Oral immunization of interleukin-4 (IL-4) knockout mice with a recombinant Salmonella strain or cholera toxin reveals that CD4+ Th2 cells producing IL-6 and IL-10 are associated with mucosal immunoglobulin A responses.

Mucosal immunoglobulin A (IgA) responses are often associated with Th2-type cells and derived cytokines, and interleukin-4 (IL-4) knockout (IL-4-/-) mice with impaired Th2 cells respond poorly to oral antigens. However, we have noted that IL-4-/- mice have normal mucosal IgA levels, which led us to query whether different oral delivery systems could elicit mucosal immunity. Two oral regimens were used: (i) a live recombinant Salmonella strain which expresses fragment C (ToxC) of tetanus toxin, and (ii) soluble tetanus toxoid (TT) with cholera toxin (CT) as an adjuvant. Oral immunization of IL-4-/- mice with recombinant Salmonella vaccine expressing ToxC induced brisk mucosal IgA and serum IgG (mainly IgG2a) anti-TT antibody responses. TT-specific CD4+ T cells from spleen or Peyer's patches produced gamma interferon, indicative of Th1 responses; however, IL-6 and IL-10 were also seen. Oral immunization of IL-4-/- mice with TT and CT induced weak mucosal IgA to TT; however, brisk IgA anti-CT-B responses and CT-B-specific CD4+ T cells producing IL-6 and IL-10 were also noted. These results show that although IL-4-dependent antibody responses are impaired, mucosal IgA responses are induced in IL-4-/- mice. These result suggest that certain cytokines, i.e., IL-6 and IL-10 from Th2-type cells, play an important compensatory role in the induction and regulation of mucosal IgA responses.     

Immunoglobulin A-deficient mice exhibit altered T helper 1-type immune responses but retain mucosal immunity to influenza virus

We have previously demonstrated that immunoglobulin A (IgA)(-/-) knockout (KO) mice exhibit levels of susceptibility to influenza virus infection that are similar to those of their normal IgA(+/+) littermates. To understand the mechanism of this apparent mucosal immunity without IgA, immunoglobulin isotype and T helper 1 (Th1)-type [interferon-gamma (IFN-gamma)] and Th2-type [interleukin (IL)-4, IL-5)] cytokine responses to influenza vaccine were evaluated. Intranasal immunization with influenza virus subunit vaccine plus cholera toxin/cholera toxin B subunit (CT/CTB) induced significant influenza virus-specific immunoglobulin G (IgG) antibody in the serum and nasal passages of both IgA(-/-) and IgA(+/+) mice, while IgA antibodies were induced only in IgA(+/+) mice. IgA KO mice exhibited an IgG1 subclass haemagglutinin (HA)-specific response but no detectable IgG2a and IgG2b responses. In contrast, IgA(+/+) mice exhibited significant IgG1 as well as IgG2a responses. This indicates a predominant Th2-type response in IgA KO mice compared to normal mice. Following stimulation with influenza virus in vitro, splenic lymphocytes from immunized IgA(-/-) mice produced significantly lower levels of IFN-gamma than IgA(+/+) mice (P < 0.001), but elaborated similar levels of IL-4 and IL-5. This was true at both protein and mRNA levels. Immunized mice were challenged intranasally with a small inoculum of influenza virus to allow deposition of virus in the nasal mucosal passages. Compared to non-immunized mice, immunized IgA(-/-) and IgA(+/+) mice exhibited significant, but similar levels of reduction in virus titres in the nose and lung. These results demonstrate that in addition to IgA deficiency, IgA gene deletion also resulted in down-regulated Th1-type immune responses and confirm our previous data that IgA antibody is not indispensable for the prevention of influenza virus infection.     

Immune response after adjuvant mucosal immunization of mice with inactivated influenza virus

Satisfactory mucosal immunity in the respiratory tract is very important for protection against influenza. It can be achieved only by mucosal immunization. Mucosal vaccination with inactivated influenza virus may not be sufficiently effective and suitable adjuvants are therefore sought. We tested intratracheal immunization of mice with inactivate B type influenza virus in a mixture with formolized G+ bacterium Bacillus firmus, whose adjuvant effects have previously been documented in another system. The treatment resulted in a marked increase of both systemic and mucosal antibody response in IgG and IgA classes. Stimulation of T lymphocytes after adjuvant immunization was very mild, no proliferation taking place after specific stimulation with antigen in vitro. However, slightly increased systemic (spleen) and local (lungs) production of cytokines without perceptible Th1/Th2 polarization was determined. B. firmus is an efficient adjuvant in respiratory tract immunization while with subcutaneous immunization it lowers the antibody response.     

