Oral administration of cholera toxin-Sendai virus conjugate potentiates gut and respiratory immunity against Sendai virus

Successful oral immunization to prevent infectious diseases in the gastrointestinal tract as well as distant mucosal tissues may depend on the effectiveness of an Ag to induce gut immune responses. We and others have previously reported that cholera toxin possesses strong adjuvant effects on the gut immune response to co-administered Ag. To explore further adjuvant effects of cholera toxin, the holotoxin or its B subunit was chemically cross-linked to Sendai virus. The resulting conjugates, which were not infectious, were evaluated for their capacity to induce gut immune responses against Sendai virus after oral administration to mice. Conjugating cholera toxin to virus significantly enhanced the adjuvant activity of cholera toxin compared to simple mixing. Cholera toxin B subunit, however, did not show an adjuvant effect either by itself or conjugated with the virus. Oral administration of the Sendai virus-cholera toxin conjugate was also able to prime for protective anti-viral responses in the respiratory tract. Mice that were orally immunized with the conjugate and intra-nasally boosted with inactivated virus alone showed virus-specific IgA titers in nasal secretions that correlated with protection against direct nasal challenge with live Sendai virus. For comparison, s.c. immunization was also studied. Systemic immunization with the virus-cholera toxin conjugate induced virus-specific antibody responses in serum as well as in the respiratory tract but failed to protect the upper respiratory tract against virus challenge. Systemic immunization plus an intra-nasal boost did, however, confer a variable degree of protection to the upper respiratory tract, which correlated primarily with bronchoalveolar lavage (lung) antibody titers.     

Modified pulmonary surfactant is a potent adjuvant that stimulates the mucosal IgA production in response to the influenza virus antigen

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

Neutralization of cholera toxin by rat IgA secretory antibodies induced by a free synthetic peptide

Secretory immunoglobulin A (sIgA) is the major immunoglobulin in the bile of several species. They contribute to local immune defences of the gut. The protection against cholera toxin (CT) is due to the presence of specific sIgA in the bile and in the gut. We have already reported that oral administration of the peptide corresponding to the sequence 50-75 of cholera toxin B subunit elicits serum antibodies neutralizing CT activity, and that IgA and local protection are observed in the intestine of P50-75 orally immunized mice. In this study, we demonstrate the potential of this synthetic peptide as immunogen without carrier or adjuvant, not only in a strain known to be sensitive to CT, but also in an outbred one. Furthermore, this peptide stimulates the mucosal immunity, since we show that P50-75 induced-sIgA purified from rats bile and serum, are capable of neutralizing CT activity in the in vivo intestinal ligated loop test.     

Combined oral/nasal immunization protects mice from Sendai virus infection

Based on the concept of a common mucosal immune system wherein mucosal associated lymphocytes traffic among the various mucous membranes, the murine gastrointestinal tract was immunized with Sendai virus antigens in order to elicit a virus-specific immune response in the respiratory tract. Multiple intragastric (oral) administration of live or killed Sendai virus induced IgA and IgG antiviral antibodies in both gastrointestinal secretions and serum. When cholera toxin as an adjuvant was included along with virus, gut IgA and IgG as well as serum IgA responses were enhanced. Antiviral antibodies induced in respiratory secretions by oral killed virus plus cholera toxin, however, were variable and protection from virus challenge was not demonstrated. Significantly higher levels of respiratory antiviral antibodies were induced if immunization with oral killed Sendai virus/cholera toxin was combined with intranasal administration of small amounts of killed virus. The combined immunization also resulted in protection of both the upper and lower respiratory tracts from virus infection. Protection of the upper respiratory tract was correlated with the presence of IgA antiviral antibodies in nasal washings. On the other hand, protection of the lower respiratory tract was correlated with IgG antiviral antibodies in bronchoalveolar lavage fluids. Immunization with intranasal killed virus alone conferred partial protection to the lower respiratory tract and no protection to the upper respiratory tract. Thus, oral immunization with killed virus antigen could prime for a protective immune response in the murine respiratory tract and this protective response included IgA antibodies.     

