interest in these vaccines is still growing and new antigens and conjugate chemistry are being studied and developed. Pneumococcal surface protein A (PspA) is one of the most studied pneumococcal antigens and is an important vaccine candidate. One approach to broaden the conjugate vaccine coverage could be the conjugation of the polysaccharide to a pneumococcal protein such as PspA. Previous results have shown that conjugated recombinant fragment of PspA (rPspA) not only maintained but also in some conjugates improved the induction of protective antibodies raised against the protein carrier. We describe here a characterization study to identify the domains of Streptococcus pneumoniae recombinant PspA (rPspA), from family 1 clade 1 and family 2 clade 3, involved in the conjugation with serotype 6B capsular polysaccharide.     

Interleukin-17A mediates acquired immunity to pneumococcal colonization

Although anticapsular antibodies confer serotype-specific immunity to pneumococci, children increase their ability to clear colonization before these antibodies appear, suggesting involvement of other mechanisms. We previously reported that intranasal immunization of mice with pneumococci confers CD4+ T cell-dependent, antibody- and serotype-independent protection against colonization. Here we show that this immunity, rather than preventing initiation of carriage, accelerates clearance over several days, accompanied by neutrophilic infiltration of the nasopharyngeal mucosa. Adoptive transfer of immune CD4+ T cells was sufficient to confer immunity to na?ve RAG1(-/-) mice. A critical role of interleukin (IL)-17A was demonstrated: mice lacking interferon-gamma or IL-4 were protected, but not mice lacking IL-17A receptor or mice with neutrophil depletion. In vitro expression of IL-17A in response to pneumococci was assayed: lymphoid tissue from vaccinated mice expressed significantly more IL-17A than controls, and IL-17A expression from peripheral blood samples from immunized mice predicted protection in vivo. IL-17A was elicited by pneumococcal stimulation of tonsillar cells of children or adult blood but not cord blood. IL-17A increased pneumococcal killing by human neutrophils both in the absence and in the presence of antibodies and complement. We conclude that IL-17A mediates pneumococcal immunity in mice and probably in humans; its elicitation in vitro could help in the development of candidate pneumococcal vaccines.     

Serum and mucosal immune responses to an inactivated influenza virus vaccine induced by epidermal powder immunization

Both circulating and mucosal antibodies are considered important for protection against infection by influenza virus in humans and animals. However, current inactivated vaccines administered by intramuscular injection using a syringe and needle elicit primarily circulating antibodies. In this study, we report that epidermal powder immunization (EPI) via a unique powder delivery system elicits both serum and mucosal antibodies to an inactivated influenza virus vaccine. Serum antibody responses to influenza vaccine following EPI were enhanced by codelivery of cholera toxin (CT), a synthetic oligodeoxynucleotide containing immunostimulatory CpG motifs (CpG DNA), or the combination of these two adjuvants. In addition, secretory immunoglobulin A (sIgA) antibodies were detected in the saliva and mucosal lavages of the small intestine, trachea, and vaginal tract, although the titers were much lower than the IgG titers. The local origin of the sIgA antibodies was further shown by measuring antibodies released from cultured tracheal and small intestinal fragments and by detecting antigen-specific IgA-secreting cells in the lamina propria using ELISPOT assays. EPI with a single dose of influenza vaccine containing CT or CT and CpG DNA conferred complete protection against lethal challenges with an influenza virus isolated 30 years ago, whereas a prime and boost immunizations were required for protection in the absence of an adjuvant. The ability to elicit augmented circulating antibody and mucosal antibody responses makes EPI a promising alternative to needle injection for administering vaccines against influenza and other diseases.     

肺炎链球菌糖疫苗的研究进展

肺炎链球菌(Streptococcus pneumoniae)是引起多种疾病的主要病原体,包括侵袭性感染(如败血症和脑膜炎菌血症),以及更常见的粘膜部位感染(如肺炎、中耳炎和鼻窦炎).根据肺炎链球菌表面荚膜多糖结构的不同可以分成不同的血清型,至今已经鉴定出98种,其中有20种具有高毒力.为了预防肺炎链球菌感染,已研制出...     

