Effect of immunization route on mucosal and systemic immune response in Atlantic salmon (Salmo salar)

This study aimed to assess systemic and mucosal immune responses of Atlantic salmon (Salmo salar) exposed to two protein-hapten antigens – dinitrophenol (DNP) and fluorescein isothiocyanate (FITC) each conjugated with keyhole limpet haemocyanin (KLH) – administered using different delivery strategies. Fish were exposed to the antigens through different routes, and were given a booster 4 weeks post initial exposure. Both systemic and mucosal antibody responses were measured for a period of 12 weeks using an enzyme-linked immunosorbent assay (ELISA). Only fish exposed to both antigens via intraperitoneal (IP) injection showed increased systemic antibody response starting 6 weeks post immunization. No treatment was able to produce a mucosal antibody response; however there was an increase in antibody levels in the tissue supernatant from skin explants obtained 12 weeks post immunization from fish injected with FITC. Western blots probed with serum and culture supernatant from skin explants showed a specific response against the antigens. In conclusion, IP injection of hapten-antigen in Atlantic salmon was the best delivery route for inducing an antibody response against these antigens in this species. Even though IP injection did not induce an increase in antibody levels in the skin mucus, there was an increased systemic antibody response and an apparent increase of antibody production in mucosal tissues as demonstrated by the increased level of specific antibody levels in supernatants from the tissue explants.     

Enhanced protective immune responses against Salmonella Enteritidis infection by Salmonella secreting an Escherichia coli heat-labile enterotoxin B subunit protein

Escherichia coli heat-labile enterotoxin B subunit (LTB) protein is a potent mucosal adjuvant. In this study, the effect of an attenuated Salmonella secreting LTB protein as an adjuvant strain (JOL1228) for a live Salmonella Enteritidis (SE) vaccine candidate (JOL919) was evaluated. In a single immunization experiment, chickens immunized with a mixture of JOL919 (5 parts) and JOL1228 (1 part) showed enhanced mucosal and cellular immune responses and efficient protection against salmonellosis as compared to those unimmunized control chickens. In further analysis, chickens were primed at one day of age and were boosted at the fifth week of age to prolong immune responses and to maximize the protection efficacy against salmonellosis. The immunized groups B (prime and booster with JOL919), C (prime with JOL919-JOL1228 mixture and booster with JOL919), and D (prime and booster with JOL919-JOL1228 mixture) showed significantly higher humoral and cellular immune responses as compared to those in the unimmunized control group A. In addition, immunized groups C and D showed fewer gross lesions in the liver and spleen and a lower number of SE-positive organs, with the lowest bacterial counts in the SE challenge strain as compared to the control group. These results indicate that SE vaccination with the LTB strain can have an adjuvant effect on the vaccine candidate by enhancing immune responses, and that a prime-boost strategy with the addition of the adjuvant strain can efficiently protect birds against salmonellosis.     

Systemic and local immune response in pigs intradermally and intramuscularly injected with inactivated Mycoplasma hyopneumoniae vaccines

The systemic and respiratory local immune response induced by the intradermal administration of a commercial inactivated Mycoplasma hyopneumoniae whole-cell vaccine (Porcilis^® MHYO ID ONCE – MSD AH) in comparison with two commercial vaccines administered via the intramuscular route and a negative control (adjuvant only) was investigated. Forty conventional M. hyopneumoniae-free pigs were randomly assigned to four groups (ten animals each): Group A = intradermal administration of the test vaccine by using the needle-less IDAL^® vaccinator at a dose of 0.2 ml; Group B = intramuscular administration of a commercially available vaccine (vaccine B); Group C = intramuscular administration of the adjuvant only (2 ml of X-solve adjuvant); Group D = intramuscular administration of a commercially available vaccine (vaccine D). Pigs were vaccinated at 28 days of age. Blood and bronchoalveolar lavage (BAL) fluid samples were collected at vaccination (blood only), 4 and 8 weeks post-vaccination. Serum and BAL fluid were tested for the presence of antibodies by ELISA test. Peripheral blood monomorphonuclear cells (PBMC) were isolated to quantify the number of IFN-γ secreting cells by ELISpot. Moreover, cytokine gene expression from the BAL fluid was performed. Total antibodies against M. hyopneumoniae and specific IgG were detected in serum of intradermally and intramuscularly (vaccine B only) vaccinated pigs at 4 and 8 weeks post-vaccination. M. hyopneumoniae specific IgA were detected in BAL fluid from vaccinated animals (Groups A and B) but not from controls and animals vaccinated with the bacterin D (p < 0.05). Significantly higher gene expression of IL-10 was observed in the BAL fluid at week 8 post-vaccination in the intradermally vaccinated pigs (p < 0.05). The results support that the intradermal administration of an adjuvanted bacterin induces both systemic and mucosal immune responses. Moreover, the intramuscularly administered commercial vaccines each had a different ability to stimulate the immune response both systemically and locally.     

