Mucosal immunization with purified flagellin from Salmonella induces systemic and mucosal immune responses in C3H/HeJ mice

This study investigated the immune response elicited in C3H/HeJ mice after oral, parenteral and nasal immunization with purified flagellin from Salmonella enterica serovar Enteritidis alone or conjugated to starch microparticles as adjuvant or together with the uptake-enhancer recombinant cholera toxin B-subunit (rCTB). Systemic (IgM-IgG, IgA, IgG2a, IgG2b, IgG1) and local (s-IgA) humoral immune responses in the mice were analyzed using enzyme-linked immunosorbent assays (ELISA). Primed splenocytes were also stimulated in vitro with flagellin and the supernatants analyzed for cytokine production. Finally, immunized mice were challenged orally with live Salmonella. A high flagellin-specific IgM-IgG response was seen in all groups, especially in mice immunized nasally with flagellin plus rCTB or subcutaneously, but a strong systemic antibody response was also induced when free antigen was given orally. Intranasal or subcutaneous immunization of mice with flagellin plus rCTB or oral immunization with flagellin plus microparticles resulted in a significantly greater mucosal response (higher s-IgA titers in feces) than seen in the control group (P <0.05). The mucosal IgA responses were significantly correlated with the serum IgA titers. The subclass profile in serum revealed a mixed Th1/Th2-type response, with a predominance of Th1-type, as indicated by the subclass ratio (IgG1/IgG2a + IgG2b). The splenocytes stimulated in vitro produced interferon (IFN)-gamma, at levels, which increased with time. The group immunized with flagellin plus rCTB subcutaneously had a relatively higher IFN-gamma response than the other groups. Interleukin (IL)-2 was also produced, especially in mice immunized nasally or subcutaneously with flagellin conjugated to microparticles. However, neither IL-4 nor IL-5 was produced in any of the groups. After oral challenge with live serovar Enteritidis, the groups immunized orally or nasally with free flagellin had significantly lower degree of infection than the control group (P <0.05).     

Development and characterization of chitosan coated poly-(ɛ-caprolactone) nanoparticulate system for effective immunization against influenza

In this study surface coated poly-(?-caprolactone) (PCL) nanoparticles with chitosan (CS) were developed as a carrier system for nasal immunization using recombinant Influenza A virus (A/California/07/2009) H1N1 hemagglutinin (HA) protein, for the induction of humoral, cellular and mucosal immunity. CS coated PCL (CS-PCL) nanoparticles were characterized in vitro for their percent yield, size, shape, entrapment efficiency, loading capacity and zeta potential. The in vitro release and antigen integrity were also evaluated. Particles were prepared by an emulsion-diffusion-solvent evaporation method. The coated cationic nanoparticles of average size 125.64 ± 6.51 nm with a narrow size distribution (pdi: 0.185 ± 0.032) and a positive surface charge (+22.88 mV) were obtained. HA antigen was efficiently entrapped in CS-PCL nanoparticles (entrapment efficiency 74.84 ± 4.51%, loading capacity 14 ± 2% (w/w)). The molecular weight and antigenicity of the entrapped HA was maintained as shown by polyacrylamide gel electrophoresis and Western blotting, respectively. In vitro release study of antigen showed that about 66.47% of entrapped antigen was released within 63 days. The immune-stimulating activity was studied by measuring hemagglutination inhibition (HAI) titer, IgG, IgG1 and IgG2a titer, secretory IgA level in nasal and lung lavage (mucosal secretions) following nasal administration of modified CS-PCL nanoparticles in Balb/c mice and compared with soluble HA antigen administered intramuscular (IM) and with PCL (uncoated) nanoparticles administered intranasal (IN). The numbers of IFN-γ or IL-4 secreting cells in spleen homogenates were also measured 21 day after third immunization. Single IN or IM immunization with antigen-loaded CS-PCL nanoparticles resulted in strong HAI and total IgG responses. These responses were higher than those achieved after booster IM administration of the subunit antigen, whereas the IgG1/IgG2a profile did not change substantially. The IN administered antigen-CS-PCL nanoparticles induced higher immune responses compared to the other IN antigen formulations, and these responses were enhanced by IN booster vaccinations. Moreover, IM administered soluble HA antigen did not elicit s-IgA in mucosal secretions as it was induced and measured in the case of nasal administration of CS-PCL nanoparticles. In contrast to IM administered antigen CS-PCL nanoparticles induced a balanced Th1 and Th2 response. CS-PCL nanoparticles (cationic nanoparticles) thus produced humoral (both systemic and mucosal) and cellular immune responses upon nasal administration. These findings demonstrate high potential of CS-PCL nanoparticles for their use as a carrier adjuvant for nasal administered influenza antigens.     

