Class specific influence of dietary Spirulina platensis on antibody production in mice

In the present study, we investigated antibody productions of IgA and other classes, such as IgE and IgG1, in mice as possible evidence of the protective effects of Spirulina toward food allergy and microbial infection. An increase of IgE antibody level in the serum was observed in the mice that were orally immunized with crude shrimp extract as an antigen (Ag group). The antibody level, however, was not further enhanced by treatment with Spirulina extract (SpHW). IgG1 antibody, on the other hand, which was increased by antigen administration, was further enhanced by Spirulina extract. It was noted that the IgA antibody level in the intestinal contents was significantly enhanced by treatment with Spirulina extract concurrently ingested with shrimp antigen, in comparison with that of the Ag group treated with shrimp antigen alone. An enhancement of IgA antibody production by Spirulina extract was also observed in culture supernatant of lymphoid cells, especially in the spleen and mesenteric lymph node from mice treated with Spirulina extract for 4 weeks before antigen stimulation. These results suggest that Spirulina may at least neither induce nor enhance allergic reaction such as food allergy dependent on an IgE antibody, and that when ingested both concurrently with antigen and before antigen stimulation, it may significantly enhance the IgA antibody level to protect against allergic reaction.     

Induction of immune responses in pigs following oral administration of purified F4 fimbriae

An effective way of stimulating the mucosal immune system was examined in piglets, using F4 fimbriae of enterotoxigenic Escherichia coli (ETEC). It was demonstrated that purified F4 fimbriae, as opposed to ovalbumin (OVA), are powerful oral immunogens. Indeed, oral administration of purified F4 induced antigen-specific antibody-secreting cells (ASC) in the Peyer's patches, mesenteric lymph nodes (LN), blood and lamina propria 4, 7, 9 and 11 days postimmunization, respectively, indicating a stimulation of the mucosal immune system, whereas upon oral administration of OVA, no immune response was observed. Moreover, the induced F4-specific IgA and IgG antibody responses were comparable with those obtained upon oral infection with viable E. coli and intramuscular (i.m.) F4 injection, respectively. Furthermore, a priming of the mucosal immune system is better obtained by oral infection (ASC localized in mesenteric LN) than by i.m. F4 injection (ASC localized in spleen and retropharyngeal LN) since an oral boost with purified F4 induced a secondary response in the orally infected animal (mainly IgA and IgG ASC, rapid increase of IgA antibodies) while in the i.m. primed animal a secondary (more circulating antigen-specific ASC than in the unprimed animal) as well as a primary IgM and IgA response (mainly IgM ASC, slow increase of IgA antibodies), suggesting a primary mucosal response, were seen. An oral challenge of the naive control displayed a primary response (mainly IgM ASC, slow increase of IgA and IgG antibodies). The capacity of purified F4 to activate the mucosal immune system on oral administration, is of importance for the development of oral vaccines against ETEC infections.     

Mucosal and systemic immune responses by intranasal immunization using archaeal lipid-adjuvanted vaccines

The utility of archaeal polar lipids as an adjuvant/delivery system for elicitation of antigen-specific mucosal immune responses in intranasally administered vaccines was investigated. Although unilamellar archaeosomes (liposomes made from archaeal polar lipids) with encapsulated ovalbumin (OVA/archaeosomes) induced anti-OVA IgG antibody responses in sera, they failed to induce anti-OVA IgA antibody responses at mucosal sites. However, the addition of CaCl2 to convert OVA/archaeosomes into an archaeal lipid mucosal vaccine adjuvant and delivery (AMVAD) vaccine (OVA/AMVAD) consisting of larger, particulate, aggregated structures resulted in an efficacious intranasal (i.n.) vaccine. Intranasal immunization of mice with OVA/AMVAD vaccines prepared from various archaeal polar lipid compositions elicited anti-OVA IgA antibody responses in sera, feces, bile, vaginal and nasal wash samples. The i.n. immunization also induced anti-OVA IgG, IgG1 and IgG2a antibody responses in sera, as well as cytotoxic T lymphocyte responses. The mucosal and systemic immune responses induced by OVA/AMVAD immunization were generally sustained over several months, and were subject to memory boost responses. Thus, polar archaeal lipids appear to be promising for developing a non-replicating mucosal adjuvant and vaccine delivery system.     

