Oral vaccine models: multiple delivery systems employing tetanus toxoid.

We have not yet directly examined the Th cell responses induced by using Salmonella/BRD 847 as a vector nor have we performed these experiments following immunization with microspheres. However, production of high serum levels of antigen-specific IgG1 may be indicative of a Th2-type response, whereas high serum levels of IgG2a may reflect a Th1-type response. An important issue in using various oral delivery systems is whether the system(s) employed affects the Th cell response to the same antigen. We therefore analyzed the serum antigen-specific IgG subclasses induced in each of the model systems we studied. Table 2 presents these results. Clearly, oral administration of soluble TT with CT as an adjuvant induced an IgG1 subclass, and encapsulation of TT within microspheres had no effect on this Th2-type response. On the other hand, oral immunization with live Salmonella expressing fragment C of tetanus toxin induced a strong IgG2a subclass response indicative of a Th1-type response. It should be noted that protection against a lethal TT challenge was afforded by elevated levels of both anti-TT IgG1 and IgG2a subclasses. We intend to examine the cytokine profiles in spleen CD4+ T cells from mice immunized with microspheres or Salmonella following in vitro antigen stimulation to confirm the T helper type responses suggested by the IgG subclass data. It will also be important to examine the cytokine patterns induced in Peyer's patch CD4+ T cells following immunization of C57BL/6 with Salmonella/BRD 847. Whereas analysis of the serum IgG subclass profile indicated a strong IgG2a response and thus a systemic Th1-type pattern, this vector also induced a good mucosal IgA response. If our current hypothesis concerning S-IgA production is correct, we would expect a predominant Th2-type profile in CD4+ T cells from Peyer's patch in these mice. Such a result would emphasize the bifurcation of T-cell responses in the systemic versus the mucosal immune environments. The data obtained to data suggest that adjuvants and various vehicle delivery systems may influence the induction of distinct T helper cell subsets to a specific antigen. The unique cytokine arrays produced by these T-cell subsets influence the immune responses in terms of systemic Ig subclasses produced, cell-mediated immune responses, and the production of mucosal S-IgA antibodies. Although additional studies are necessary, the manipulation of T-cell subsets employing adjuvants, antigen packaging, or perhaps even the addition of individual cytokines to various formulations holds significant promise for optimizing immune responses to orally administered vaccines.

Helper Th1 and Th2 cell responses following mucosal or systemic immunization with cholera toxin.

We have used the potent mucosal immunogen cholera toxin (CT) to assess antigen-specific CD4+ T-cell responses, including Th1- and Th2-type cells in mucosa-associated tissues, e.g. Peyer's patches (PP), and systemic tissue, e.g. spleen (SP), for their regulatory role in the induction of CT-specific B-cell antibody responses in the gastrointestinal (GI) tract as well as in systemic sites. The CT was given by either oral or intravenous (i.v.) routes and the mice orally immunized with CT exhibited brisk IgA anti-CT antibody responses in faecal extracts and elevated IgG anti-CT antibody responses in serum. Further, significant IgA anti-CT spot-forming cells (SFCs) were seen in lamina propria lymphocytes (LPLs) from mice orally immunized with CT. In contrast, i.v. immunization with CT induced IgM and IgG anti-CT SFC responses in SP, and serum anti-CT antibodies of these two isotypes; no anti-CT responses were induced in the GI tract after immunization by this route. The CD4+ T cells isolated from PP and SP of mice orally immunized with CT were stimulated in vitro with CT-B-coated latex microspheres for 1-6 days, and the induction of IL-2 and interferon gamma (IFN-gamma) (Th1-type) or IL-4 and IL-5 (Th2-type) producing SFCs were analysed by a cytokine-specific ELISPOT and cytokine-specific mRNA was detected by reverse transcriptase (RT)-PCR assays.(ABSTRACT TRUNCATED AT 250 WORDS)

The common mucosal immune system: from basic principles to enteric vaccines with relevance for the female reproductive tract.