Rectal immunization with antigen-containing microspheres induces stronger Th2 responses than oral immunization: a new method for vaccination.

The rectum as an effective site for induction of systemic and local immunity has received little attention. Rectal immunization with microspheres-containing ovalbumin (MS-OVA) was tested for its ability to elicit systemic and mucosal immune responses. Rectal immunization with MS-OVA enhanced both Th2 dominant OVA-specific IgG levels in the serum and OVA-specific IgA levels in fecal extracts more prominently than did oral immunization. Cytokine analysis of CD4(+) T cells indicated a predominant induction of Th2-type responses compared to Th1-type responses following rectal immunization compared to oral immunization. These results demonstrate that rectal immunization with microspheres could be an effective new vaccination method.     

Recombinant interleukin 6 with M cell-targeting moiety produced in Lactococcus lactis IL1403 as a potent mucosal adjuvant for peroral immunization

Development and application of safe and effective mucosal adjuvants are important to improve immunization efficiency in oral vaccine. Here, we report a novel mucosal adjuvant, IL-6-CKS9, a recombinant cytokine generated by conjugating an M cell-targeting peptide (CKS9) with c-terminus of the murine interleukin 6 (IL-6), which facilitated enhancement of mucosal immune response. Lactococcus lactis IL1403, a food-grade strain of lactic acid bacteria (LAB) which is widely used in dairy industry, was used as a host cell to express and secrete the IL-6-CKS9 for a mucosal vaccine adjuvant. The recombinant L. lactis IL1403 secreting IL-6-CKS9 was orally administered with a model antigen protein, M-BmpB (Brachyspira membrane protein B conjugated with CKS9), to BALB/c mice for mucosal immunization. ELISA analyses showed consistent enhancement tendencies in induction of anti-M-BmpB antibody levels both with mucosal (IgA) and systemic (IgG) immune responses in IL-6-CKS9-LAB treated group compared with other groups tested by conducting two separated mice immunization assays. In addition, we characterized that the oral administration of model protein antigen with live LAB producing IL-6-CKS9 could induce both Th1 and Th2 type immune responses by analysis of the specific anti-BmpB IgG1 and IgG2a isotypes in the sera and also investigated possible oral tolerance in our vaccine strategy. Collectively, our results showed successful production and secretion of recombinant murine IL-6 with M cell-targeting moiety (IL-6-CKS9) from L. lactis IL1403 and demonstrated the live recombinant LAB producing IL-6-CKS9 could have a potential to be used as an efficient adjuvant for peroral vaccination.     

Effectiveness of intranasal immunization with HIV-gp160 and an HIV-1 env CTL epitope peptide (E7) in combination with the mucosal adjuvant LT(R192G)

LT(R192G) is a novel mucosal adjuvant that induces protective immunity when co-administered with certain whole inactivated bacteria or viruses or with subunits of relevant virulence determinants from these pathogens. LT(R192G) stimulates antigen-specific humoral and cellular immune responses, both systemically and in mucosal compartments, and is safe and nontoxic at adjuvant effective doses. Intranasal (IN) immunization of mice with LT(R192G) in conjunction with oligomeric HIV-1 gp160 elevates antigen-specific systemic and mucosal IgG and IgA production and Th1- and Th2-type cytokine responses. Isotype characterization of induced IgG reveals that gp160 alone fails to stimulate IgG2a responses in the absence of adjuvant. Both IgG1 and IgG2a are induced by immunization in the presence of LT(R192G). Additionally, intranasal immunization with a 15-amino acid peptide corresponding to an HIV-1 Env CTL determinant and LT(R192G) induces systemic, peptide-specific CTL activity and Th1 and Th2 cytokine responses that are absent when the adjuvant is excluded from the immunizations. These studies show that LT(R192G) quantitatively and qualitatively enhances cellular and humoral HIV-specific immune responses and that this adjuvant may offer significant advantages toward vaccine development against HIV.     