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.     

Characterisation of the oral adjuvant effect of lemnan, a pectic polysaccharide of Lemna minor L

Lemnan LM, apiogalacturonanic pectin of duckweed Lemna minor L. was tested for adjuvant properties following oral administration with protein antigen. Male Swiss mice were orally immunized thrice with weekly intervals with free OVA or OVA with lemnan (LM). Lemnan LM was shown to increase delayed type hypersensitivity (DTH) and serum anti OVA IgG responses. LM was established to increase levels of both serum IgG1 and IgG2a subclasses, intestinal IgA and failed to elevate levels of serum IgE. Lemnan was found to increase the adhesion of macrophages and to enhance the generation of oxygen radicals by macrophages in response to phorbol 12-myristate 13-acetate. Serum OVA levels were four-fold higher in mice immunized with the mixture of OVA and LM in comparison with those in mice immunized with OVA only. Thus, substantial systemic and local mucosal immune responses were attained by oral immunization with the mixture of OVA and lemnan. Lemnan appeared to elicit adjuvant activity via induction of both Th1- and Th2-type responses. The immunopotentiating effect of lemnan may result from enhanced antigen ingestion and stimulation of macrophage activity.     

OVA-bound nanoparticles induce OVA-specific IgG1, IgG2a,and IgG2b responses with low IgE synthesis

There is an urgent requirement for a novel vaccine that can stimulate immune responses without unwanted toxicity, including IgE elevation. We examined whether antigen ovalbumin (OVA) conjugated to the surface of nanoparticles (NPs) (OVA-NPs) with average diameter of 110 nm would serve as an immune adjuvant. When BALB/c mice were immunized with OVA-NPs, they developed sufficient levels of OVA-specific IgG1 antibody responses with low levels of IgE synthesis, representing helper T (Th)2-mediated humoral immunity. OVA-specific IgG2a and IgG2b responses (i.e., Th1-mediated immunity) were also induced by secondary immunization with OVA-NPs. As expected, immunization with OVA in alum (OVA-alum) stimulated humoral immune responses, including IgG1 and IgE antibodies, with only low levels of IgG2a/IgG2b antibodies. CD4-positive T cells from mice primed with OVA-NPs produced substantial levels of IL-21 and IL-4, comparable to those from OVA-alum group. The irradiated mice receiving OVA-NPs-primed B cells together with OVA-alum-primed T cells exhibited enhanced anti-OVA IgG2b responses relative to OVA-alum-primed B cells and T cells following stimulation with OVA-NPs. Moreover, when OVA-NPs-primed, but not OVA-alum-primed, B cells were cultured in the presence of anti-CD40 monoclonal antibody, IL-4, and IL-21, or LPS plus TGF-β in vitro, OVA-specific IgG1 or IgG2b antibody responses were elicited, suggesting that immunization with OVA-NPs modulates B cells to generate IgG1 and IgG2b responses. Thus, OVA-NPs might exert their adjuvant action on B cells, and they represent a promising potential vaccine for generating both IgG1 and IgG2a/IgG2b antibody responses with low IgE synthesis.     

Induction of systemic and mucosal antibody responses in mice immunized intranasally with aluminium-non-adsorbed diphtheria toxoid together with recombinant cholera toxin B subunit as an adjuvant

Nasal mucosal immunization is very attractive for vaccination to prevent various bacterial and viral infectious diseases because of induction of systemic and mucosal immune responses. The aim of the present study was to investigate the possibility of changing the immunization procedure of diphtheria toxoid (Dt) from intramuscular or subcutaneous injection to intranasal administration. Intranasal immunization with aluminium-non-adsorbed diphtheria toxoid (nDt) together with recombinant cholera toxin B subunit (rCTB, 10 μg) induced, at a concentration of 5 Lf, high levels of serum Dt-specific IgG antibody responses and high or moderate levels of the specific IgA antibody responses in all mice and only a slight level of the specific IgE antibody responses in some mice. Furthermore, sufficiently high diphtheria antitoxin titres more than 0.1 international units (IU) ml⁻¹ were obtained from mice which showed high levels of serum Dt-specific IgG antibody responses. Under the same experimental conditions, induction of significant levels of mucosal Dt-specific IgA antibody responses occurred in the nasal cavity, the lung, the saliva and vaginal secretions and the small and large intestines of all mice, although there were different titres between individual mice. Similar results were also obtained with rCTB-specific serum IgG and IgA and mucosal IgA antibody responses; serum rCTB-specific IgE antibody titres were not detected. These results show that intranasal administration of nDt with rCTB must be a very useful means for vaccination against diphtheria.     