Expression of recombinant enterotoxigenic Escherichia coli colonization factor antigen I by Salmonella typhimurium elicits a biphasic T helper cell response

Protective immunity to enterotoxigenic Escherichia coli (ETEC) is antibody (Ab) dependent; however, oral immunization with purified ETEC fimbriae fails to elicit protective immunity as a consequence of antigenic alteration by the gastrointestinal (GI) tract. Unless unaltered ETEC fimbriae can reach the inductive lymphoid tissues of the GI tract, immunity to ETEC cannot be induced. To produce immunity, live vectors, such as Salmonella typhimurium, can effectively target passenger antigens to the inductive lymphoid tissues of the GI tract. By convention, oral immunizations with Salmonella vectors induce CD4(+) T helper (Th) cell responses by gamma interferon (IFN-gamma)-dominated pathways both to the vector and passenger antigen, resulting in serum immunoglobulin G2a (IgG2a) and modest mucosal IgA Ab responses. In the present study, mice orally immunized with a Salmonella vector engineered to stably express ETEC colonization factor antigen I (CFA/I) showed initially elevated serum IgG1 and mucosal IgA anti-CFA/I Ab responses. As expected, mice orally immunized with an E. coli-CFA/I construct elicited poor anti-CFA/I Ab responses. In fact, the addition of cholera toxin during oral E. coli-CFA/I immunization failed to greatly enhance mucosal IgA Ab responses. Seven days after immunization with the Salmonella-CFA/I construct, cytokine-specific ELISPOT showed induction of predominant Th2-type responses in both mucosal and systemic immune compartments supporting the early IgG1 and IgA anti-CFA/I Abs. By 4 weeks, the Th cell response became Th1 cell dominant from the earlier Th2-type responses, as evidenced by increased mucosal and systemic IFN-gamma-producing T cells and a concomitant elevation of serum IgG2a Ab responses. This biphasic response offers an alternative strategy for directing Salmonella vector-induced host immunity along a Th2 cell-dependent pathway, allowing for early promotion of mucosal and systemic Abs.     

Enhancement of mucosal immune responses by chimeric influenza HA/SHIV virus-like particles

To enhance mucosal immune responses using simian-human immunodeficiency virus-like particles (SHIV VLPs) as a mucosal HIV vaccine, we have produced phenotypically mixed, chimeric influenza HA/SHIV 89.6 VLPs and used them to immunize C57B/6J mice intranasally. Systemic and mucosal antibody responses, as well as cytotoxic T cell (CTL) responses, were compared in groups immunized with SHIV 89.6 VLPs or HA/SHIV 89.6 VLPs. Intranasal immunizations were given using VLPs either with or without the addition of the mucosal adjuvant cholera toxin. Total serum IgG, IgG1 and IgG2a, and IgA in saliva, vaginal lavage, lung wash, and fecal extracts were evaluated by enzyme-linked immunosorbent assay (ELISA). The level of serum IgG production to HIV Env was highest in the group immunized with chimeric HA/SHIV 89.6 VLPs. Similarly, mucosal IgA production was also enhanced in the mucosal HA/SHIV 89.6 VLP-immunized group. Analysis of the IgG1/IgG2a ratio indicated that a Th1-oriented immune response resulted from these VLP immunizations. High levels of serum IgG and mucosal IgA against influenza virus were also detected in mice immunized with HA/SHIV VLPs. HA/SHIV 89.6 VLP-immunized mice also showed significantly higher CTL responses than those observed in SHIV 89.6 VLP-immunized mice. Furthermore, a Major Histocompatibility Complex (MHC)-class-I-restricted T cell activation ELISPOT assay showed elevated interferon-gamma, interleukin-2, and interleukin-12 production in HA/SHIV 89.6 VLP-immunized mice, indicating that phenotypically mixed HA/SHIV 89.6 VLPs can enhance both humoral and cellular immune responses at multiple mucosal sites. Therefore, chimeric HA-containing VLPs represent a potential approach for mucosal immunization for prevention of HIV infection.     

Protective and cross-protective mucosal immunization of mice by influenza virus type A with bacterial adjuvant.