Comparison of mucosal and systemic humoral immune responses after transcutaneous and oral immunization strategies

In order to compare the ability of transcutaneous and oral immunization strategies to induce mucosal and systemic immune responses, we inoculated mice transcutaneously with cholera toxin (CT) or the non-toxic B subunit of cholera toxin (CtxB), or orally with Peru2(pETR1), an attenuated vaccine strain of Vibrio cholerae expressing CtxB. In addition, we also evaluated dual immunization regimens (oral inoculation with transcutaneous boosting, and transcutaneous immunization with oral boosting) in an attempt to optimize induction of both mucosal and systemic immune responses. We found that transcutaneous immunization with purified CtxB or CT induces much more prominent systemic IgG anti-CtxB responses than does oral inoculation with a vaccine vector strain of V. cholerae expressing CtxB. In comparison, anti-CtxB IgA in serum, stool and bile were comparable in mice either transcutaneously or orally immunized. Overall, the most prominent systemic and mucosal anti-CtxB responses occurred in mice that were orally primed with Peru2(pETR1) and transcutaneously boosted with CT. Our results suggest that combination oral and transcutaneous immunization strategies may most prominently induce both mucosal and systemic humoral responses.     

Induction of Th2-related immune responses and production of systemic IgA in mice intranasally immunized with Brucella abortus malate dehydrogenase loaded chitosan nanoparticles

The aim of this study was to investigate the induction of mucosal immune responses by an important Brucella abortus antigen, malate dehydrogenase (Mdh), loaded in mucoadhesive chitosan nanoparticles (CNs) and immunized intranasally in a BALB/c mouse model. The production of cytokines was investigated in human leukemic monocyte cells (THP-1 cells) after stimulation with the nanoparticles. Mdh-loaded CNs (CNs-Mdh) induced higher interleukin (IL)-6 production than unloaded antigens and TF loaded CNs (CNs-TF). Using ELISpot to quantify cytokines and antibody-secreting cells in the intranasally immunized mice, IL-4 and IgG-secreting cells were found to be significantly increased at 4?weeks and 6?weeks post-immunization in the CNs-Mdh immunized group, respectively. Increases in Mdh-specific IgG, IgG1, and IgG2a antibodies were confirmed at 6?weeks after immunization, indicating a predominant IgG1 response. Analysis of the mucosal immune response in the intranasally immunized mice revealed, Mdh-specific IgA and total IgA in the nasal washes, genital secretions, fecal extracts and sera that were remarkably increased in the CNs-Mdh-immunized group compared to the CNs-TF-immunized group except total IgA of nasal wash. Therefore, the results indicated that the intranasal immunization of CNs-loaded B. abortus Mdh antigen effectively induced antigen-specific mucosal immune responses through the elicitation of Th2-related immune responses.     

Immunogenic properties of the Salmonella atypical fimbriae in BALB/c mice

Components of the Salmonella atypical fimbriae (Saf) were investigated for potential inclusion in a Salmonella vaccine. Recombinant histidine-tagged SafB chaperone complexed with SafD adhesin was expressed in Escherichia coli and purified. Starch microparticles were used, as an adjuvant and recombinant cholera toxin B subunit (rCTB) was included as a mucosal antigen-uptake enhancer. BALB/c mice were immunized orally or subcutaneously with SafB/D- and rCTB-conjugated microparticles and nasally or subcutaneously with SafB/D mixed with rCTB. The systemic and mucosal immune responses were studied, and an oral challenge with Salmonella enteritidis was performed. All immunized groups except that receiving oral immunization responded with high IgM-IgG titers to SafB/D. Analysis of the subclass ratio (IgG1/IgG2a+IgG2b) indicated a mixed Th1 and Th2 response, with Th1 predominating. The mucosal response, measured as specific IgA/total IgA (from fecal samples), was significantly greater than that in the untreated control group only in the group receiving intranasal immunization (P<0.05). Spleens were removed 6 days after oral challenge and Salmonella colony-forming units (CFU) were counted. The group immunized subcutaneously with SafB/D- and rCTB-conjugated microparticles had significantly lower CFU counts than the untreated control group (P<0.05).     