Abrogation of the oral tolerance to ovalbumin in mice by citrus pectin

ObjectiveWe studied the effects of dietary pectins (citrus pectin [CP] and apple pectin) on oral tolerance in mice.MethodsPectins (1 mg/d) were administered orally for 2 wk. Tolerance was induced with 20 mg of ovalbumin (OVA). Levels of serum antibodies (immunoglobulin [Ig] G, IgG1, IgG2a, IgE) and delayed type hypersensitivity response determined in footpad tests were measured after subcutaneous injection of OVA with complete Freund's adjuvant. Concentrations of immunoreactive OVA in blood were measured by enzyme-linked immunosorbent assay after feeding the animals 20 mg of OVA. Adhesion and cytokine production (tumor necrosis factor-α, interferon-γ) were measured in peritoneal macrophages.ResultsOral administration of CP was found to prevent the induction of immune hyporesponsiveness induced by OVA feeding. Animals fed OVA and CP were found to produce similar titers of antigen-specific serum IgG and levels of delayed type hypersensitivity response as those animals not fed OVA. CP increased levels of serum IgG1 and IgE. CP was found to enhance the penetration of immunogenic OVA into the serum. CP (1 mg/d) administered orally for 1 wk was also observed to enhance the adhesion and production of cytokines (tumor necrosis factor-α, interferon-γ) in peritoneal macrophages.ConclusionCP administered orally was shown to inhibit oral tolerance. Enhancement of protein antigen penetration to the blood and activation of macrophages were found to precede the inhibitory effect and appeared to mediate it.     

Dietary ribonucleotides modulate type 1 and type 2 T-helper cell responses against ovalbumin in young BALB/cJ mice

Dietary ribonucleotides have been shown to augment type 1 T-helper cell (Th1) responses to a protein antigen (Ag) in Th1-prone C57BL/6 mice, but their effects on type 2 Th (Th2)-prone mice are unknown. BALB/cJ mice have skewed Th2 responses against ovalbumin (OVA), characterized by augmented production of Th2 cytokines and immunoglobulin (Ig)G1/IgE antibodies (Ab); Th1 responses augment IgG2a Ab production, whereas Th2 responses augment IgG1/IgE Ab production. In this study, we determined the effects of dietary ribonucleotides obtained from yeast on the balance of Th1/Th2 responses against OVA in young BALB/cJ mice. Mice were fed a ribonucleotide-free (NF) or ribonucleotide-supplemented (NS) diet (4.74 g nucleotides/kg diet) and given OVA (10 microg/dose) with incomplete Freund's adjuvant (IFA) at 3 and 6 wk. We assessed T-cell responses in the regional draining lymph nodes (LN) by measuring production and expression of Th1/Th2 cytokines, interferon-gamma (IFN-gamma) and interleukin-5 (IL-5), respectively. Anti-OVA IgG subclass and IgE Ab levels were determined 3 wk after the first OVA challenge and 5 d and 2 wk after the second OVA challenge. Dietary ribonucleotides significantly augmented OVA-specific IFN-gamma production by the regional draining LN cells after the first and second OVA challenges. The NS diet increased anti-OVA IgG2a Ab levels after the first OVA challenge and both anti-OVA IgG2a and anti-OVA IgG2b after the second challenge. OVA-specific IgG1 and IgE Ab levels were lower (P < 0.05) after the second OVA challenge in mice fed the NS diet. Dietary ribonucleotides did not affect production or expression of IL-5. Our findings thus indicate that in Th2-prone BALB/c J mice, dietary ribonucleotides modulated skewed Th2 responses against OVA toward Th1 as measured by production of IFN-gamma, a Th1 cytokine, and changes in anti-OVA Ab isotype levels.     