Intranasal vaccination with murabutide enhances humoral and mucosal immune responses to a virus-like particle vaccine

Murabutide (MB) is a synthetic immunomodulator recognized by the nucleotide-binding oligomerization domain-containing protein 2 (NOD2) receptor on mammalian cells. MB has previously been approved for testing in multiple human clinical trials to determine its value as an antiviral therapeutic, and as an adjuvant for injected vaccines. We have found a new use for this immunomodulator; it functions as a mucosal adjuvant that enhances immunogenicity of virus-like particles (VLP) administered intranasally. MB enhanced Norwalk virus (NV) VLP-specific IgG systemically and IgA production at distal mucosal sites following intranasal (IN) vaccination. A dose escalation study identified 100 μg as the optimal MB dosage in mice, based on the magnitude of VLP-specific IgG, IgG1, IgG2a and IgA production in serum and VLP-specific IgA production at distal mucosal sites. IN vaccination using VLP with MB was compared to IN delivery VLP with cholera toxin (CT) or gardiquimod (GARD) and to parenteral VLP delivery with alum; the MB groups were equivalent to CT and GARD and superior to alum in inducing mucosal immune responses and stimulated equivalent systemic VLP-specific antibodies. These data support the further testing of MB as a potent mucosal adjuvant for inducing robust and durable antibody responses to non-replicating subunit vaccines.     

Suppression of an already established tumor growing through activated mucosal CTLs induced by oral administration of tumor antigen with cholera toxin

Priming of CTLs at mucosal sites, where various tumors are originated, seems critical for controlling tumors. In the present study, the effect of the oral administration of OVA plus adjuvant cholera toxin (CT) on the induction of Ag-specific mucosal CTLs as well as their effect on tumor regression was investigated. Although OVA-specific TCRs expressing lymphocytes requiring in vitro restimulation to gain specific cytotoxicity could be detected by OVA peptide-bearing tetramers in both freshly isolated intraepithelial lymphocytes and spleen cells when OVA was orally administered CT, those showing direct cytotoxic activity without requiring in vitro restimulation were dominantly observed in intraepithelial lymphocytes. The magnitude of such direct cytotoxicity at mucosal sites was drastically enhanced after the second oral administration of OVA with intact whole CT but not with its subcomponent, an A subunit (CTA) or a B subunit (CTB). When OVA plus CT were orally administrated to C57BL/6 mice bearing OVA-expressing syngeneic tumor cells, E.G7-OVA, in either gastric tissue or the dermis, tumor growth was significantly suppressed after the second oral treatment; however, s.c. or i.p. injection of OVA plus CT did not show any remarkable suppression. Those mucosal OVA-specific CTLs having direct cytotoxicity expressed CD8alphabeta but not CD8alphaalpha, suggesting that they originated from thymus-educated cells. Moreover, the infiltration of such OVA-specific CD8(+) CTLs was observed in suppressed tumor tissues. These results indicate that the growth of ongoing tumor cells can be suppressed by activated CD8alphabeta CTLs with tumor-specific cytotoxicity via an orally administered tumor Ag with a suitable mucosal adjuvant.     

IL-1 is an effective adjuvant for mucosal and systemic immune responses when coadministered with protein immunogens

Mucosal immunization with soluble protein Ag alone may induce Ag-specific tolerance, whereas mucosal immunization with Ag in the presence of a mucosal adjuvant may induce Ag-specific systemic and mucosal humoral and cell-mediated immune responses. The most widely used and studied mucosal adjuvant is cholera toxin (CT). Although the mechanism of adjuvanticity of CT is not completely understood, it is known that CT induces mucosal epithelial cells to produce the proinflammatory cytokines IL-1, IL-6, and IL-8 and up-regulates macrophage production of IL-1 and the costimulatory molecule B7.2. Because IL-1 may duplicate many of the activities of CT, we evaluated IL-1alpha and IL-1beta for their ability to serve as mucosal adjuvants when intranasally administered with soluble protein Ags. IL-1alpha and IL-1beta were as effective as CT for the induction of Ag-specific serum IgG, vaginal IgG and IgA, systemic delayed-type hypersensitivity, and lymphocyte proliferative responses when intranasally administered with soluble protein Ag. Our results indicate that IL-1alpha and IL-1beta may be useful as mucosal vaccine adjuvants. Such an adjuvant may be useful, and possibly required, for vaccine-mediated protection against pathogens that infect via the mucosal surfaces of the host such as HIV.     

Lack of J chain inhibits the transport of gut IgA and abrogates the development of intestinal antitoxic protection.