The realization that induction of immune responses at mucosal surfaces may prevent colonization, invasion or dissemination of pathogenic microorganisms has spurred intensive efforts to develop vaccines which elicit effective mucosal immunity. In this paper, recent results are discussed for mice given cholera toxin as both an immunogen and as an adjuvant for inducing both humoral and gastrointestinal mucosal immune responses. Oral administration of cholera toxin alone or with a co-administered protein vaccine tetanus toxoid induces a strong T helper type 2 (TH2) cell response in both Peyer's patches and spleen. Both serum IgG and secretory IgA antibodies specific for cholera toxin or for the co-administered protein tetanus toxoid were induced. When administered parentally, however, no mucosal antibody responses were evident and a mixed TH1- and TH2-type CD4+ T cell response was noted in the spleen. Various vectors are being employed in an effort not only to induce mucosal immune responses but also to direct the response to a TH1-type response, thought to promote strong cell-mediated immune responses, or to a TH2-type response for maximum B cell antibody responses. The ability to manipulate the TH cell responses may provide a more rational approach for the design of vaccines. Although lymphoid tissues of the female reproductive tract differ from that of the gut, many of the strategies and evolving principles may be directly applicable to the development of vaccines designed to prevent sexually transmitted diseases.

Optimizing oral vaccines: induction of systemic and mucosal B-cell and antibody responses to tetanus toxoid by use of cholera toxin as an adjuvant.

Cholera toxin (CT) is an effective mucosal antigen and acts as an adjuvant when given orally with various antigens; however, few studies have compared the levels of antibody responses to CT and coadministered protein in systemic and mucosal tissues. In this study, we used tetanus toxoid (TT) for assessment of immune responses. Time course and dose-response studies established that 250 micrograms of TT given orally with 10 micrograms of CT three times at weekly intervals induced high serum and gastrointestinal tract anti-TT and anti-CT antibody responses. Oral immunization with TT alone induced no detectable mucosal immunoglobulin A (IgA) antibodies in fecal extracts and only weak serum IgG anti-TT responses. The coadministration of CT and TT induced peak serum IgG anti-TT responses following two oral doses that remained constant after the third oral immunization, while optimal mucosal IgA responses were seen after the third oral immunization. The serum anti-TT response obtained with CT and TT proved protective against TT challenge (100 minimum lethal doses), whereas mice orally given CT or TT alone died. Antigen-specific B-cell responses were assessed with an isotype-specific Elispot assay of isolated lymphoid cells from the spleen, Peyer's patches, and the small intestinal lamina propria. Interestingly, approximately fourfold-higher numbers of IgA anti-CT than of anti-TT antibody-producing (spot-forming) cells occurred in lymphocytes from the lamina propria of mice orally immunized with both TT and CT. The adjuvant CT did not induce polyclonal B-cell responses in mice given CT by the oral route, since no significant differences in total numbers of B cells producing IgA, IgG, or IgM were found compared with the numbers in mice given TT alone. The results clearly indicate that serum and mucosal antibody responses develop with different kinetics and that protective TT-specific antibody responses are generated in the systemic compartment when TT is administered with CT via the oral route.

Helper T cell subsets for immunoglobulin A responses: oral immunization with tetanus toxoid and cholera toxin as adjuvant selectively induces Th2 cells in mucosa associated tissues.