Attenuated Salmonella typhimurium delivering DNA vaccine encoding duck enteritis virus UL24 induced systemic and mucosal immune responses and conferred good protection against challenge

Orally delivered DNA vaccines against duck enteritis virus (DEV) were developed using live attenuated Salmonella typhimurium (SL7207) as a carrier and Escherichia coli heat labile enterotoxin B subunit (LTB) as a mucosal adjuvant. DNA vaccine plasmids pVAX-UL24 and pVAX-LTB-UL24 were constructed and transformed into attenuated Salmonella typhimurium SL7207 resulting SL7207 (pVAX-UL24) and SL7207 (pVAX-LTB-UL24) respectively. After ducklings were orally inoculated with SL7207 (pVAX-UL24) or SL7207 (pVAX-LTB-UL24), the anti-DEV mucosal and systemic immune responses were recorded. To identify the optimum dose that confers maximum protection, we used different doses of the candidate vaccine SL7207 (pVAX-LTB-UL24) during oral immunization. The strongest mucosal and systemic immune responses developed in the SL7207 (pVAX-LTB-UL24) (10¹¹ CFU) immunized group. Accordingly, oral immunization of ducklings with SL7207 (pVAX-LTB-UL24) showed superior efficacy of protection (60-80%) against a lethal DEV challenge (1000 LD₅₀), compared with the limited survival rate (40%) of ducklings immunized with SL7207 (pVAX-UL24). Our study suggests that the SL7207 (pVAX-LTB-UL24) can be a candidate DEV vaccine.     

Evaluating the immune responses of mice to subcutaneous immunization with Helicobacter pylori urease B subunit

Background

Helicobacter pylori, a gram-negative bacterial pathogen that expresses a strong urease activity, is associated with the development of gastroduodenal disease. Urease B subunit, one of the two structural subunits of urease, was expressed in E. coli BL21 (DE3) strain. The objective of this study was to evaluate the effects of Helicobacter pylori urease B subunit on the immune responses in mice by subcutaneous immunization.

Methods

The mice were immunized and boosted with Helicobacter pylori urease B subunit antigen subcutaneously three times with 2-wk intervals between the immunizations and boosters. The mice in the control group were immunized with PBS. The adjuvant group received PBS containing complete/incomplete freund’s adjuvant identical to antigen group without Helicobacter pylori urease B subunit antigen. Four weeks after the final booster, all the mice were sacrificed. Blood was collected on d 0, 14, 28 and 56 before immunization, booster and sacrifice, respectively. Immediately after sacrifice, gastric liquid and spleen were collected for antibody and cytokine analyses.

Results

Urease B subunit increased the concentrations of serum and gastric anti-urease B antigen specific IgG, and the levels of interleukin-4 and interferon-γ in splenocytes of the mice (P < 0.05).

Conclusions

This study demonstrated that recombinant urease B subunit can induce systemic and local immune responses in mice by subcutaneous immunization, which might be used as the effective component of vaccine against Helicobacter pylori.     

Effect of booster shot and investigation of vaccination efficacy period against herpesviral haematopoietic necrosis (HVHN) in goldfish Carassius auratus