Controlled release microparticles for vaccine development.

The primary and secondary sera IgG antibody responses to ovalbumin (OVA) entrapped in biodegradable poly(lactide-co-glycolide) (PLGA) microparticles were compared with the responses obtained with soluble OVA. In addition, OVA in PLGA microparticles was also administered after dispersion in an immunostimulatory vehicle, Freund's incomplete adjuvant (FIA). The primary IgG responses to OVA in microparticles/FIA were significantly greater than the responses to soluble OVA from day 14 to day 42, when booster immunizations were administered. From day 49 to the end of the study at day 84, the responses to OVA, both in microparticles alone and in microparticles/FIA, were significantly greater than the responses to soluble OVA. Nevertheless, the responses obtained for OVA in microparticles or microparticles/FIA were, in general, not as high as those obtained with OVA in Freund's complete adjuvant.     

Functional differences in IgG anti-polysaccharide antibodies elicited by immunization of mice with C3d versus ovalbumin conjugates of pneumococcal serotype 14 capsular polysaccharide

We previously have shown that conjugation of C3d to pneumococcal serotype type 14 capsular polysaccharide (PPS14) significantly enhances anti-PPS14 antibody production to a degree similar to that found when the T-dependent protein carrier ovalbumin (OVA) is coupled to PPS14. However, the anti-PPS14 antibody response to PPS14–C3d conjugates is characterized by less switching from IgM to IgG and lower serum concentrations of anti-PPS14 IgG after secondary immunization. To determine if these quantitative differences in anti-PPS14 IgG are accompanied by qualitative differences in the IgG anti-PPS14 antibodies, we performed several functional assays on serum IgG anti-PPS14 antibodies from mice immunized with PPS14–C3d or PPS14–OVA. Compared with antibodies elicited by immunization with PPS14–C3d, IgG anti-PPS14 antibodies produced after immunization with PPS14–OVA were found to have higher avidity and enhanced function as opsonins. Comparisons of avidity for IgG from serum samples obtained after primary and secondary immunization demonstrated a higher degree of avidity maturation after immunization with PPS14–OVA than with PPS14–C3d. These results suggest that PPS14–C3d conjugates are unlikely to be more efficacious than PPS14 conjugate vaccines incorporating T-dependent protein carriers.     

PLG microparticles stabilised using enteric coating polymers as oral vaccine delivery systems.

Novel poly(dl-lactide-co-glycolide) microparticles for oral vaccine delivery were formulated using the enteric polymers Eudragit L100-55 and carboxymethylethylcellulose (CMEC) as stabilisers. To serve as a control, microparticles were also produced using the conventional PVA surfactant. In all three cases the antigen, ovalbumin (OVA)-loaded microparticles produced were less than 5 microm in diameter and had a spherical, smooth rounded appearance. The presence of surfactants at the microparticle surface was demonstrated by the surface analysis techniques, XPS and SSIMS. Incubation of microparticles with solutions of pepsin or trypsin led to the removal of a proportion of the antigen associated with all three systems. However, in three CMEC-stabilised microparticle formulations and one of three Eudragit formulations, a high percentage of the associated antigen was protected from removal by a solution of pepsin at pH 1.2 compared with the PVA-stabilised microparticles. In addition, with certain CMEC and Eudragit formulations a degree of protection was also afforded to the associated OVA against removal by trypsin at pH 7.4. Following the incubation of microparticles in simulated gastric fluid a higher percentage of intact antigenic OVA was detected in microparticles stabilised using CMEC than in the PVA- and Eudragit- stabilised formulations. Oral immunisation of mice with OVA-loaded microparticles stabilised using either of the three surfactants led to the induction of specific serum IgG and salivary IgA antibodies. Significantly higher levels of specific salivary IgA antibody to OVA were measured in mice immunised with the CMEC-stabilised microparticles than with the other two formulations. This novel approach in PLG microparticle formulation may have potential in increasing the efficacy of microparticulate systems for the oral administration of vaccines.     