Mucosal immunization by inactivated viruses often fails to evoke a sufficient immune response. Intensive efforts have been made to enhance the response by suitable adjuvants. We used the G+ nonpathogenic delipidated bacterium Bacillus firmus with pronounced immunostimulatory properties as an adjuvant for immunizing mice with inactivated influenza virus type A. BALB/c mice were immunized intratracheally with inactivated influenza A H1N1 and H3N2 viruses. The production of antibodies in sera and secretions was determined by the ELISA. The local situation in the lungs was assessed histologically and by testing the cytokine expression. The protective and cross-protective effect against infection was tested in in vivo experiments after infection with influenza virus A H1N1. B. firmus as adjuvant increased both systemic and mucosal antibody responses, improved protection against homologous virus and induced cross-protection against virus H1N1 after immunization with virus H3N2.     

Effect of oral immunization with recombinant urease on murine Helicobacter felis gastritis.

The ability of oral immunization to interfere with the establishment of infection with Helicobacter felis was examined. Groups of Swiss Webster mice were immunized orally with 250 micrograms of Helicobacter pylori recombinant urease (rUrease) and 10 micrograms of cholera toxin (CT) adjuvant, 1 mg of H. felis sonicate antigens and CT, or phosphate-buffered saline (PBS) and CT. Oral immunization with rUrease resulted in markedly elevated serum immunoglobulin G (IgG), serum IgA, and intestinal IgA antibody responses. Challenge with live H. felis further stimulated the urease-specific intestinal IgA and serum IgG and IgA antibody levels in mice previously immunized with rUrease but activated primarily the serum IgG compartment of PBS-treated and H. felis-immunized mice. Intestinal IgA and serum IgG and IgA anti-urease antibody responses were highest in rUrease-immunized mice at the termination of the experiment. Mice immunized with rUrease were significantly protected (P < or = 0.0476) against infection when challenged with H. felis 2 or 6 weeks post-oral immunization in comparison with PBS-treated mice. Whereas H. felis-infected mice displayed multifocal gastric mucosal lymphoid follicles consisting of CD45R+ B cells surrounded by clusters of Thy1.2+ T cells, gastric tissue from rUrease-immunized mice contained few CD45R+ B cells and infrequent mucosal follicles. These observations show that oral immunization with rUrease confers protection against H. felis infection and suggest that gastric tissue may function as an effector organ of the mucosal immune system which reflects the extent of local antigenic stimulation.     

Comparison of long-term systemic and secretory antibody responses in children given live, attenuated, or inactivated influenza A vaccine

A comparison of inactivated intramuscular and live intranasal influenza A vaccines in young children undergoing primary immunization might be expected to show differences in serum and local mucosal antibody responses. To demonstrate such differences, serum and local respiratory tract antibody responses of young children vaccinated with intranasal live, attenuated, cold-adapted (H3N2 or H1N1), or intramuscular inactivated (H3N2) influenza A vaccines were examined for one year after vaccination. Antibody responses were measured by hemagglutination-inhibition (HAI) and class-specific enzyme-linked immunosorbent assay (ELISA). One year after vaccination, live intranasal vaccinees had significantly less decay of serum HAI (p = 0.025) and IgG antibody (p = 0.01) directed against the influenza hemagglutinin and neuraminidase than did intramuscular inactivated vaccinees. Nasal secretory IgA developed almost exclusively in live vaccinees and persisted for up to one year. Persistent nasal secretory IgG was detected in both live and inactivated vaccinees. Live vaccination not only stimulates a more durable serum antibody response, but also induces long-lasting local respiratory tract IgA antibody that may play an important role in host protection.     

CD19-deficient mice exhibit poor responsiveness to oral immunization despite evidence of unaltered total IgA levels, germinal centers and IgA-isotype switching in Peyer's patches