Induction of immune memory by a multisubunit chlamydial vaccine

We tested the hypothesis that intramuscular immunization with a multisubunit chlamydial vaccine candidate will induce long lasting immune responses in mice. Accordingly, groups of female C57BL/6 mice were immunized intramuscularly with Vibrio cholerae ghosts (VCG) expressing the Poring B and polymorphic membrane protein-D proteins of Chlamydia trachomatis or a control antigen. Humoral and cell-mediated immune responses were evaluated following immunization and after live chlamydial infection. Immunization induced an anamnestic response characterized by chlamydial-specific IgG2a and IgA antibodies in sera and vaginal lavage as well as specific genital and splenic T cell responses. The results also revealed that the local mucosal and systemic cellular and humoral immune effectors induced in mice following immunization with the vaccine candidate are long lasting. Vaccinated mice cleared intravaginal challenge with 10⁵ chlamydial inclusion forming units within 12 days compared to control mice, which shed up to 2 × 10³ IFUs at this time point. Moreover, rechallenge of mice 98 days after resolution of the primary infection resulted in the recall and retention of a relatively high frequency of chlamydial-specific Th1 cells and IgG2a in the genital mucosa. These results provide the first evidence that a VCG-based multisubunit chlamydial vaccine is capable of effectively stimulating anamnestic systemic and mucosal immune responses in mice. The data support further vaccine evaluation and testing for induction of long-term protective immunity.     

Role of CD80 and CD86 in host immune responses to the recombinant hemagglutinin domain of Porphyromonas gingivalis gingipain and in the adjuvanticity of cholera toxin B and monophosphoryl lipid A

The gingipains of Porphyromonas gingivalis have been implicated in the virulence of this bacterium, and antibodies to the hemagglutinin/adhesin domain (HArep) of the gingipains have been shown to protect against P. gingivalis colonization. However, the cellular mechanisms involved in host responses to HArep have not been elucidated. The purpose of the present study was to determine the functional role of CD80 and CD86 in mediating systemic and mucosal immune responses to the recombinant HArep derived from the gingipain Kgp (Kgp-HArep) after intranasal (i.n.) immunization. We also investigated the effect of the mucosal adjuvants the B subunit of cholera toxin (CTB) and monophosphoryl lipid A (MPL) on the functional role of the costimulatory molecules for the induction of systemic and mucosal responses to Kgp-HArep. The in vivo functional roles of CD80 and CD86 were assessed in C57BL/6 wild-type (wt), CD80(-/-), CD86(-/-) and CD80/CD86(-/-) mice following intranasal immunization with Kgp-HArep with or without adjuvant. Serum IgG and mucosal IgA antibody responses were induced following i.n. immunization of mice with Kgp-HArep, and were potentiated by CTB or MPL. A differential requirement of CD80and/or CD86 was observed for systemic IgG anti-Kgp-HArep responses following the primary and secondary immunization with antigen alone or antigen+adjuvant. Compared to wt and CD80(-/-) mice, CD86(-/-) mice had reduced serum IgG anti-Kgp-HArep responses following the second immunization with antigen alone or antigen+CTB, whereas similar levels of serum IgG anti-Kgp-HArep antibody activity were observed in wt, CD80(-/-) and CD86(-/-) mice immunized with antigen+MPL. Analysis of the serum IgG subclass responses revealed that CD80 influenced both Th1- and Th2-like IgG subclass responses, while CD86 preferentially influenced a Th2-associated IgG subclass response to Kgp-HArep. Mucosal IgA anti-Kgp-HArep responses in saliva and vaginal washes were diminished in CD86(-/-) mice. In vitro stimulation of murine bone marrow-derived dendritic cells with Kgp-HArep, CTB and MPL resulted in an up-regulation of CD80 and especially CD86 expression. Taken together, our results demonstrate that CD80 and CD86 can play distinct as well as redundant roles in mediating a systemic immune response and that CD86 plays a unique role in mediating a mucosal response to Kgp-HArep following immunization via the i.n. route alone or with adjuvant.     

Oral administration of polymer-grafted starch microparticles activates gut-associated lymphocytes and primes mice for a subsequent systemic antigen challenge