In this study, the efficacy period of an intraperitoneal vaccination and effect of a booster shot of vaccine against herpesviral haematopoietic necrosis (HVHN) in goldfish Carassius auratus were investigated. Cell culture supernatant of cyprinid herpesvirus 2 (CyHV-2), causative agent of HVHN, propagated in goldfish fin (GFF) cells was inactivated with formalin (0.1%, v/v) for 2 days at 4 °C. Three groups of the variety Ryukin were individually intraperitoneally injected with the vaccine and each group was separately maintained in replicate tanks. After 4 weeks (Vaccinated-4w-1 and 2) and 8 weeks (Vaccinated-8w-1 and 2) from the first vaccination, the fish were CyHV-2-challenged by the immersion route (10 TCID₅₀ l⁻¹). In addition, the other vaccinated group of fish were injected with a booster vaccine 4 weeks after the first vaccination as the Vaccinated-booster groups, then the fish of these groups were CyHV-2-challenged by the immersion route (10 TCID₅₀ l⁻¹) after 8 weeks from the first vaccination. The mean of the relative percentage survival (RPS) values of the Vaccinated-4w and 8w groups showed 42.5% and 57.6%, respectively. In addition, the mean RPS value of Vaccinated-booster groups showed 63.6%. Statistical analysis showed significantly higher survival rates in all the vaccinated groups than those of the respective negative control groups using Fisher's exact test. Moreover, the survival rates of vaccinated-booster groups were significantly higher (p = 0.036) compared with the respective control groups by Student's t test. The present study shows the efficacy period of the vaccine is at least 8 weeks and a booster shot showed a tendency to enhance the protection against HVHN in goldfish.     

Maternal immunity enhances systemic recall immune responses upon oral immunization of piglets with F4 fimbriae

F4 enterotoxigenic Escherichia coli (ETEC) cause diarrhoea and mortality in piglets leading to severe economic losses. Oral immunization of piglets with F4 fimbriae induces a protective intestinal immune response evidenced by an F4-specific serum and intestinal IgA response. However, successful oral immunization of pigs with F4 fimbriae in the presence of maternal immunity has not been demonstrated yet. In the present study we aimed to evaluate the effect of maternal immunity on the induction of a systemic immune response upon oral immunization of piglets. Whereas F4-specific IgG and IgA could be induced by oral immunization of pigs without maternal antibodies and by intramuscular immunization of pigs with maternal antibodies, no such response was seen in the orally immunized animals with maternal antibodies. Since maternal antibodies can mask an antibody response, we also looked by ELIspot assays for circulating F4-specific antibody secreting cells (ASCs). Enumerating the F4-specific ASCs within the circulating peripheral blood mononuclear cells, and the number of F4-specific IgA ASCs within the circulating IgA⁺ B-cells revealed an F4-specific immune response in the orally immunized animals with maternal antibodies. Interestingly, results suggest a more robust IgA booster response by oral immunization of pigs with than without maternal antibodies. These results demonstrate that oral immunization of piglets with F4-specific maternal antibodies is feasible and that these maternal antibodies seem to enhance the secondary systemic immune response. Furthermore, our ELIspot assay on enriched IgA⁺ B-cells could be used as a screening procedure to optimize mucosal immunization protocols in pigs with maternal immunity.     

Enhancement of protective immune responses by oral vaccination with Saccharomyces cerevisiae expressing recombinant Actinobacillus pleuropneumoniae ApxIA or ApxIIA in mice

We previously induced protective immune response by oral immunization with yeast expressing the ApxIIA antigen. The ApxI antigen is also an important factor in the protection against Actinobacillus pleuropneumoniae serotype 5 infection; therefore, the protective immunity in mice following oral immunization with Saccharomyces cerevisiae expressing either ApxIA (group C) or ApxIIA (group D) alone or both (group E) was compared with that in two control groups (group A and B). The immunogenicity of the rApxIA antigen derived from the yeast was confirmed by a high survival rate and an ApxIA-specific IgG antibody response (p < 0.01). The highest systemic (IgG) and local (IgA) humoral immune responses to ApxIA and ApxIIA were detected in group E after the third immunization (p < 0.05). The levels of IL-1β and IL-6 after challenge with an A. pleuropneumoniae field isolate did not change significantly in the vaccinated groups. The level of TNF-α increased in a time-dependent manner in group E but was not significantly different after the challenge. After the challenge, the mice in group E had a significantly lower infectious burden and a higher level of protection than the mice in the other groups (p < 0.05). The survival rate in each group was closely correlated to the immune response and histopathological observations in the lung following the challenge. These results suggested that immunity to the ApxIA antigen is required for optimal protection.     

Passive protection of mice pups through oral or intranasal immunization of dams with recombinant Lactobacillus casei vaccine against ETEC F41

Mucosal immunization is advantageous over other routes of antigen delivery because it can induce both mucosal and systemic immune responses. In this study, we have developed fimbriae protein of enterotoxigenic Escherichia coli (ETEC) F41 was stably expressed on the surface Lactobacillus casei 525. The method of expressing vaccine antigens in L. casei induces both systemic and mucosal immunity after oral or intranasal administration. We demonstrate that an oral or intranasal vaccine based on live recombinant L. casei 525 protects infant mice from ETEC F41 infection. This platform technology can be applied to design oral or intranasal vaccine delivery vehicles against several microbial pathogens.     