CD19 exhibits a critical role as a response regulator in B cells, influencing activation, differentiation and survival. Accordingly, CD19-deficient mice largely lack B-1 cells, and their conventional B-2 cells are poor responders to thymus-dependent antigen. Since both B-1 and B-2 cells may contribute to the total intestinal IgA production, we investigated whether lack of CD19 negatively affected mucosal immunity. We found that CD19(-/-) mice have near normal total IgA levels in serum and gut mucosa and, contrary to systemic lymphoid tissues, Peyer's patches (PP) had germinal centers to which also IgA+ B cells localized. However, the mice demonstrated severely impaired responses to oral immunization with keyhole limpet hemocyanin plus cholera toxin adjuvant. Mucosal responses to oral immunization were significantly more impaired than systemic responses. Despite normal specific IL-4 production, a selective defect in Th2-regulated B cell isotypes was observed, with poor or no mucosal IgA, low serum IgG1 and no IgE, but intact serum IgA and IgG2a production. Ex vivo experiments revealed strongly inhibited CD40-stimulated proliferation and IgA differentiation in CD19-deficient PP B cells. Taken together, an impaired CD40 responsiveness selectively affected Th2, but not Th1, coordinated B cell responses. The CD19(-/-) mice provide compelling evidence for the differential regulation of serum and mucosal IgA immunity.     

Determination of cytokine co-expression in individual splenic CD4+ and CD8+ T cells from influenza virus-immune mice.

We have studied the patterns of interleukin-2 (IL-2), IL-4 and interferon-gamma (IFN-gamma) co-expression displayed by individual splenic CD4+ and CD8+ T cells in response to influenza virus immunization. Unseparated spleen cells obtained from mice intraperitoneally (i.p.) injected with A/PR8 (H1N1) influenza virus (PR8) were cultured for 24 hr in the presence of ultraviolet-inactivated PR8. As controls, cultures of both naive spleen cells stimulated with PR8 or of immune cells lacking the inactivated virus were used. The frequencies of CD4+ and CD8+ T cells expressing IL-2, IL-4 and IFN-gamma were determined by three-colour flow cytometric analysis of fixed and saponin-permeabilized cells fluorescent-stained for either CD4 or CD8 surface molecules and for one of the following combinations of two intracellular cytokines: IL-2/IL-4, IL-2/IFN-gamma and IL-4/IFN-gamma. The results showed that immunization with influenza virus induces in both CD4+ and CD8+ T cells a heterogeneity of cytokine response patterns that do not follow the type 1/type 2 polarized response model, but with substantial differences between the two populations. In fact, the analysis of the phenotypes of virus-immune CD8+ T cells revealed similar significant proportions of cells either expressing any one of the three cytokines or co-expressing combinations of them (i.e. IL-4/IL-2, IL-4/IFN-gamma and IL-2/IFN-gamma), whereas immune CD4+ T cells were seen to express almost exclusively a single cytokine per cell. The observed patterns of cytokine production suggest that influenza virus immunization induces the expression of a type 0 cytokine pattern at both population and single cell levels in CD8+ T cells and exclusively at the population level in CD4+ T cells.     

Effects of Different Adjuvants in the Context of Intramuscular and Intranasal Routes on Humoral and Cellular Immune Responses Induced by Detergent-Split A/H3N2 Influenza Vaccines in Mice

Influenza A/H3N2 viruses have caused the most severe epidemics since 1968 despite current immunization programs with inactivated vaccines. We undertook a side-by-side preclinical evaluation of different adjuvants (Alum, AS03, and Protollin) and routes of administration (intramuscular [i.m.] and intranasal [i.n.]) for assessing their effect on the immunogenicity and cross-reactivity of inactivated split vaccines (A/H3N2/New York/55/2004). Humoral and T cell-mediated immune responses against the homologous virus and a heterologous drifted strain (A/H3N2/Wisconsin/67/2005) were measured in BALB/c mice at 2, 6, and 19 weeks postboost. The AS03- and Alum-adjuvanted i.m. vaccines induced at least an 8-fold increase over the nonadjuvanted vaccine in functional antibody titers against both the homotypic and heterotypic strains and low IgG2a and high IgG1 levels, suggesting a mixed Th1/Th2 response with a Th2 trend. The Protollin-adjuvanted i.n. vaccine induced the lowest IgG1/IgG2a ratio, which is indicative of a mixed Th1/Th2-type profile with a Th1 trend. This adjuvanted vaccine was the only vaccine to stimulate a mucosal IgA response. Whatever the timing after the boost, both hemagglutination inhibition (HAI) and microneutralization (MN) titers were higher with the AS03-adjuvanted i.m. vaccine than with the protollin-adjuvanted i.n. vaccine. Finally, the Alum-adjuvanted i.m. vaccine and the lower-dose Protollin-adjuvanted i.n. vaccine elicited significantly higher CD4⁺ Th1 and Th2 responses and more gamma interferon (IFN-γ)-producing CD8⁺ T cells than the nonadjuvanted vaccine. Our data indicate that the adjuvanted vaccines tested in this study can elicit stronger, more persistent, and broader immune responses against A/H3N2 strains than nonadjuvanted inactivated influenza vaccines.     