Antigen-specific B cell responses to mucosally delivered proteins are dependent upon CD4-positive T helper (Th) cells, and the frequency of Th1 and Th2 cell responses after oral immunization may determine the level and isotype of mucosal antibody responses. We have used a protein-based vaccine, tetanus toxoid (TT), together with the mucosal adjuvant cholera toxin (CT), for oral immunization of mice to study the nature of antigen-specific Th cell subsets induced in Peyer's patches (PP) of the gastrointestinal (GI) tract and in the spleen (SP) during peak antibody responses. Mice orally immunized with TT and CT responded with antigen-specific secretory immunoglobulin A (S-IgA) antibodies in the GI tract, and with both IgG and IgA antibody responses in serum. PP and SP CD4+ T cells from mice orally immunized with TT plus CT were cultured with antigen-coated latex microspheres for induction of proliferative responses and for enumeration of cytokine producing CD4+ T cells. Interestingly, both PP and SP CD4+ T cell cultures showed increased numbers of IL-4- and IL-5 (Th2-type)-producing, spot-forming cells (SFCs) after 21 d of immunization, while essentially no interferon-gamma (IFN-gamma) or IL-2 (Th1-type) SFCs were noted. Cytokine-specific Northern blots and RT-PCR also revealed that significant IL-4 and IL-5 mRNA levels, but not IFN-gamma or IL-2 mRNA, were present in CD4+ T cells isolated from antigen-stimulated cultures. However, systemic immunization with TT and CT induced antigen-specific IgG and IgM but not IgA antibodies in serum. Further, both IL-2 and IFN-gamma-producing Th1-type cells as well as IL-4- and IL-5-secreting Th2-type cells were generated in SP. Our results show that oral immunization with TT and the mucosal adjuvant CT selectively induced antigen-specific Th2-type responses which may represent the major helper cell phenotype involved in mucosal IgA responses in the GI tract.

New perspectives in mucosal immunity with emphasis on vaccine development.

In this review, we have purposely focused on five areas that are currently receiving extensive research attention and will be of major importance for development of mucosal and systemic immunity to oral vaccines. These five areas include the following: (1) helper T-cell (Th) subsets and cytokines for mucosal IgA responses; (2) Th1- and Th2-type subsets in regulation of mucosal IgA responses; (3) antigen uptake and presentation in the mucosal immune system; (4) the importance of memory in mucosal immunity to vaccines; and (5) the determination of whether oral immunization alone induces immunity in all mucosal effector tissues. It is now established that the mucosal immune system can be divided into discrete mucosal inductive sites where vaccines/antigens are encountered and taken up, processed, and presented to B and T cells, and separate areas where immune cells actually function (mucosal effector tissues). Further, through the help provided by Th cells and cytokines, the B cells respond to antigen and undergo expansion including memory cell formation. Following the migration of memory B cells to mucosal effector tissues, the cells rapidly develop into IgA plasma cells, and the prevalence of the latter cell type represents a major characteristic of mucosal effector tissues. It also appears that antigen-specific Th cells and perhaps even CD8+ cytotoxic T lymphocytes can make this circular journey (along with memory/activated B cells) from inductive to mucosal effector sites, and this is termed the common mucosal immune system (CMIS). The major implications of the CMIS for development of vaccines would include each of the five components that are discussed.

Gingival mononuclear cells from chronic inflammatory periodontal tissues produce interleukin (IL)-5 and IL-6 but not IL-2 and IL-4.

A unique characteristic of the localized inflammatory tissue in the periodontium (e.g., adult periodontitis [AP]) is the accumulation of IgG (IgG1 > IgG2 > IgG3 > or = IgG4) followed by IgA plasma cells (IgA1 > IgA2). However, the exact molecular mechanisms contributing to these elevated B-cell responses at the local disease site are still unknown. Thus, this study has examined the production of cytokines of importance in B-cell responses, e.g., interleukin (IL)-2, IL-4, IL-5, and IL-6 by gingival mononuclear cells (GMC) isolated from patients in severe stages of AP. These cytokines were assessed at the protein and messenger (m)RNA levels to understand their importance for the observed increased B-cell responses present in these tissues. Among the four cytokines tested by respective cytokine-specific, polymerase chain reaction and dot-blot hybridization, high levels of IL-5- and IL-6-specific mRNA were noted in GMC freshly isolated from AP patients. On the other hand, specific message for IL-2 and IL-4 were not present. Further, the analysis of culture supernatants of GMC also revealed that cells from AP patients spontaneously produced IL-5 and IL-6 but not IL-2 and IL-4. In contrast, when peripheral blood mononuclear cells isolated from the same patients were examined for these cytokines, no detectable levels of mRNA or secreted cytokines were noted. These results showed that GMC from localized inflammatory tissues in severe stages of AP possess a distinct cytokine profile represented by high levels of IL-5 and IL-6 mRNA expression and protein synthesis, whereas IL-2 and IL-4 were not detected. Further, this study supports the concept that AP is a localized inflammatory disease, because GMC from the inflamed tissue actively produce IL-5 and IL-6, whereas peripheral blood mononuclear cells from the same patients do not.