Induction of heterosubtypic immunity to influenza virus by intranasal immunization

Recovery from live influenza virus infection is known to induce heterosubtypic immunity. In contrast, immunity induced by inactivated vaccines is predominantly subtype specific. In this study, we investigated the heterosubtypic protective immunity induced by inactivated influenza virus. Intranasal immunization of mice with inactivated influenza virus A/PR8 (H1N1) provided complete protection against the homologous virus and a drift virus within the same subtype, A/WSN (H1N1), but not against the heterosubtypic virus A/Philippines (H3N2). However, coadministration of inactivated virus with cholera toxin as an adjuvant conferred complete heterosubtypic protection, without observed illness, even under conditions of CD4⁺ or CD8⁺ T-cell depletion. Analysis of immune correlates prior to challenge and postchallenge indicated that humoral immune responses with cross-neutralizing activity in lungs and in sera play a major role in conferring protective immunity against heterosubtypic challenge. This study has significant implications for developing broadly cross-reactive vaccines against newly emerging pathogenic influenza viruses.     

T(H)17-based vaccine design for prevention of Streptococcus pneumoniae colonization

Streptococcus pneumoniae is a leading cause of mortality in young children. While successful conjugate polysaccharide vaccines exist, a less expensive serotype-independent protein-based pneumococcal vaccine offers a major advancement for preventing life-threatening pneumococcal infections, particularly in developing nations. IL-17A-secreting CD4+ T cells (T(H)17) mediate resistance to mucosal colonization by multiple pathogens including S. pneumoniae. Screening an expression library containing >96% of predicted pneumococcal proteins, we identified antigens recognized by T(H)17 cells from mice immune to pneumococcal colonization. The identified antigens also elicited IL-17A secretion from colonized mouse splenocytes and human PBMCs suggesting that similar responses are primed during natural exposure. Immunization of two mouse strains with identified antigens provided protection from pneumococcal colonization that was significantly diminished in animals treated with blocking CD4 or IL-17A antibodies. This work demonstrates the potential of proteomic screening approaches to identify specific antigens for the design of subunit vaccines against mucosal pathogens via harnessing T(H)17-mediated immunity.     

Effect of vaccination against pneumococcal infection and influenza in children with asthma

Assessment of clinical course of asthma and IgG response in children with asthma immunized with pneumococcal polysaccharide vaccine (Pneumo 23) and influenza vaccine (Vaxigrip). 78 children aged 4 - 17 years old were allocated to two groups. Children from the 1st group were immunized against pneumococcal infection and influenza; children from 2nd group were immunized against pneumococcal infection only. Rate of asthma exacerbations in the 1st group of children decreased by 1.7 times compared with the period before vaccination, whereas the same rate in the 2nd group of children decreased by 1.5 times. It was accompanied by the increase of IgG level to antigens of pneumococcalvaccine in blood, which was observed in both groups. Vaccination did not result in increase of IgE levels. Immunization of children with asthma against pneumococcal infection with polysaccharide vaccine or combined immunization against pneumococcal infection and influenza reduced rate of asthma exacerbations and led to formation of immunity to vaccine strains of Streptococcus pneumoniae. Vaccination did not lead to sensitization of children.     

Efficacy of pneumococcal vaccination in adults: a meta-analysis

Background

Clinical trials and meta-analyses have produced conflicting results of the efficacy of unconjugated pneumococcal polysaccharide vaccine in adults. We sought to evaluate the vaccine''s efficacy on clinical outcomes as well as the methodologic quality of the trials.

Methods

We searched several databases and all bibliographies of reviews and meta-analyses for clinical trials that compared pneumococcal polysaccharide vaccine with a control. We examined rates of pneumonia and death, taking the methodologic quality of the trials into consideration.