Enhanced induction of the IgA response in pigs by calcitriol after intramuscular immunization

In this study, the immunomodulating effect of two steroid hormones namely 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and dehydroepiandrosterone (DHEA) was examined on the antigen-specific antibody responses by intramuscular immunization of pigs with human serum albumin alone (HSA) or supplemented with 2 microg of 1,25(OH)(2)D(3,) 40 microg of DHEA or the combination of both steroids. 1,25(OH)(2)D(3) significantly enhanced the antigen-specific IgA and IgM serum response. Higher HSA-specific IgA titers were also found in the mucosal secretions (saliva, feces and nasal) of the steroid treated animals, especially in the 1,25(OH)(2)D(3) group. Furthermore, 1,25(OH)(2)D(3) and DHEA increased the number of antigen-specific IgA and IgG antibody-secreting cells in the local draining lymph nodes, but only low numbers were detected in lymph nodes draining the mucosa. DHEA decreased the IgM serum response and had a tendency to enhance the IgG2 and IgG serum responses. Strong and comparable IgG, IgG1 and IgG2 serum responses were seen in all groups. Combining both steroids did not result in a higher IgA serum response. On the contrary DHEA seems to neutralize the effect of 1,25(OH)(2)D(3) on the IgA response. In conclusion, 1,25(OH)(2)D(3) significantly enhanced the antigen-specific IgA and IgM response in serum and the number of antigen-specific IgA and IgG ASC in the local draining lymph nodes following intramuscular immunization.     

PCPP (poly[di(carboxylatophenoxy)-phosphazene]) microparticles co-encapsulating ovalbumin and CpG oligo-deoxynucleotides are potent enhancers of antigen specific Th1 immune responses in mice

We generated poly[di(carboxylatophenoxy)-phosphazene] (PCPP) microparticles encapsulating ovalbumin (OVA) and CpG of 0.5-2.5 μm in diameter with an encapsulation efficiency of approximately 63% and 95% respectively. In mice the microparticles generated high antigen-specific IgG, IgG1 and IgG2a titers with higher IgG2a/IgG1 ratios. Whole body in vivo imaging of mice subcutaneously injected with MPs showed several fold increase of OVA and CpG in draining inguinal lymph nodes compared to soluble formulations. We conclude that PCPP MPs are more effective in enhancing immune responses compared to soluble formulations, due to co-delivery of OVA and CpG resulting in a Th1 type of immune response.     

Mucosal and systemic antibody responses against an acellular pertussis vaccine in mice after intranasal co-administration with recombinant cholera toxin B subunit as an adjuvant

To investigate the possibility of intranasal immunization with an acellular pertussis vaccine, groups of mice were administered intranasally with aluminium-non-adsorbed pertussis toxoid (PTd; 0.5 or 5 microg) and formalin-treated filamentous hemagglutinin (fFHA; 5 microg) with and without recombinant cholera toxin B subunit (rCTB; 10 microg) as a mucosal adjuvant. At a low concentration of PTd, the following things became clear: (1) earlier and higher elevation of serum anti-PTd and anti-FHA IgG antibody titres in the presence of rCTB than in its absence, (2) higher serum anti-PTd and anti-FHA IgG antibody titres than 200 and 100 ELISA units ml(-1) (EU ml(-1)) in all mice, respectively, in the presence of rCTB, which were obtained by calibration against a reference anti-pertussis mouse serum, and (3) in an intranasal challenge experiment with Bordetella pertussis, slightly more rapid elimination of the bacteria from the lungs of mice intranasally immunized in the presence of rCTB, suggesting the effectiveness of rCTB as a mucosal adjuvant. However, irrespective of rCTB and dose of PTd, mice which were immunized four times and sacrificed on day 35 developed high levels of anti-PTd serum IgG antibodies, high or moderate levels of anti-FHA serum IgG antibodies and mucosal anti-PTd IgA antibodies in the lungs; only a slight or no increase of anti-FHA mucosal IgA antibodies was observed in the lung. These facts suggested the immunogenicity and mucosal adjuvanticity of PTd, and therefore, the mucosal adjuvanticity of rCTB seemed to be inconspicuous. Moreover, the addition of rCTB induced higher anti-PTd serum IgE antibody responses than no addition of it depending on dose of PTd. These results show that dose of PTd included in an acellular pertussis vaccine had better be low as possible and the addition of rCTB may not be always necessary in case of this nasal vaccine alone unlike tetanus and diphtheria toxoids and hepatitis B virus vaccine reported before.     