Recent publications have provided confusing information on the importance of the J chain for secretion of dimeric IgA at mucosal surfaces. Using J chain-deficient (J chain-/-) mice, we addressed whether a lack of J chain had any functional consequence for the ability to resist challenge with cholera toxin (CT) in intestinal loops. J chain-/- mice had normal levels of IgA plasma cells in the gut mucosa, and the Peyer's patches exhibited normal IgA B cell differentiation and germinal center reactions. The total IgA levels in gut lavage were reduced by roughly 90% as compared with that in wild-type controls, while concomitantly serum IgA levels were significantly increased. Total serum IgM levels were depressed, whereas IgG concentrations were normal. Following oral immunizations with CT, J chain-/- mice developed 10-fold increased serum antitoxin IgA titers, but gut lavage anti-CT IgA levels were substantially reduced. However, anti-CT IgA spot-forming cell frequencies in the gut lamina propria were normal. Anti-CT IgM concentrations were low in serum and gut lavage, whereas anti-CT IgG titers were unaltered. Challenge of small intestinal ligated loops with CT caused dramatic fluid accumulation in immunized J chain-/- mice, and only 20% protection was detected compared with unimmunized controls. In contrast, wild-type mice demonstrated 80% protection against CT challenge. Mice heterozygous for the J chain deletion exhibited intermediate gut lavage anti-CT IgA and intestinal protection levels, arguing for a J chain gene-dosage effect on the transport of secretory IgA. This study unequivocally demonstrates a direct relationship between mucosal transport of secretory SIgA and intestinal immune protection.     

Effective mucosal immunity to anthrax: neutralizing antibodies and Th cell responses following nasal immunization with protective antigen

Mucosal, but not parenteral, immunization induces immune responses in both systemic and secretory immune compartments. Thus, despite the reports that Abs to the protective Ag of anthrax (PA) have both anti-toxin and anti-spore activities, a vaccine administered parenterally, such as the aluminum-adsorbed anthrax vaccine, will most likely not induce the needed mucosal immunity to efficiently protect the initial site of infection with inhaled anthrax spores. We therefore took a nasal anthrax vaccine approach to attempt to induce protective immunity both at mucosal surfaces and in the peripheral immune compartment. Mice nasally immunized with recombinant PA (rPA) and cholera toxin (CT) as mucosal adjuvant developed high plasma PA-specific IgG Ab responses. Plasma IgA Abs as well as secretory IgA anti-PA Abs in saliva, nasal washes, and fecal extracts were also induced when a higher dose of rPA was used. The anti-PA IgG subclass responses to nasal rPA plus CT consisted of IgG1 and IgG2b Abs. A more balanced profile of IgG subclasses with IgG1, IgG2a, and IgG2b Abs was seen when rPA was given with a CpG oligodeoxynucleotide as adjuvant, suggesting a role for the adjuvants in the nasal rPA-induced immunity. The PA-specific CD4(+) T cells from mice nasally immunized with rPA and CT as adjuvant secreted low levels of CD4(+) Th1-type cytokines in vitro, but exhibited elevated IL-4, IL-5, IL-6, and IL-10 responses. The functional significance of the anti-PA Ab responses was established in an in vitro macrophage toxicity assay in which both plasma and mucosal secretions neutralized the lethal effects of Bacillus anthracis toxin.     

The combined CTA1-DD/ISCOM adjuvant vector promotes priming of mucosal and systemic immunity to incorporated antigens by specific targeting of B cells

The cholera toxin A1 (CTA1)-DD/QuilA-containing, immune-stimulating complex (ISCOM) vector is a rationally designed mucosal adjuvant that greatly potentiates humoral and cellular immune responses. It was developed to incorporate the distinctive properties of either adjuvant alone in a combination that exerted additive enhancing effects on mucosal immune responses. In this study we demonstrate that CTA1-DD and an unrelated Ag can be incorporated together into the ISCOM, resulting in greatly augmented immunogenicity of the Ag. To demonstrate its relevance for protection against infectious diseases, we tested the vector incorporating PR8 Ag from the influenza virus. After intranasal immunization we found that the immunogenicity of the PR8 proteins were significantly augmented by a mechanism that was enzyme dependent, because the presence of the enzymatically inactive CTA1R7K-DD mutant largely failed to enhance the response over that seen with ISCOMs alone. The combined vector was a highly effective enhancer of a broad range of immune responses, including specific serum Abs and balanced Th1 and Th2 CD4(+) T cell priming as well as a strong mucosal IgA response. Unlike unmodified ISCOMs, Ag incorporated into the combined vector could be presented by B cells in vitro and in vivo as well as by dendritic cells; it also accumulated in B cell follicles of draining lymph nodes when given s.c. and stimulated much enhanced germinal center reactions. Strikingly, the enhanced adjuvant activity of the combined vector was absent in B cell-deficient mice, supporting the idea that B cells are important for the adjuvant effects of the combined CTA1-DD/ISCOM vector.     