Comparison of immune responses to intranasal and intrapulmonary vaccinations with the attenuated Mycoplasma hyopneumoniae 168 strain in pigs

The aim of this study was to evaluate the immune responses to intranasal and intrapulmonary vaccinations with the attenuated Mycoplasma hyopneumoniae (Mhp) 168 strain in the local respiratory tract in pigs. Twenty-four pigs were randomly divided into 4 groups: an intranasal immunization group, an intrapulmonary immunization group, an intramuscular immunization group and a control group. The levels of local respiratory tract cellular and humoral immune responses were investigated. The levels of interleukin (IL)-6 in the early stage of immunization (P<0.01), local specific secretory IgA (sIgA) in nasal swab samples (P<0.01); and IgA- and IgG-secreting cells in the nasal mucosa and trachea were higher after intranasal vaccination (P<0.01) than in the control group. Interestingly, intrapulmonary immunization induced much stronger immune responses than intranasal immunization. Intrapulmonary immunization also significantly increased the secretion of IL-6 and local specific sIgA and the numbers of IgA- and IgG-secreting cells. The levels of IL-10 and interferon-γ in the nasal swab samples and the numbers of CD4⁺ and CD8⁺ T lymphocytes in the lung and hilar lymph nodes were significantly increased by intrapulmonary immunization compared with those in the control group (P<0.01). These data suggest that intrapulmonary immunization with attenuated Mhp is effective in evoking local cellular and humoral immune responses in the respiratory tract. Intrapulmonary immunization with Mhp may be a promising route for defense against Mhp in pigs.     

Nasal immunization with major epitope-containing ApxIIA toxin fragment induces protective immunity against challenge infection with Actinobacillus pleuropneumoniae in a murine model

Actinobacillus pleuropneumoniae is an infective agent that leads to porcine pleuropneumonia, a disease that causes severe economic losses in the swine industry. Based on the fact that the respiratory tract is the primary site for bacterial infection, it has been suggested that bacterial exclusion in the respiratory tract through mucosal immune induction is the most effective disease prevention strategy. ApxIIA is a vaccine candidate against A. pleuropneumoniae infection, and fragment #5 (aa. 439–801) of ApxIIA contains the major epitopes for effective vaccination. In this study, we used mice to verify the efficacy of intranasal immunization with fragment #5 in the induction of protective immunity against nasal challenge with A. pleuropneumoniae and compared its efficacy with that of subcutaneous immunization. Intranasal immunization of the fragment induced significantly higher systemic and mucosal immune responses measured at the levels of antigen-specific antibodies, cytokine-secreting cells after antigen exposure, and antigen-specific lymphocyte proliferation. Intranasal immunization not only efficiently inhibited the bacterial colonization in respiratory organs, but also prevented alveolar tissue damage in infectious condition similar to that of a contaminated pig. Moreover, intranasal immunization with fragment #5 provided acquired protective immunity against intranasal challenge with A. pleuropneumoniae serotype 2. In addition, it conferred cross-protection against serotype 5, a heterologous pathogen that causes severe disease by ApxI and ApxII secretion. Collectively, intranasal immunization with fragment #5 of ApxIIA can be considered an efficient protective immunization procedure against A. pleuropneumoniae infection.     

Systemic and mucosal immunity induced by attenuated Salmonella enterica serovar Typhimurium expressing ORF7 of porcine reproductive and respiratory syndrome virus

Oral administration of attenuated Salmonella vaccine may provide valuable advantages such as low cost, easy preparation, and safety. Attenuated Salmonella vaccines also serve as carriers of foreign antigens and immunomodulatory cytokines. Presently, an attenuated Salmonella enterica serovar Typhimurium strain was used as a carrier for open reading frame 7 (ORF7) protein of porcine reproductive and respiratory syndrome virus (PRRSV), a swine pathogen of significant global economic importance. Initially, an attenuated S. enterica serovar Typhimurium expressing ORF7 gene derived from PRRSV Korean isolate was constructed. Following oral administration of a single dose of the attenuated Salmonella vaccine expressing PRRSV ORF7, humoral and cell-mediated immune responses specific for ORF7 were induced at both systemic and mucosal sites including spleen, mesenteric lymph node, Peyer's patch, and laminar propria, as evaluated by determining serum ORF7-specific IgG and mucosal IgA responses, as well as Th1- and Th2-type cytokine production from antigen-stimulated T cells. The induced humoral responses were sustained for at least 12 weeks post-immunization. In particular, the immunized mice displayed immune responses to both the foreign ORF7 antigen and Salmonella itself. The results indicate the value of attenuated S. enterica serovar Typhimurium as an oral carrier of PRRSV antigenic proteins to induce effective systemic and mucosal immunity.     