The immune response to influenza vaccines

Specific immunity to influenza is associated with a systemic immune response (serum haemagglutination inhibition antibody), local respiratory immune response (virus-specific local IgA and IgG antibodies in nasal wash), and with the cell-mediated immune response. Both inactivated and live influenza vaccines induce virus-specific serum antibody which can protect against infection with influenza virus possessing the same antigenic specificity. In the absence of serum antibodies, local antibodies in nasal wash are a major determinant of resistance to infection with influenza virus. In comparative studies in humans it was shown that nasal secretory IgA develops chiefly after immunization with live cold-adapted (CA) vaccine, but persistent nasal secretory IgG was detected in both CA live and inactivated vaccines. The origin of nasal wash haemagglutination inhibition (HI) antibodies is not completely known. Recently it was found that cytotoxic T-cells (CTL) play an important role in immunity against influenza and in clearance of influenza virus from the body. In primed humans, inactivated influenza vaccine stimulates a cross-reactive T-cell response, whereas the ability of inactivated vaccine to stimulate such immunity in unprimed humans has not been determined. Data on the T-cell response to live vaccine in humans are limited to the development of secondary T-cell responses in primed individuals vaccinated with a host-range (HR) attenuated vaccine. The data obtained have shown that immunity induced by inactivated influenza vaccines is presumably dependent on the stimulation of serum antibody. Live CA vaccines not only stimulate a durable serum antibody response, but also induce long-lasting local respiratory tract IgA antibody that plays an important role in host protection.     

Estimation of human and animal immunological memory by testing the trogocytosis of virus-specific T lymphocytes

This study is the first attempt to evaluate the immunogenicity of Russian live attenuated influenza reassortant influenza vaccine (LAIV), by using a modified T-cell recognition of antigen presenting cells by protein capture (TRAP) method. Single vaccination of 18-20-year-old volunteers with LAIV causes an increase in the peripheral blood levels of virus-specific memory CD4+ T lymphocytes. Some (40-60%) LAIV-vaccination volunteers respond to immunization by showing a significant elevation in the peripheral blood level of memory CD4+ T cells without a systemic humoral immune response recorded in the passive hemagglutination test. Vaccination of mice with live attenuated A (H1N1) influenza reassortant virus stimulates the production of memory CD8+CD44hi T lymphocytes in the nasal-associated lymphoid tissue, the entry of infection, so does influenza infection. Vaccination with inactivated A (H1N1) influenza virus practically fails to induce these cells. A (H1N1) influenza virus-specific CD8+CD44hi T lymphocytes remain within at least 2 months (observation time). The authors' modified TRAP may be used to evaluate virus-specific immunological T-cell memory after vaccination.     

Immunologic uniqueness of the genital tract: challenge for vaccine development

Although the genital tract is considered to be a component of the mucosal immune system, it displays several distinct features not shared by other typical mucosal tissues and external secretions. Both male and female genital tract tissues lack inductive mucosal sites analogous to intestinal Peyer's patches. Consequently, local humoral and cellular immune responses stimulated by infections [with e.g. Neisseria gonorrhoeae, Chlamydia trachomatis, papilloma virus, and human immunodeficiency virus (HIV-1)] are weak or absent, and repeated local intravaginal immunizations result in minimal humoral responses. In contrast to typical external secretions such as intestinal fluid that contain secretory immunoglobulin A (S-IgA) as the dominant isotype, semen and cervico-vaginal fluid contain more IgG than IgA. Furthermore, irrespective of the route of infection, humoral immune responses to HIV-1 are dominated by specific IgG and low or absent IgA antibodies in all external secretions. Because a significant proportion of IgG in genital tract secretions is derived from the circulation, systemic immunization may provide protective IgG antibody-mediated immunity in the genital tract. Furthermore, combined systemic and mucosal (oral, rectal, and especially intranasal) immunization may induce protective humoral responses in both the systemic and mucosal compartments of the immune system.     