Selective induction of Th2 cells in murine Peyer's patches by oral immunization.

We have used three different methods to determine the T helper (Th) cell response, including Th1 and Th2 types, in murine Peyer's patches (PP) following oral immunization with sheep red blood cells (SRBC). These include: (i) use of cytokine-specific (IFN-gamma and IL-5), single cell assays to estimate the frequencies of Th1 and Th2 cells respectively, (ii) cytokine-specific mRNA--cDNA dot blot and Northern gel hybridizations to detect levels of specific mRNA, and (iii) T cell cloning techniques to determine the frequency of Th1 and Th2 clones. Mice were immunized with SRBC by either the oral or i.p. route. The PP and splenic (SP) CD3+ and CD3+ CD4+ T cell subsets were isolated and cultured with antigen, feeder cells, and IL-2, and were assessed at various intervals (days 0, 1, 3, and 6) for numbers of T cells producing either IFN-gamma or IL-5 by use of an enzyme-linked immunospot (ELISPOT) procedure. Cultures of T cells from PP or SP of mice given SRBC by the oral route had a high frequency of IL-5 spot forming cells (SFC), with lower numbers of IFN-gamma SFC. However, cultures of CD3+ T cells and CD3+ CD4+ Th cells from spleens of i.p. immunized mice exhibited predominantly IFN-gamma SFC, with smaller but significant numbers of IL-5 SFC. This distinct pattern of cytokine production was supported by mRNA analysis where high IL-5 specific mRNA levels were noted in PP T cell cultures of orally primed mice, while IFN-gamma mRNA was predominant in the SP CD3+ T cell and CD3+ CD4+ Th cell cultures from i.p. immunized mice. When the frequencies of IFN-gamma or IL-5 SFC were assessed among cloned Th cells from orally- or systemically-immunized mice, 74% of Th cell clones from PP of mice orally immunized with SRBC were IL-5 producers (Th2 type), while 67% of Th cell clones from SP of mice immunized by the i.p. route were IFN-gamma producers (Th1 type). Our studies show that higher frequencies of IFN-gamma producing Th1-type cells occur in SP of mice given antigen by the systemic route, while oral immunization results in predominantly IL-5 producing, Th2-type cells in PP.

The cytokine-like action of substance P upon B cell differentiation.

B cells respond to a variety of effector molecules that can induce these cells to differentiate. One such molecule is the neuropeptide, substance P (SP). Previous studies have demonstrated the presence of SP receptors on lymphocytes while limited studies have been able to demonstrate the biological significance of their expression. SP has been shown to enhance IgA and IgM responses by Peyer's patch and splenic B cells. A limitation of these studies was that the direct effect of SP upon B cells was not ascertained, suggesting these B cells were stimulated via alternate mechanisms. To this end, evidence here will be discussed that SP can directly interact with clonal B lymphoma cells and highly purified splenic B cells. The data implicate SP as a late-acting B cell differentiation factor that requires an additional triggering mechanism to initiate the B cell differentiation process.

Induction of T helper cells and cytokines for mucosal IgA responses.