Effect of calf age and Salmonella bacterin type on ability to produce immunoglobulins directed against Salmonella whole cells or lipopolysaccharide.

A commercially available Salmonella bacterin was administered to Holstein calves starting at 1 to 19 weeks of age. Serum samples were obtained before administering bacterin and at 2-week intervals thereafter. An ELISA with Salmonella dublin lipopolysaccharide (LPS) or S dublin whole cells as antigen, was used to measure specific IgG and IgM responses. Antibody responses to LPS were not detected from calves < 12 weeks old inoculated with killed bacterin. Immunoglobulin responses to whole-cell antigen were detected from all age groups of calves inoculated with the same killed Salmonella bacterin. Calves < 11 weeks old are able to produce immunoglobulins to some whole-cell antigens, but are unable to produce anti-LPS immunoglobulins when inoculated with killed Salmonella bacterin. This age-related response to killed Salmonella antigens may account, in part, for increased susceptibility to salmonellosis in calves < 12 weeks old. In comparison to the response for killed antigen, 8 calves given modified-live aromatic-dependent S dublin bacterin at 1 to 3 weeks of age had detectable anti-LPS immunoglobulins after immunization, although the response was not as rapid and was of a lesser magnitude than that of older calves given killed Salmonella bacterin.     

Tulathromycin enhances humoral but not cellular immune response in pigs vaccinated against swine influenza

The effect of a standard, single dose therapy with tulathromycin was investigated on the postvaccinal humoral and cellular immune response in pigs vaccinated against swine influenza. Forty‐five pigs, divided into 3 groups, were used (control not vaccinated (C, n = 15), control vaccinated (CV, n = 15), and experimentally received tulathromycin (TUL, n = 15)). For vaccination of pigs, an inactivated, commercial vaccine was used. Pigs from TUL group received single dose of tulathromycin intramuscularly, at the recommended dose (2.5 mg/kg body weight). Pigs from TUL and CV groups were vaccinated at 8 and 10 weeks of age. The specific humoral and cellular immune response against swine influenza virus (SIV) was evaluated. The results of present study showed that humoral postvaccinal response after vaccination against SIV can be modulated by treatment with tulathromycin. In pigs from TUL group, the significantly higher titers of anti‐SIV‐specific antibodies were observed 4 and 6 weeks after booster dose of vaccine. Simultaneously, T‐cell‐mediated immune response against SIV was not affected by tulathromycin. Our recent study confirmed the importance of defining the modulatory activity of tulathromycin because of its influence on the immune response to vaccines. Since the antibodies against hemagglutinin are crucial for the protection against SIV, the present observations should prompt further studies on the practical significance of recent results in terms of clinical implications (postvaccinal protection) in the field conditions.     

Immunity to bovine reproductive infections.

Protective immune responses in the genital tract are robust, as shown by convalescent and vaccine-induced immunity. Systemic immunity is crucial for systemic infections that result in reproductive failure (such as brucellosis, leptospirosis, and the systemic forms of C. fetus and H. somnus infection). Although IgA responses can protect against sexually transmitted or venereal infections, systemically induced IgG antibody responses also protect. IgA responses can be induced by immunization of the genital tract, where inductive sites develop after antigenic stimulation. The common mucosal immune system can also be used to induce a genital IgA response, as shown by intranasal vaccination. Lastly, it is necessary to determine which antigens of each infectious agent are protective and which types of immune responses protect best.     