Enhancement of mucosal immune responses to the influenza virus HA protein by alternative approaches to DNA immunization

DNA immunization provides many advantages as an approach to prevent infectious diseases. However, although previous studies using this approach have demonstrated immune responses in serum, they were not successful in inducing significant levels of antibodies in secretions. In this study, plasmid DNAs expressing the influenza virus hemagglutinin glycoprotein have been evaluated for their ability to induce antibody responses in serum and saliva when used alone or along with either liposomes or bioadhesive polymers as mucosal delivery vehicles. Significant levels of virus-specific Ig in serum as well as secretory IgA in saliva were detected in mice following mucosal DNA immunization. These antibodies were found to block the infectivity of the virus using a plaque reduction assay. Our findings thus indicate that mucosal DNA immunization with specific delivery systems can elicit virus-specific antibody responses in serum as well as IgA responses at mucosal surfaces.     

Mucosal homeostasis: role of interleukins, isotype-specific factors and contrasuppression in the IgA response

Oral ingestion of antigen elicits immune responses at mucosal sites where humoral immunity is largely due to antibodies of the IgA isotype. This is often accompanied by suppression of systemic responses to the same antigen, a state termed oral tolerance. This IgA response is regulated by interactions between T cell subsets found at IgA inductive tissues, i.e., the gut-associated lymphoreticular tissue (GALT) or Peyer's patches (PP). PP T helper (Th) cells support IgA responses, and interleukins 5 (IL-5) and IL-6 can augment secretion of this isotype. Subsets of Th cells may also express Fc receptors for IgA (Fc alpha R) and secrete Fc alpha R as an IgA-binding factor (IBF alpha). Membrane-derived Fc alpha R is a glycoprotein of 38,000 M.W. and this molecule induces selective increases in IgA secreting cells (as determined by the ELISPOT assay) in PP B cell cultures. Fc alpha R+ T cell lines have been shown to secrete IBF alpha as well as IL-5 both of which promote IgA synthesis. Recombinant IL-5 (rIL-5) and rIL-6 induce IgA synthesis mainly by PP B cell blasts, and principally act on surface IgA-positive (sIgA+) B cells for these responses. Another form of mucosal regulation is provided by T contrasuppressor (Tcs) cells, which abrogate oral tolerance when adoptively transferred to mice and restore systemic responsiveness to the antigen sheep erythrocyte (SRBC). Tcs cells from mice systemically primed with SRBC support IgM and IgG subclass responses, while Tcs cells from orally primed mice support IgM, IgG subclass and IgA anti-SRBC responses. These Tcs cells are CD3+, CD4-, 8- and are antigen-specific. These regulatory cells may use the gamma-delta (gamma-delta) form of T cell receptor for antigen recognition.     

Comparison of in vitro immunostimulatory potential of live and inactivated influenza viruses

Live influenza viruses, heat-inactivated virus, and a trivalent formalin-inactivated influenza vaccine were analyzed for their in vitro stimulatory properties on immune cells from healthy donors. Lymphocyte proliferation induced by each influenza antigen was comparable. Influenza vaccine stimulated significantly lower production of interferon-gamma (IFN-gamma) compared with live and heat inactivated viruses, whereas both vaccine and heat-inactivated influenza induced lower levels of IFN-alpha compared with live virus. Furthermore, only live virus generated influenza-specific cytotoxic T lymphocyte (CTL) activity. A significant increase in monocyte expression of CD80, CD86, CD40, and human leukocyte antigen-DR (HLA-DR) was also induced by live influenza virus. Our results suggest that immunization with live influenza vaccines might induce immune responses that would not be induced by conventional inactivated vaccines, including CTL generation, antiviral IFN-gamma and IFN-alpha cytokine production, and increased antigen presentation and costimulatory capacity on antigen presenting cells (APC).     

Enhancement of mucosal antibody responses to Salmonella typhimurium and the microbial hapten phosphorylcholine in mice with X-linked immunodeficiency by B-cell precursors from the peritoneal cavity.