The mucosal and systemic humoral immune systems can function essentially independent of each other, responding to mucosal and parenteral antigens, respectively. Nevertheless, antigen administered by one route can modify responsiveness to subsequent immunization by an alternate route. Here we demonstrated, in mice, in addition to stimulating rapid and robust sera antibody responses, intragastric (i.g.) immunization with human serum albumin (HSA)-containing starch microparticles (MP) grafted with 3-(triethoxysilyl)-propyl-terminated polydimethylsiloxane (TS-PDMS) primed for enhanced specific sera IgG following a parenteral antigen boost. After as little as one i.g. immunization with microentrapped, but not with soluble, HSA antigen-specific proliferation and antibody secretion were detected in Peyer's pathches (PP); this activity peaked after three i.g. MP immunizations. We observed a progressive dissemination of antigen-specific lymphocyte reactivity from PP to splenic tissue following oral MP immunization. Similarly, we observed a shift in HSA-specific antibody-secreting cells from PP and mesenteric lymph nodes to splenic tissue following i.g. MP immunization. We also demonstrated that oral immunization with microentrapped, but not with soluble HSA, resulted in enhanced numbers of spontaneous Th2-cytokine secreting lymphocytes which disseminated from mucosal to systemic lymphoid compartments. This observation coincided with our findings that HSA-specific sera IgG1 responses in animals given HSA in MP were significantly higher than those detected in the sera of mice given soluble HSA i.g., both before and after parenteral antigen challenge. These findings suggest that orally-administered TS-PDMS-grafted MP, by stimulating elements of the mucosal immune system, are a valuable addition to mucosal and systemic vaccine delivery systems.     

Flagellin produced in plants is a potent adjuvant for oral immunization

The aim of this study was to produce adjuvant with high biosafety, efficacy and low cost. Towards this goal, the plant Nicotiana benthamiana transient expression system was successfully used to express Salmonella typhimurium's flagellin (FljB). The yield of the expressed FljB was 280 mg per kg of fresh weight (FW) leaves. The lyophilized plant powder containing plant expressing FljB was mixed with ovalbumin (OVA) and used for oral immunization of BALB/c mice. The ELISA analysis showed higher and accelerated OVA-specific serum antibody responses in mice given the mixture when compared to animals receiving OVA alone. Furthermore, FljB elicited a mixed Th1/Th2 response as shown by the presence of specific anti-OVA IgG1, IgG2a and IgG2b isotypes. OVA-specific IgAs were also detected in mice given the mixture. Cell-mediated immune response to OVA was induced by FljB as determined by a spleen lymphocyte specific proliferation test. No immune response was generated against FljB. In conclusion, our results showed for the first time the production of FljB in plants and the efficient use of the crude lyophilized extract as an adjuvant for oral immunization.     

Oral immunization of mice with a multivalent therapeutic subunit vaccine protects against Helicobacter pylori infection

Helicobacter pylori is a human class I carcinogen and no effective prophylactic or therapeutic H. pylori vaccine has yet been marketed. H. pylori can escape the host immune response, but the precise immune protection mechanisms in humans remain unknown. In this study, we developed a multivalent, subunit H. pylori vaccine candidate by formulating three commonly used H. pylori antigens, neutrophil-activating protein (NAP), urease subunit A (UreA) and subunit B (UreB) with the mucosal adjuvant, a double-mutant heat-labile toxin (dmLT) from Escherichia coli, and evaluated its immunogenicity and therapeutic efficacy in a mouse model of H. pylori infection. We found that oral immunization of H. pylori-infected mice significantly reduced gastric bacterial colonization at both 2 and 8 weeks after immunization. The reduction in bacterial burdens was accompanied with significantly increased serum antigen-specific IgG responses and mucosal IgA responses. Moreover, oral immunization also induced Th1/Th17 immune responses, which may play a synergistic role with the specific antibodies in the elimination of H. pylori. Thus, our vaccine candidate appears able to overcome the immune evasion mechanism of H. pylori, restore the suppression of Th2 immune responses with the induction of a strong humoral immune response. These results lay the foundation for the development of an optimized oral therapeutic H. pylori vaccine with increased immunogenicity of UreA and UreB, as well as providing long-term immunity.     

Salmonella flagellins are potent adjuvants for intranasally administered whole inactivated influenza vaccine

Bacterial flagellins are potent inducers of innate immune responses in the mouse lung because they bind to TLR5 expressed on the apical surfaces of airway epithelial cells. TLR engagement leads to the initiation of a signaling cascade that results in a pro-inflammatory response with subsequent up-regulation of several cytokines and leads to adaptive immune responses. We examined the ability of two soluble flagellins, a monomeric flagellin expressed in Escherichia coli and a highly purified polymeric flagellin directly isolated from Salmonella, to enhance the efficacy of influenza vaccines in mice. Here we demonstrate that both flagellins co-administered intranasally with inactivated A/PR/8/34 (PR8) virus induced robust increases of systemic influenza-specific IgA and IgG titers and resulted in a more comprehensive humoral response as indicated by the increase of IgG2a and IgG2b subclass responses. Groups immunized with the adjuvanted vaccines were fully protected against high dose lethal challenge by homologous virus whereas inactivated PR8 alone conferred only partial protection. Finally we show that shortly after immunization the adjuvanted vaccines induced a dramatic increase in pro-inflammatory cytokines in the lung, resulting in extensive lung infiltration by granulocytes and monocytes/macrophages. Our results reveal a promising perspective for the use of both soluble monomeric and polymeric flagellin as mucosal vaccine adjuvants to improve protection against influenza epidemics as well as a range of other infectious diseases.     