Strong differential regulation of serum and mucosal IgA responses as revealed in CD28-deficient mice using cholera toxin adjuvant

In this study, we show that costimulation required for mucosal IgA responses is strikingly different from that needed for systemic responses, including serum IgA. Following oral immunization with cholera toxin (CT) adjuvant we found that whereas CTLA4-H1 transgenic mice largely failed to respond, CD28-/- mice developed near normal gut mucosal IgA responses but poor serum Ab responses. The local IgA response was functional in that strong antitoxic protection developed in CT-immunized CD28-/- mice. This was in spite of the fact that no germinal centers (GC) were observed in the Peyer's patches, spleen, or other peripheral lymph nodes. Moreover, significant somatic hypermutation was found in isolated IgA plasma cells from gut lamina propria of CD28-/- mice. Thus, differentiation to functional gut mucosal IgA responses against T cell-dependent Ags does not require signaling through CD28 and can be independent of GC formations and isotype-switching in Peyer's patches. By contrast, serum IgA responses, similar to IgG-responses, are dependent on GC and CD28. However, both local and systemic responses are impaired in CTLA4-Hgamma1 transgenic mice, indicating that mucosal IgA responses are dependent on the B7-family ligands, but require signaling via CTLA4 or more likely a third related receptor. Therefore, T-B cell interactions leading to mucosal as opposed to serum IgA responses are uniquely regulated and appear to represent separate events. Although CT is known to strongly up-regulate B7-molecules, we have demonstrated that it acts as a potent mucosal adjuvant in the absence of CD28, suggesting that alternative costimulatory pathways are involved.     

Intraperitoneal delivery of cholera toxin B subunit enhances systemic and mucosal antibody responses

Although cholera toxin (CT) is a potent mucosal adjuvant, its activity in systemic immunity is relatively undocumented. In the present study, we investigated its adjuvant effect on systemic and mucosal antibody responses following intraperitoneal immunization of mice with BSA. CT increased levels of anti-BSA specific IgG1, IgM, and IgA antibodies in the peritoneum and serum, as well as IgA and IgG1 antibodies in the intestinal fluids. The B subunit of CT (CTB) was as potent as CT itself, with potency comparable to that of incomplete Freund's adjuvant. CTB also increased the number of BSA-specific Ig secreting cells in the spleen and mesenteric lymph node, and stimulated expression of B7.2 but not of MHC class II molecules on peritoneal macrophages, particularly in the presence of IFN-gamma. Our results imply that intraperitoneally administered CTB enhances systemic and mucosal antibody responses, in part at least via effects on macrophages.     

Modulating dendritic cells to optimize mucosal immunization protocols.

Oral administration of soluble protein Ag induces tolerance, a phenomenon that has hampered mucosal vaccine design. To provoke active immunity, orally administered Ag must be fed together with a mucosal adjuvant such as cholera toxin (CT). Unfortunately, CT is not suitable for clinical use because of its associated toxicity. There is, therefore, a need to develop alternative mucosal immunization regimens. Here we have attempted to alter the intrinsically tolerogenic nature of the intestine and improve immunization potential by expanding and activating intestinal APC in vivo. Previous studies have indicated that intestinal dendritic cells (DC) present oral Ag, but do so in a tolerogenic manner. In the present study we investigated whether DC can be converted from tolerogenic into immunogenic APC by treating mice with Flt3 ligand (Flt3L), a DC growth factor, and then immunizing with CT. We observed increased local and systemic responses to CT in the presence of elevated numbers of intestinal DC. In parallel, CT induced up-regulation of CD80 and CD86 on these Flt3L-expanded DC. In an attempt to develop a toxin-free adjuvant system, we investigated whether IL-1 could be used as an alternative DC-activating stimulus. Using a combination of Flt3L and IL-1alpha, we observed a potent active response to fed soluble Ag, rather than the tolerogenic response normally observed. These data suggest that Flt3L-expanded DC are well positioned to regulate intestinal responses depending on the presence or the absence of inflammatory signals. Flt3L may therefore be a reagent useful for the design of mucosal immunization strategies.     