The observation that approximately half of the B cells in the murine intestinal lamina propria are derived from peritoneal CD5 B-cell precursors raises the question of their contribution to mucosal protection. Using mice with X-linked immunodeficiency which are deficient in CD5+ B cells, we showed that they mount little serum and virtually no intestinal immunoglobulin M (IgM), IgG, and IgA antibody responses following oral inoculation with live Salmonella typhimurium. Nonresponsive Xid mice were reconstituted with responsive CBA/Ca donor cell preparations which were constitutively enriched or depleted of CD5 B-cell precursors. Reconstitution of irradiated Xid mice with CD5 B-cell-deficient bone marrow from CBA/Ca donors marginally improved IgM responses in the intestinal mucosa but had no effect on IgG or IgA in response to oral immunization with live S. typhimurium. Whenever Xid mice were reconstituted with donor cells from the peritoneal cavity, which are enriched for CD5 B-cell precursors, strong IgA and in some cases IgG responses in the intestinal mucosa were stimulated in response to oral immunization. When mucosal and serum antibody responses were compared, the peritoneal donor cells again reinstated maximal serum antibody responses to S. typhimurium. Serum and mucosal responses to the bacterial hapten phosphorylcholine could be induced in Xid mice after immunization with S. typhimurium or hapten-carrier conjugates but only following reconstitution with donor cells containing CD5 B-cell precursors. These observations suggest that different lymphoid compartments are enriched for regulatory or effector cells which vary in their contributions to the mucosal antibody response against epitopes on S. typhimurium.     

The immunostimulating complex (ISCOM) is an efficient mucosal delivery system for respiratory syncytial virus (RSV) envelope antigens inducing high local and systemic antibody responses

ISCOM is an efficient mucosal delivery system for RSV envelope proteins as measured by antibody responses in respiratory tract secretions and in sera of mice following two intranasal (i.n.) administrations. Intranasally administered RSV ISCOMs induced high levels of IgA antibodies both in the upper respiratory tract and in the lungs. In the lungs, a prominent and long-lasting IgA response was recorded, which still persisted 22 weeks after the second i.n. immunization when the experiment ended. Subcutaneous (s.c.) immunization only induced low IgA titres in the upper respiratory tract and no measurable response to RSV was found in the lungs. Differences were also noticed in serum between the i.n. and s.c. modes of immunization. ISCOMs given intranasally induced earlier, higher and longer lasting IgM and IgG1 serum anti-RSV antibody responses than those induced by the s.c. mode of administration. A low serum IgE response was only detectable at 2 weeks after i.n. immunization with ISCOMs and after s.c. immunization with an inactivated virus, but no IgE response was detectable after s.c. injection of ISCOMs. The serum IgA response was more pronounced following s.c. injection of inactivated virus than after i.n. application of ISCOMs, and a clear-cut booster effect was obtained with a second immunization. Virtually no serum IgA response was detected after the s.c. administration of ISCOMs. In conclusion, the high immune responses induced by RSV ISCOMs in the respiratory tract and serum after i.n. administration indicate prominent mucosal delivery and adjuvant properties of the ISCOMs, warranting further studies.     

Interleukin 12 and innate molecules for enhanced mucosal immunity

Recent strategies for understanding the mechanisms underlying mucosal immune responses and subsequent development of mucosal vaccines for induction of targeted immunity now include cytokines and molecules of innate immunity. These studies have shown that cytokines influencing the development of T helper (Th) cells differentially affect the outcome of mucosal vs. systemic immune responses to mucosal vaccines. Serum antigen-specific antibody (Ab) responses were enhanced when either IL-6 or IL-12 was mucosally administered with a protein antigen, while only IL-12 induced antigen-specific mucosal IgA Ab responses. Mucosal IL-6 and IL-12 also affected the type of Th cell responses induced by CD4+ T cells from mice that received IL-12 secreted larger amounts of IFN-gamma and IL-6 when compared with mice nasally treated with IL-6. Discrepancies in the ability to enhance mucosal or systemic immune responses were also observed when human neutrophil peptide (HNP) defensins or lymphotactin were nasally coadministered with protein antigens. Only lymphotactin promoted mucosal secretory IgA (S-IgA) Ab responses while both lymphotactin and defensins enhanced systemic immunity to mucosally co-administered protein antigens. Mixed antigen-specific Th1 -and Th2-type CD4+ T cell responses were induced in the systemic compartment by both lymphotactin and the mixture of HNP-1, HNP-2, and HNP-3 defensins. However, HNPs failed to significantly enhance cytokine secretion by mucosally derived, antigen-specific CD4+ T cells relative to those isolated from the systemic compartment. In summary, these studies clearly show that IL-12 and lymphotactin are able to trigger S-IgA Ab responses and provide new avenues for the design of safe and targeted mucosal vaccines.