Phosphorylcholine intranasal immunization with a 13-valent pneumococcal conjugate vaccine can boost immune response against Streptococcus pneumoniae

ObjectiveThis study aimed to investigate whether systemic immunization with a 13-valent pneumococcal conjugate vaccine (PCV13) followed by intranasal (IN) immunization with phosphorylcholine (PC) can boost immune response against Streptococcus pneumoniae.Materials and methodsTwo weeks after the intraperitoneal (IP) injection of PCV13, mice were divided into two groups (mice requiring another IP injection of PCV13 and mice requiring PC-keyhole limpet hemocyanin IN immunization in combination with cholera toxin as a mucosal adjuvant) to compare the magnitude of systemic and mucosal immune responses against S. pneumoniae and PC.ResultsSerum immunoglobulin (Ig) G antibody titer against the vaccine strains of S. pneumoniae was similar between the PCV13 systemic immunization group and PC IN immunization group, while the serum IgG antibody titer against PC was significantly higher in the PC IN immunization group. PC-specific IgA antibody titer in the nasal lavage and PC-specific IgA-producing cell number in the nasal mucosa were also significantly higher in the PC IN immunization group. Induction of PC-specific IgA in the PC IN immunization group enhanced the clearance of bacteria from the middle ear.ConclusionAdditional IN immunization with PC after PCV13 immunization, which is currently conducted under a periodic vaccination program, can produce a booster effect comparable to that achieved by additional systemic immunization as well as PC-specific mucosal immune response, thereby providing protection against S. pneumoniae serotypes not contained in PCV13.     

Safety and immunogenicity of phoP/phoQ-deleted Salmonella typhi expressing Helicobacter pylori urease in adult volunteers.

Salmonella typhi Ty800, deleted for the Salmonella phoP/phoQ virulence regulon has been shown to be a safe and immunogenic single dose oral typhoid fever vaccine in volunteers. This promising vaccine strain was modified to constitutively express a heterologous protein of Gram negative bacterial origin, Helicobacter pylori urease subunits A and B, yielding S. typhi strain Ty1033. Seven volunteers received single oral doses of > or = 10(10) colony forming units of Ty1033; an eighth volunteer received two doses 3 months apart. Side effects were similar to those observed previously in volunteers who received the unmodified vector Ty800. All volunteers had strong mucosal immune responses to vaccination as measured by increases in IgA-secreting cells in peripheral blood directed against S. typhi antigens. Seven of eight volunteers had convincing seroconversion as measured by increases in serum IgG directed against S. typhi flagella and lipopolysaccharide antigens by ELISA. No volunteer had detectable mucosal or humoral immune responses to the urease antigen after immunization with single doses of Ty1033. A subset of three volunteers received an oral booster vaccination consisting of recombinant purified H. pylori urease A/B and E. coli heat labile toxin adjuvant 15 days after immunization with Ty1033. None of three had detectable humoral or mucosal immune responses to urease. Expression of a stable immunogenic protein in an appropriately attenuated S. typhi vector did not engender detectable mucosal or systemic antibody responses; additional work will be needed to define variables important for immunogenicity of heterologous antigens carried by live S. typhi vectors in humans.     

IgA and IgG antibody responses following systemic immunization of cattle with native H7 flagellin differ in epitope recognition and capacity to neutralise TLR5 signalling

Systemic immunization of cattle with H7 flagellin results in induction of both H7-specific IgA and IgG antibodies but only partially protects against subsequent colonization with Escherichia coli O157:H7. Recent studies indicate that anti-flagellin antibodies directed against TLR5 binding domains located in the conserved N- and C-terminal domains of flagellin can neutralise TLR5 activation and impair vaccine efficacy. In the current study we determined whether systemic immunization of cattle with H7 flagellin induces antibodies capable of interfering with flagellin-mediated TLR5 activation. Both anti-H7 IgG1 and IgG2 but not IgA antibodies recognised epitopes within the conserved N- and C-terminal domains of H7 flagellin, and purified H7-specific IgG but not IgA was capable of inhibiting H7-mediated TLR5 activation in vitro. These results suggest that (i) IgA and IgG isotypes originated from different populations of B cells and (ii) systemically induced H7-specific IgG but not IgA may impair innate immune responses to E. coli O157:H7 via neutralisation of TLR5 activation and subsequently reduce vaccine efficacy.     