Peroral immunization of microencapsulated human VP8 in combination with cholera toxin induces intestinal antibody responses

To develop an orally delivered subunit vaccine for rotavirus infection, a trypsin cleavage product of VP4, recombinant VP8*, was expressed in Escherichia coli. The recombinant VP8* (rVP8*), purified by affinity chromatography, was reactive against human rotavirus positive serum in Western-blot analysis. To further evaluate the immunogenicity of the oral-delivered rVP8*, it was encapsulated with alginate-microsphere and administered in combination with cholera toxin (CT) as a mucosal adjuvant perorally into mice. The ELISPOT assay showed that the number of rVP8*-specific IgG1 antibody secreting cells increased about 3-fold and about 2-fold in spleen and Peyer's patch, respectively as compared to non-immune mice. In addition, the number of rVP8*-specific IgA antibody secreting cells increased about 2-fold in Peyer's patch. Finally, rVP8*-specific IgA antibody response was significantly enhanced in the intestinal fluids from the mice immunized perorally with encapsulated rVP8* and CT. Taken together, these results indicate that rVP8* possessed proper immunogenicity and it would be potentially useful as a subunit vaccine against rotavirus-associated disease through peroral immunization.     

Rotavirus 2/6 Viruslike Particles Administered Intranasally with Cholera Toxin, Escherichia coli Heat-Labile Toxin (LT), and LT-R192G Induce Protection from Rotavirus Challenge

We have shown that rotavirus 2/6 viruslike particles composed of proteins VP2 and VP6 (2/6-VLPs) administered to mice intranasally with cholera toxin (CT) induced protection from rotavirus challenge, as measured by virus shedding. Since it is unclear if CT will be approved for human use, we evaluated the adjuvanticity of Escherichia coli heat-labile toxin (LT) and LT-R192G. Mice were inoculated intranasally with 10 μg of 2/6-VLPs combined with CT, LT, or LT-R192G. All three adjuvants induced equivalent geometric mean titers of rotavirus-specific serum antibody and intestinal immunoglobulin G (IgG). Mice inoculated with 2/6-VLPs with LT produced significantly higher titers of intestinal IgA than mice given CT as the adjuvant. All mice inoculated with 2/6-VLPs mixed with LT and LT-R192G were totally protected (100%) from rotavirus challenge, while mice inoculated with 2/6-VLPs mixed with CT showed a mean 91% protection from challenge. The availability of a safe, effective mucosal adjuvant such as LT-R192G will increase the practicality of administering recombinant vaccines mucosally.     

A Functional Antigen in a Practical Crop: LT-B Producing Maize Protects Mice against Escherichia coli Heat Labile Enterotoxin (LT) and Cholera Toxin (CT)

We have produced a functional heat labile enterotoxin (LT-) B subunit of Escherichia coli in maize. LT-B is a multimeric protein that presents an ideal model for an edible vaccine, displaying stability in the gut and inducing mucosal and systemic immune responses. Transgenic maize was engineered to synthesize the LT-B polypeptides, which assembled into oligomeric structures with affinity for G_M1 gangliosides. We orally immunized BALB/c mice by feeding transgenic maize meal expressing LT-B or non-transgenic maize meal spiked with bacterial LT-B. Both treatments stimulated elevated IgA and IgG antibodies against LT-B and the closely related cholera toxin B subunit (CT-B) in serum, and elevated IgA in fecal pellets. The transgenic maize induced a higher anti-LT-B and anti-CT-B mucosal and serum IgA response compared to the equivalent amount of bacterial LT-B spiked into maize. Following challenge by oral administration of the diarrhea inducing toxins LT and CT, transgenic maize-fed mice displayed reduced fluid accumulation in the gut compared to non-immunized mice. Moreover, the gut to carcass ratio of immunized mice was not significantly different from the PBS (non-toxin) challenged control group. We concluded that maize-synthesized LT-B had features of the native bacterial LT-B such as molecular weight, G_M1 binding ability, and induction of serum and mucosal immunity. We have demonstrated that maize, a major food and feed ingredient, can be efficiently transformed to produce, accumulate, and store a fully assembled and functional candidate vaccine antigen.     