A comparison of non-toxin vaccine adjuvants for their ability to enhance the immunogenicity of nasally-administered anthrax recombinant protective antigen

Development of nasal immunization for human use is hindered by the lack of acceptable adjuvants. Although CT is an effective adjuvant, its toxicity will likely prevent its use in nasal vaccines. This study compared non-toxin adjuvants to CT for their ability to induce protective antibody responses with nasal immunization. C3H/HeN and C57BL/6 mice were immunized with rPA formulated with the following adjuvants: CT, IL-1α, LPS, CpG, Pam3CSK4, 3M-019, resiquimod/R848 or c48/80. Serum and nasal wash cytokine concentrations were monitored 6 h post-vaccination as biomarkers for acute activation of the innate immune system. Not all of the adjuvants induced significant changes in innate serum or nasal wash cytokines, but when changes were observed, the cytokine signatures were unique for each adjuvant. All adjuvants except Pam3CSK4 induced significantly increased anti-rPA serum IgG titers in both strains of mice, while only IL-1α, c48/80 and CpG enhanced mucosal anti-rPA IgA. Pam3CSK4 was the only adjuvant unable to enhance the induction of serum LeTx-neutralizing antibodies in C3H/HeN mice while c48/80 was the only adjuvant to induce increased serum LeTx-neutralizing antibodies in C57BL/6 mice. Only CT enhanced total serum IgE in C3H/HeN mice while IL-1α enhanced total serum IgE in C57BL/6 mice. The adjuvant influenced antigen-specific serum IgG subclass and T cell cytokine profiles, but these responses did not correlate with the induction of LeTx-neutralizing activity. Our results demonstrate the induction of diverse innate and adaptive immune responses by non-toxin nasal vaccine adjuvants that lead to protective humoral immunity comparable to CT and that these responses may be influenced by the host strain.     

Rectal immunization of mice with hepatitis A vaccine induces stronger systemic and local immune responses than parenteral immunization

Systemic (spleen cell (SPLC), serum antibodies) and intestinal mucosal (Peyer's patch cells (PPC), lamina propria lymphocytes (LPLs), coproantibodies) immune responses were compared in mice immunized with varying doses (144, 72, 36, 18 ELISA units [EU]) of HAVRIX, an alum-adsorbed killed hepatitis A virus (HAV) vaccine, delivered either intrarectally (i.r.) or intraperitoneally (i.p.) in three doses at weekly intervals. HAV-specific IgG, IgM, and IgA antibody responses were evaluated by ELISPOT and EIA and HAV-responsive lymphocytes by lymphocyte stimulation assays. Systemic IgG responses were greater in mice immunized intraperitoneally with 144, 72, and 36EU of HAVRIX, while IgM and IgA responses were greater in PPC and LPL cell populations, serum and coproantibodies of rectally immunized mice, particularly at HAVRIX doses of 36 and 18EU. Rectal immunization at lower doses (36, 18EU) also elicited strong cellular responses in all cell populations while parenteral (i.p.) vaccination, did not. Results suggest that rectal immunization may be a highly effective way of inducing both local and systemic immunity to HAV.     