Induction of mucosal IgA by a novel jet delivery technique for HIV-1 DNA

Novel ways of delivering plasmid DNA to elicit humoral IgA, IgG and cell-mediated immune responses in mice were investigated. Intraoral administration of DNA in the cheek, using a jet immunization technique, elicited the highest IgA mucosal responses. Intranasal immunization gave strong mucosal IgA responses and persistent systemic IgG. Immunoglobulin isotype analysis revealed an IgG1 profile for intramuscular tongue and gene gun immunizations and an IgG2a profile following oral jet injection and intranasal application. The route of delivery was of importance for the characteristics and quality of the mucosal immune response following DNA immunization. For DNA vaccine delivery, the intraoral jet injection technique has the advantages of being a simple and rapid way of administering the DNA in solution and of provoking specific mucosal IgA when administered in the mucosal associated lymphoid tissue.     

Sublingual immunization induces broad-based systemic and mucosal immune responses in mice

The potential of sublingual (s.l.) delivery of vaccine was examined in mice. We show the existence of a dense network of dendritic cells (DCs) in the s.l. epithelium and a rapid and transient increase in the frequency of s.l. DCs after topical application of cholera toxin (CT) adjuvant under the tongue. S.l. immunization with ovalbumin and CT induced vigorous systemic and mucosal antibody responses. Such treatment promoted mixed Th1 and Th2 cytokine responses and induced cytotoxic CD8(+) T cells in lung tissues and in systemic lymphoid organs. S.l. immunization was comparable to intranasal immunization and was superior to oral immunization regarding the magnitude and anatomic dissemination of the induced immune responses. S.l. administration of live influenza virus at a dose lethal by the nasal route was well tolerated and did not redirect virus to the olfactory bulb. These features underscore the potential of the s.l. mucosa to serve as an alternative vaccine delivery route.     

Immunization with recombinant Lactobacillus casei strains producing K99, K88 fimbrial protein protects mice against enterotoxigenic Escherichia coli

To exploit a safe and effective vaccine for the prevention against K99 or K88 infections of enterotoxigenic Escherichia coli (ETEC), we have developed a mucosal delivery vehicle based on Lactobacillus casei CICC 6105 using poly-γ-glutamate synthetase A (PgsA) as an anchoring matrix. To evaluate the immunization effect of the recombinant strains (harboring plasmids pLA-K99-K88-LTB, pLA-K99, and pLA-K88), anti-ETEC K99 or K88 antibody responses, T-cell proliferation, and cytokines by intracellular staining (ICS) were investigated after specific pathogen-free (SPF) C57BL/6 mice orally inoculated with these recombinant strains. After oral vaccination into C57BL/6 mice, all recombinant strains were proved to be immunogenic and able to elicit high levels of mucosal immunoglobulin A (IgA) titers in bronchoalveolar lavage fluids, intestinal fluids and prominent systemic immunoglobulin G and IgG subclasses (IgG1, IgG2b, and IgG2a) responses in sera. Using the T-cell proliferation assay, the stimulation index (SI) of groups immunized with pLA-K99/L. casei and pLA-K88/L. casei reached to 2.73 and 2.64, respectively, versus 2.56 in a group immunized with pLA-K99-K88-LTB/L. casei. A detailed analysis of the cell-mediated immune responses by ICS showed the number of specific CD8(+) T cells expressing cytokines (IFN-γ, TNF-α, and IL-2) and granule-associated proteins (CD107a) was higher than that of specific CD4(+) T cells secreted by immune spleen cells upon restimulation in vitro with peptides. Next, the results showed that DCs activated in vitro with recombinant L. casei enhance specific T-cell proliferation and promote T cells to produce both Th1 and Th2 cytokines. More than 80% of the vaccinated mice were protected after challenge with a lethal dose of standard strains C83912 and C83902. These results demonstrate that recombinant L. casei can induce specific humoral and mucosal antibodies and cellular immune response against protective antigens upon oral administration.