Cytokines as Adjuvants for the Induction of Anti-Human Immunodeficiency Virus Peptide Immunoglobulin G (IgG) and IgA Antibodies in Serum and Mucosal Secretions after Nasal Immunization

Safe and potent new adjuvants are needed for vaccines that are administered to mucosal surfaces. This study was performed to determine if interleukin-1alpha (IL-1alpha) combined with other proinflammatory cytokines provided mucosal adjuvant activity for induction of systemic and mucosal anti-human immunodeficiency virus (HIV) peptide antibody when intranasally administered with an HIV peptide immunogen. Nasal immunization of BALB/c mice with 10 microg of an HIV env peptide immunogen with IL-1alpha, IL-12, and IL-18 on days 0, 7, 14, and 28 induced peak serum anti-HIV peptide immunoglobulin G1 (IgG1) and IgA titers of 1:131,072 and 1:7,131, respectively (P = 0.05 versus no adjuvant). The use of cholera toxin (CT) as a mucosal adjuvant induced serum IgG1 and IgA titers of 1:32,768 and 1:776, respectively. The adjuvant combination of IL-1alpha, IL-12, and IL-18 induced anti-HIV peptide IgA titers of 1:1,176, 1:7,131, and 1:4,705 in saliva, fecal extracts and vaginal lavage, respectively. Titers induced by the use of CT as an adjuvant were 1:223, 1:1,176, and 1:675, respectively. These results indicate that the proinflammatory cytokines IL-1alpha, IL-12, and IL-18 can replace CT as a mucosal adjuvant for antibody induction and are important candidates for use as mucosal adjuvants with HIV and other vaccines.     

Cholera toxin prevents Th1-mediated autoimmune disease by inducing immune deviation

Cholera toxin (CT), a major enterotoxin produced by Vibrio cholerae, is known for its properties as a mucosal adjuvant that promotes Th2 or mixed Th1 + Th2 responses. In this study, we explore the ability of CT to act as a systemic adjuvant to counteract the Th1 response leading to experimental autoimmune uveitis. We report that susceptible B10.RIII mice immunized with a uveitogenic regimen of the retinal Ag interphotoreceptor retinoid-binding protein could be protected from disease by a single systemic injection of as little as 2 micro g of CT at the time of immunization. The protected mice were not immunosuppressed, but rather displayed evidence of immune deviation. Subsequent adaptive responses to interphotoreceptor retinoid-binding protein showed evidence of Th2 enhancement, as indicated by reduced delayed-type hypersensitivity in the context of enhanced Ag-specific lymphocyte proliferation and IL-4 production. Ag-specific production of several other cytokines, including IFN-gamma, was not appreciably altered. The inhibitory effect of CT was dependent on the enzymatic A subunit of CT, because the cell-binding B subunit alone could not block disease development. Mice given CT displayed detectable IL-4 levels in their serum within hours of CT administration. This innate IL-4 production was critical for protection, as infusion of neutralizing Ab against IL-4 to mice, given a uveitogenic immunization and treated with CT, counteracted immune deviation and abrogated protection. Our data indicate that systemic administration of CT inhibits experimental autoimmune uveitis by skewing the response to the uveitogenic autoantigen to a nonpathogenic phenotype.     

Induction of HIV immunity in the genital tract after intranasal delivery of a MVA vector: enhanced immunogenicity after DNA prime-modified vaccinia virus Ankara boost immunization schedule

Vaccines intended to prevent mucosal transmission of HIV should be able to induce multiple immune effectors in the host including Abs and cell-mediated immune responses at mucosal sites. The aim of this study was to characterize and to enhance the immunogenicity of a recombinant modified vaccinia virus Ankara (MVA) expressing HIV-1 Env IIIB Ag (MVAenv) inoculated in BALB/c mice by mucosal routes. Intravaginal inoculation of MVAenv was not immunogenic, whereas intranasally it induced a significant immune response to the HIV Ag. Intranasal codelivery of MVAenv plus cholera toxin (CT) significantly enhanced the cellular and humoral immune response against Env in the spleen and genitorectal draining lymph nodes, respectively. Heterologous DNAenv prime-MVAenv boost by intranasal immunization, together with CT, produced a cellular immune response in the spleen 10-fold superior to that in the absence of CT. A key finding of these studies was that both MVAenv/MVAenv and DNAenv/MVAenv schemes, plus CT, induced a specific mucosal CD8(+) T cell response in genital tissue and draining lymph nodes. In addition, both immunizations also generated systemic Abs, and more importantly, mucosal IgA and IgG Abs in vaginal washings. Specific secretion of beta-chemokines was also generated by both immunizations, with a stronger response in mice immunized by the DNA-CT/MVA-CT regimen. Our findings are of relevance in the area of vaccine development and support the optimization of protocols of immunization based on MVA as vaccine vectors to induce mucosal immune responses against HIV.