CD4+ Th cells and their derived cytokines play an important role in the regulation of IgA responses in the mucosal immune system. Th1 and Th2 cells induce different Ig isotype and IgG-subclass responses. Further, cytokines produced by Th2-type cells (e.g., IL-5 and IL-6) have been shown to induce PP sIgA+ B cells to secrete IgA. Our studies have now shown that oral immunization with SRBC selectively induces Th2-type cells in PP while systemic (I.P.) immunization with SRBC predominantly induces Th1-type cells. It is tempting to suggest that Th2 cells which produce IL-5 and IL-6 tend to be predominant in mucosal effector regions, such as the salivary glands and LP tissues and account for the predominant IgA responses which characterize these tissues. The PP contain B cell subsets which respond to IL-5 and IL-6, and these are largely restricted to the PNALo non-GC (memory) sIgA+ B cells. The importance of CD4+ Th cells in the regulation of IgA responses has also been shown by the depletion of CD4+ Th cells in anti-L3T4 (CD4)-treated mice. Loss of CD4+ Th cells from mucosal tissues resulted in dramatically decreased numbers of IgA plasma cells in the small intestine and led to a reduction in IgA SFC in isolated LP cells. The overall size of PP was reduced and the GCs were absent; however, the relative frequency of sIgA+ B cells in PP did not change, possibly suggesting that CD4+ Th cells do not influence switches to IgA.

The regulation of antibody responses by mini-cytokines.

The presence of adrenocorticotropic hormone (ACTH) and substance P (SP) receptors on leukocytes is suggestive that these cells can respond to these ligands. To address this possibility, we have investigated the consequences of ACTH and SP stimulation of B cells. As a result, enhanced immunoglobulin synthesis mimicking an IL-4-like mechanism was noted. Importantly, this stimulation could be induced at ligand concentrations at or near the kD for their receptors. Herein these effects by ACTH and SP were described using B cell lymphoma cell lines and normal B cells.

Substance P acts directly upon cloned B lymphoma cells to enhance IgA and IgM production.

The IgA producing murine B lymphoma, CH12.LX.C4.4F10 (4F10) and the IgM producing murine lymphoma, CH12.LX.C4.5F5 (5F5) were found to express substantial numbers of substance P (SP) receptors having dissociation constants equal to 0.69 nM. Binding of SP by these B lymphoma cells was via the tachykinin-specific C-terminus sequence, Phe-X-Gly-Leu-Met-NH2, because SP, SP antagonist (D-Pro2-D-Phe7-D-Trp9-SP), eledoisin, and substance K could effectively inhibit radiolabeled SP binding, whereas the SP N-terminus fragment, SP (1-4), could not. The functionality of these receptors could be demonstrated by the ability of subnanomolar concentrations of SP to induce Ig secretion in a dose-dependent fashion. However, the presence of a second stimulus in these cultures was required to obtain maximal increases. IgA secretion by 4F10 cells was elevated only 25 to 37%, and IgM secretion by 5F5 cells was not significantly increased in cultures in which nanomolar concentrations of SP were present. Conversely, coculturing 5F5 cells with a suboptimal concentration of LPS (50 ng/ml) and 10(-10)M SP resulted in an approximate threefold increase in supernatant IgM when compared to control cultures stimulated with LPS alone. While not as dramatic, 10(-10) M SP also enhanced IgA secretion of LPS-stimulated 4F10 cells by approximately 45%. This enhancement of Ig secretion was SP-specific, as evidenced by the ability of 1000-fold excess of SP antagonist to block SP-induced, but not LPS-induced, Ig production. Clearly, SP could act synergistically with LPS to enhance Ig secretion; therefore, we questioned whether this augmentation was also reflected at the level of H chain mRNA expression. 10(-9)M SP induced modest increases (50 to 60%) in mu-chain mRNA expression by LPS-stimulated 5F5 cells when compared with cells stimulated with LPS alone. The 4F10 cells did not display this magnitude of difference for alpha-chain mRNA expression. Thus, although SP-induced increases of mu-chain mRNA by 5F5 cells may contribute to the increased Ig secretion observed by these LPS-activated lymphocytes, it is unlikely that increased mRNA expression can totally account for the threefold increases in secretion that were observed.