Results

We included 22 trials involving 101 507 participants: 11 trials reported on presumptive pneumococcal pneumonia, 19 on all-cause pneumonia and 12 on all-cause mortality. The current 23-valent vaccine was used in 8 trials. The relative risk (RR) was 0.64 (95% confidence interval [CI] 0.43–0.96) for presumptive pneumococcal pneumonia and 0.73 (95% CI 0.56–0.94) for all-cause pneumonia. There was significant heterogeneity between the trials reporting on presumptive pneumonia (I² = 74%, p < 0.001) and between those reporting on all-cause pneumonia (I² = 90%, p < 0.001). The RR for all-cause mortality was 0.97 (95% CI 0.87–1.09), with moderate heterogeneity between trials (I² = 44%, p = 0.053). Trial quality, especially regarding double blinding, explained a substantial proportion of the heterogeneity in the trials reporting on presumptive pneumonia and all-cause pneumonia. There was little evidence of vaccine protection in trials of higher methodologic quality (RR 1.20, 95% CI 0.75–1.92, for presumptive pneumonia; and 1.19, 95% CI 0.95–1.49, for all-cause pneumonia in double-blind trials; p for heterogeneity > 0.05). The results for all-cause mortality in double-blind trials were similar to those in all trials combined. There was little evidence of vaccine protection among elderly patients or adults with chronic illness in analyses of all trials (RR 1.04, 95% CI 0.78–1.38, for presumptive pneumococcal pneumonia; 0.89, 95% CI 0.69–1.14, for all-cause pneumonia; and 1.00, 95% CI 0.87–1.14, for all-cause mortality).

Interpretation

Pneumococcal vaccination does not appear to be effective in preventing pneumonia, even in populations for whom the vaccine is currently recommended.The burden of disease due to Streptococcus pneumoniae falls mainly on children, elderly people and people with underlying conditions such as HIV infection.¹ Pneumococcal polysaccharide vaccines were developed more than 50 years ago and have progressed from 2-valent vaccines to the current 23-valent vaccine, which has been available since the early 1980s. The 23-valent vaccine includes serotypes accounting for 72%² to 95%³ of invasive pneumococcal disease, depending on the geographic area. In many industrialized countries, pneumococcal vaccination is currently recommended for people aged 65 years and older and for individuals aged 2–64 who are at increased risk of pneumococcal disease.^4–6Meta-analyses of controlled clinical trials have produced conflicting results of the efficacy of unconjugated pneumococcal polysaccharide vaccine.^7–22 The lack of consistency between results reported from observational studies and controlled trials is another reason why the efficacy of the vaccine remains controversial. Empirical studies have shown that inadequate quality of clinical trials can lead to biases that distort their results.²³ For example, inadequate allocation concealment or failure to blind patients, caregivers or those assessing outcomes may exaggerate treatment effects.²³ Despite this, none of the previous reviews formally compared effect sizes in trials of high methodologic quality with effect sizes in trials of lower quality. We conducted a systematic review and meta-analysis of clinical trials examining the efficacy of pneumococcal polysaccharide vaccination on clinical outcomes, taking the quality of trials into account.     

Maternal immunization with pneumococcal surface protein A protects against pneumococcal infections among derived offspring

Pathogen-specific antibody plays an important role in protection against pneumococcal carriage and infections. However, neonates and infants exhibit impaired innate and adaptive immune responses, which result in their high susceptibility to pneumococci. To protect neonates and infants against pneumococcal infection it is important to elicit specific protective immune responses at very young ages. In this study, we investigated the protective immunity against pneumococcal carriage, pneumonia, and sepsis induced by maternal immunization with pneumococcal surface protein A (PspA). Mother mice were intranasally immunized with recombinant PspA (rPspA) and cholera toxin B subunit (CTB) prior to being mated. Anti-PspA specific IgG, predominantly IgG1, was present at a high level in the serum and milk of immunized mothers and in the sera of their pups. The pneumococcal densities in washed nasal tissues and in lung homogenate were significantly reduced in pups delivered from and/or breast-fed by PspA-immunized mothers. Survival after fatal systemic infections with various types of pneumococci was significantly extended in the pups, which had received anti-PspA antibody via the placenta or through their milk. The current findings strongly suggest that maternal immunization with PspA is an attractive strategy against pneumococcal infections during early childhood.     