Immune response to liposome-associated recombinant SEF21 following oral immunization in chickens

In order to generate Salmonella enterica serovar Enteritidis fimbriae antigens (rSEF21), the intact region encoding SEF21 was amplified from Salmonella Enteritidis by PCR and subcloned into a prokaryotic expression vector pET-28a(+) to yield pET-28a(+)-SEF21. The rSEF21 protein was highly expressed and purified by nickel affinity chromatography. Liposomeassociated rSEF21 was prepared for oral immunization to seek protective efficacy for intestinal infection with Salmonella Enteritidis. Evidence of IgA and IgG responses were found in the intestinal tracts and in the sera of a group of chickens immunized. Two weeks after the booster immunization, the chickens were challenged orally with 2 x 10(6) colony-forming units of live Salmonella Enteritidis, and fecal samples were examined for bacterial excretion from the intestinal tract. Significantly less fecal excretion of bacteria was observed in immunized chickens for 4 wk after challenge. The numbers of bacteria in the intestinal contents (cecum and rectum) were also significantly lower in immunized chickens than in unimmunized controls. Therefore, oral immunization with liposome-associated rSEF21 elicits both systemic and mucosal antibody responses, leading to a reduction in bacterial colonization in the intestinal tract and excretion of Salmonella Enteritidis in the feces.     

The Mycoplasma hyopneumoniae recombinant heat shock protein P42 induces an immune response in pigs under field conditions

Enzootic pneumonia (EP), resulting from Mycoplasma hyopneumoniae infection is one of the most prevalent diseases in pigs and is a major cause of economic losses to the swine industry worldwide. EP is often controlled by vaccination with inactivated, adjuvanted whole-cell bacterin. However, these bacterins provide only partial protection and do not prevent M. hyopneumoniae colonization. Attempts to develop vaccines that are more efficient have made use of the recombinant DNA technology. The objective of this study was to assess the potential of recombinant M. hyopneumoniae heat shock protein P42 in vaccine preparations against EP, using piglets housed under field conditions in a M. hyopneumoniae-positive farm. The cellular and humoral immune responses were elicited after a single intramuscular inoculation of rP42 in an oil-based adjuvant, or in conjunction with whole-cell vaccine preparation. The production of INF-γ and IL-10 cytokines was quantified in the supernatant of the cultured mononuclear cells. The rP42 emulsified in oil-based adjuvant was able to trigger a strong humoral immune response. Further, it induced a cellular immune response, accompanied by the production of antibodies that reacted with the native M. hyopneumoniae protein. The rP42 mediated induction of cellular and humoral immune response in the host suggests that rP42 emulsified in an oil-based adjuvant holds promise as an effective recombinant subunit vaccine against EP.     

Cross-protection against Salmonella Typhimurium infection conferred by a live attenuated Salmonella Enteritidis vaccine

In this study, a genetically engineered live attenuated Salmonella Enteritidis (SE) vaccine was evaluated for its ability to protect against Salmonella Typhimurium (ST) infection in chickens. The birds were orally primed with the vaccine on the 1st day of life and given an oral booster at 5 wk of age. Control birds were orally inoculated with phosphate-buffered saline. Both groups of birds were orally challenged with a virulent ST strain at 9 wk of age. Compared with the control chickens, the vaccinated chickens had significantly higher levels of systemic IgG and mucosal IgA against specific ST antigens and a significantly greater lymphoproliferative response to ST antigens. The excretion of ST into the feces was significantly lower in the vaccinated group than in the control group on days 9 and 13 d after challenge. In addition, the vaccinated group had significantly fewer pronounced gross lesions in the liver and spleen and lower bacterial counts in the internal organs than the control group after challenge. These data indicate that genetically engineered live attenuated SE may induce humoral and cellular immune responses against ST antigens and may confer protection against virulent ST challenge.     

Adjuvants modulating mucosal immune responses or directing systemic responses towards the mucosa

In developing veterinary mucosal vaccines and vaccination strategies, mucosal adjuvants are one of the key players for inducing protective immune responses. Most of the mucosal adjuvants seem to exert their effect via binding to a receptor/or target cells and these properties were used to classify the mucosal adjuvants reviewed in the present paper: (1) ganglioside receptor-binding toxins (cholera toxin, LT enterotoxin, their B subunits and mutants); (2) surface immunoglobulin binding complex CTA1-DD; (3) TLR4 binding lipopolysaccharide; (4) TLR2-binding muramyl dipeptide; (5) Mannose receptor-binding mannan; (6) Dectin-1-binding ss 1,3/1,6 glucans; (7) TLR9-binding CpG-oligodeoxynucleotides; (8) Cytokines and chemokines; (9) Antigen-presenting cell targeting ISCOMATRIX and ISCOM. In addition, attention is given to two adjuvants able to prime the mucosal immune system following a systemic immunization, namely 1alpha, 25(OH)2D3 and cholera toxin.