A virus-like particle vaccine platform elicits heightened and hastened local lung mucosal antibody production after a single dose

We show that a model antigen, ovalbumin (OVA), can be chemically conjugated to the exterior of a small heat shock protein (sHsp) cage that has structural similarities to virus-like particles (VLPs). OVA–sHsp conjugation efficiency was dependent upon the stoichiometry and the length of the small molecule linker utilized, and the attachment position on the sHsp cage. When conjugated OVA–sHsp was delivered intranasally to naïve mice, the resulting immune response to OVA was accelerated and intensified, and OVA-specific IgG1 responses were apparent within 5 days after a single immunizing dose, illustrating its utility for vaccine development. If animals were pretreated with a disparate VLP, P22 (a non-replicative bacteriophage capsid), before OVA–sHsp conjugate immunization, OVA-specific IgG1 responses were apparent already by 4 days after a single immunizing dose of conjugate in OVA-naïve mice. Additionally, the mice pretreated with P22 produced high titer mucosal IgA, and isotype-switched OVA-specific serum IgG. Similarly, sHsp pretreatment enhanced the accumulation of lung germinal center B cells, T follicular helper cells, and increased polymeric Ig receptor expression, priming the lungs for subsequent IgG and IgA responses to influenza virus challenge. Thus, sHsp nanoparticles elicited quick and intense antibody responses and these accelerated responses could similarly be induced to antigen chemically conjugated to the sHsp. Pretreatment of mice with P22 further accelerated the onset of the antibody response to OVA–sHsp, demonstrating the utility of conjugating antigens to VLPs for pre-, or possibly post-exposure prophylaxis of lung, all without the need for adjuvant.     

Nasal or intramuscular immunization of mice with influenza subunit antigen and the B subunit of Escherichia coli heat-labile toxin induces IgA- or IgG-mediated protective mucosal immunity

Local mucosal IgA antibodies play a central role in protection of the respiratory tract against influenza virus infection. Therefore, new-generation influenza vaccines should aim at stimulating not only systemic, but also local antibody responses. Previously, we demonstrated that the recombinant B subunit of the Escherichia coli heat-labile toxin (LTB) is a potent adjuvant towards nasally administered influenza subunit antigen. Here, we investigated the protection conferred by LTB-supplemented influenza subunit antigen given intranasally (i.n.) or intramuscularly (i.m.) to mice. Both i.n. and i.m. immunization with subunit antigen and LTB completely protected the animals against viral infection. Protection upon i.n. immunization was associated with the induction of antigen-specific serum IgG and mucosal IgA, whereas protection upon i.m. immunization correlated with strong serum and mucosal IgG, but not IgA responses. We conclude that LTB-supplemented influenza subunit antigen, given either i.n. or i.m, induces protective antibody-mediated mucosal immunity and thus represents a promising novel flu vaccine candidate.     

Low-protein diet induces oral tolerance to ovalbumin in mice

The suitable development of oral tolerance against ingested dietary foods is of critical importance to escaping food allergy. Using mice as an animal model for oral tolerance against ovalbumin (OVA) as a dietary antigen, we investigated the effects of dietary protein on their immunological tolerance. Female BALB/c mice fed either a 20% or 5% protein diet were orally administered 5 mg of OVA for four consecutive days, then immunized intraperitoneally with 100 microg of OVA. The immunized group of mice were fed and treated in the same manner, except that they received orally distilled water for four consecutive days before receiving intraperitoneal immunization with the antigen. Immunization alone with OVA elevated the total IgE and induced the production of OVA-specific antibodies IgE, IgG, IgG1, and IgG2a in the sera of both the 20% and 5% protein diet groups. The oral administration of OVA to mice before intraperitoneal immunization significantly reduced the total IgE and OVA-specific antibodies in mice fed 5% protein diet, but it had hardly any effect on those in mice fed a 20% protein diet. When spleen cells from these groups of mice were cultured with OVA as a mitogen, they responded substantially to OVA in the immunized groups fed 20% and 5% protein diets and in the presensitized group fed 20% protein, but those from the presensitized group fed a 5% protein diet did not respond. Furthermore, when IL-4 was assayed in the spleen cell cultures of the 20% and 5% groups, mice in the presensitized group fed a 5% protein diet produced a significantly less amount of IL-4 than those fed a 20% protein diet. Moreover, irrelevant to the protein amount in the diet, the production of IFN-gamma from spleen cell cultures dramatically decreased in the group without presensitization and profoundly increased in the presensitized group of mice fed a 5% protein diet. These findings suggest that a low-protein diet leads to an induction of oral tolerance against dietary antigens; this appears to involve a clear down-regulation of Th2 cytokine, IL-4.