Evaluation of Genetically Inactivated Alpha Toxin for Protection in Multiple Mouse Models of Staphylococcus aureus Infection

Staphylococcus aureus is a major human pathogen and a leading cause of nosocomial and community-acquired infections. Development of a vaccine against this pathogen is an important goal. While S. aureus protective antigens have been identified in the literature, the majority have only been tested in a single animal model of disease. We wished to evaluate the ability of one S. aureus vaccine antigen to protect in multiple mouse models, thus assessing whether protection in one model translates to protection in other models encompassing the full breadth of infections the pathogen can cause. We chose to focus on genetically inactivated alpha toxin mutant HlaH35L. We evaluated the protection afforded by this antigen in three models of infection using the same vaccine dose, regimen, route of immunization, adjuvant, and challenge strain. When mice were immunized with HlaH35L and challenged via a skin and soft tissue infection model, HlaH35L immunization led to a less severe infection and decreased S. aureus levels at the challenge site when compared to controls. Challenge of HlaH35L-immunized mice using a systemic infection model resulted in a limited, but statistically significant decrease in bacterial colonization as compared to that observed with control mice. In contrast, in a prosthetic implant model of chronic biofilm infection, there was no significant difference in bacterial levels when compared to controls. These results demonstrate that vaccines may confer protection against one form of S. aureus disease without conferring protection against other disease presentations and thus underscore a significant challenge in S. aureus vaccine development.     

C-Terminal Clostridium perfringens Enterotoxin-Mediated Antigen Delivery for Nasal Pneumococcal Vaccine

Efficient vaccine delivery to mucosal tissues including mucosa-associated lymphoid tissues is essential for the development of mucosal vaccine. We previously reported that claudin-4 was highly expressed on the epithelium of nasopharynx-associated lymphoid tissue (NALT) and thus claudin-4-targeting using C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) effectively delivered fused antigen to NALT and consequently induced antigen-specific immune responses. In this study, we applied the C-CPE-based vaccine delivery system to develop a nasal pneumococcal vaccine. We fused C-CPE with pneumococcal surface protein A (PspA), an important antigen for the induction of protective immunity against Streptococcus pneumoniae infection, (PspA-C-CPE). PspA-C-CPE binds to claudin-4 and thus efficiently attaches to NALT epithelium, including antigen-sampling M cells. Nasal immunization with PspA-C-CPE induced PspA-specific IgG in the serum and bronchoalveolar lavage fluid (BALF) as well as IgA in the nasal wash and BALF. These immune responses were sufficient to protect against pneumococcal infection. These results suggest that C-CPE is an efficient vaccine delivery system for the development of nasal vaccines against pneumococcal infection.     

Loss of discrete memory B cell subsets is associated with impaired immunization responses in HIV-1 infection and may be a risk factor for invasive pneumococcal disease

Invasive pneumococcal infection is an important cause of morbidity and mortality in HIV-1-infected individuals. B cells play an important role in maintaining serologic memory after infection. IgM memory B cells are significantly reduced in HIV-1-infected patients and their frequency is similar to that observed in other patient groups (splenectomized individuals and patients with primary Ab deficiency) who are also known to have an increased risk of invasive pneumococcal infection. Antiretroviral therapy does not restore marginal zone B cell percentages. Immunization with the 23-valent polysaccharide pneumococcal vaccine shows that HIV-1-infected patients have impaired total IgM and IgG pneumococcal vaccines compared with healthy controls. Loss of switched memory B cells was associated with impaired tetanus toxoid IgG vaccine responses. Results of this study demonstrate that defects in B cell memory subsets are associated with impaired humoral immune responses in HIV-1 patients who are receiving antiretroviral therapy and may be a contributory factor to the increased risk of invasive pneumococcal infection observed in HIV-1 infection.     

3′,5′-Cyclic diguanylic acid elicits mucosal immunity against bacterial infection

3′,5′-Cyclic diguanylic acid (cdiGMP) is emerging as a universal bacterial second messenger in regulating bacterial growth on surfaces. It has been recently shown that cdiGMP stimulates innate immunity and enhances antigen-specific humoral and cellular immune responses. We herein report that intranasal (i.n.) administration with cdiGMP induces an acute but transient inflammatory response and activation of dendritic cells in the lungs. Moreover, i.n. immunization of mice with pneumococcal surface adhesion A (PsaA) in conjunction with cdiGMP elicited strong antigen-specific serum immunoglobulin G (IgG) and secretory IgA antibody responses at multiple mucosal surfaces. More importantly, the immunized mice showed significantly reduced nasopharyngeal Streptococcus pneumoniae colonization. These results, for the first time, provide direct evidence for the induction of protection against mucosal bacterial infections by cdiGMP as an adjuvant.     

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.