Lipopeptides as immunoadjuvants and immunostimulants in mucosal immunization

In previous studies we have shown that lipopeptides constitute potent immunoadjuvants in mice, rabbits and other species: in parenteral immunization, lipopeptide adjuvants were comparable, or in some cases superior to Freund's adjuvant, and were devoid of the side effects of this additive. Here we demonstrate that lipopeptides also constitute adjuvants for mucosal immunizations. The serum antibody responses against the wheat storage protein gliadin, the bee venom constituent melittin, or the hen egg protein ovalbumin could in most cases be enhanced more than 100-fold by the lipopeptide P3CSK4, applied via the nasal route. An enhanced specific antibody level could also be detected in supernatants of cell cultures prepared from spleens, Peyer's patches, lungs and mesenteric lymph nodes of immunized mice. Moreover, the lipopeptide P3CSK4 enhanced chemiluminescence in mouse spleen cells and peritoneal macrophages in vitro, indicating a macrophage-activating effect. Finally, nasal application of lipopeptide increased protection against a lethal infection of influenza. Our findings are of importance for the improvement of immunizations and might lead to more effective vaccines.     

Effects of the Nature of Adjuvant and Site of Parenteral Immunization on the Serum and Mucosal Immune Responses Induced by a Nasal Boost with a Vaccine Alone

Outbred OF1 mice were immunized subcutaneously with flu vaccine, either in the neck or in the lumbar region (back), in combination with adjuvants inducing either a Th1- or a Th2-type response, referred to as adjuvants A1 and A2, respectively. After two parenteral immunizations, the mice were boosted intranasally with nonadjuvanted vaccine. The serum response was analyzed after each immunization by measuring specific immunoglobulin A (IgA), IgG1, and IgG2a antibody levels, while the local response (same isotypes) was measured in the salivary glands after the mucosal boost by ELISPOTs. We observed that systemic priming at any of the two sites with a Th2 rather than a Th1 adjuvant dramatically enhanced the mucosal IgG1 and IgA responses following a mucosal boost with unadjuvanted vaccine. In addition, as judged by the IgG2a/IgG1 ratios and serum IgA levels, immunization of mice in the back induced a rise in Th2 response compared to neck immunization with adjuvant A1. In contrast, such back immunization with adjuvant A2 reversed the Th1-Th2 balance in favor of the Th1 response compared to neck immunization. Similar differences were observed in mucosal antibody levels according to the site of priming with one given adjuvant; priming in the back with adjuvant A1 increased the mucosal IgA and IgG1 responses compared to neck priming, while the local IgG2a levels were decreased. The reverse was true for adjuvant A2. Back versus neck priming with this latter adjuvant decreased the mucosal IgG1 response, while local IgG2a levels were increased. The different lymphatic drainages of the two sites of parenteral immunization may explain these differences, due to the targeting of particular lymphoid inductive sites. Some of these sites may represent crossroads between systemic and mucosal immunity.     

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.     

Elicitation of Th1/Th2 related responses in mice by chitosan nanoparticles loaded with Brucella abortus malate dehydrogenase,outer membrane proteins 10 and 19

Brucella spp. is the causative agent of brucellosis, one of the worldwide diseases. The pathogen infects humans and animals mainly through the digestive or respiratory tract. Therefore, induction of mucosal immunity is required as the first line of defense. In this study, three Brucella abortus recombinant proteins, malate dehydrogenase (rMdh), outer membrane proteins (rOmp) 10 and 19 were loaded in mucoadhesive chitosan nanoparticles (CNs) and induction of mucosal and systemic immunity were investigated after intranasal immunization of BALB/c mice. These antigens were also coimmunized as cocktail (rCocktail) to evaluate multiple antigen specific vaccine candidates. At 6-weeks post-immunization (wpi), antigen specific total IgG was increased in all of the immunized groups, predominantly IgG1. In addition, spleenocyte from rMdh-, rOmp19-, and rCocktail-immunized groups significantly produced IFN-γ and IL-4 suggesting the induction of a mixed Th1-Th2 response. For mucosal immunity, anti-Mdh IgA from nasal washes and fecal excretions, and anti-Omps IgA from sera, nasal washes, genital secretions and fecal excretions were significantly increased in single antigen immunized groups. In the rCocktail-immunized group, anti-Mdh IgA were significantly increased while anti-Omps IgA was not. Collectively, this study indicates that comprise of B. abortus antigen-loaded CNs elicited the antigen-specific IgA with a Th2-polarized immune responses and combination of the highly immunogenic antigens elicited IgG specific to each type of antigen.     

The bacterial second messenger cdiGMP exhibits promising activity as a mucosal adjuvant

The development of mucosal adjuvants is still a critical need in vaccinology. In the present work, we show that bis(3',5')-cyclic dimeric GMP (cdiGMP), a second messenger that modulates cell surface properties of several microorganisms, exerts potent activity as a mucosal adjuvant. BALB/c mice were immunized intranasally with the model antigen beta-galactosidase (beta-Gal) coadministered with cdiGMP. Animals receiving cdiGMP as an adjuvant showed significantly higher anti-beta-Gal immunoglobulin G (IgG) titers in sera than controls (i.e., 512-fold [P < 0.05]). Coadministration of cdiGMP also stimulated efficient beta-Gal-specific secretory IgA production in the lung (P < 0.016) and vagina (P < 0.036). Cellular immune responses were observed in response to both the beta-Gal protein and a peptide encompassing its major histocompatibility complex class I-restricted epitope. The IgG1-to-IgG2a ratio of anti-beta-Gal antibodies and the observed profiles of secreted cytokines suggest that a dominant Th1 response pattern is promoted by mucosal coadministration of cdiGMP. Finally, the use of cdiGMP as a mucosal adjuvant also led to the stimulation of in vivo cytotoxic T-lymphocyte responses in C57BL/6 mice intranasally immunized with ovalbumin and cdiGMP (up to 30% of specific lysis). The results obtained indicate that cdiGMP is a promising tool for the development of mucosal vaccines.     

Vaccine-Induced Th1-Type Responses are Dominant over Th2-Type Responses in the Short Term whereas Pre-existing Th2 Responses are Dominant in the Longer Term

The effect of adjuvant on induction of human papillomavirus type 16 E7 protein-specific cytotoxic T lymphocytes (CTL) and immunoglobulin G (IgG)_2a antibody was studied in C57BL/6 J mice immunized with various adjuvants and E7 protein. Quil-A adjuvant, but not complete Freund's adjuvant (CFA) or Algammulin, induced a T-helper 1 (Th1)-type response to E7, which was characterized by CTL activity against a tumour cell line transfected with E7 protein and by E7-specific IgG_2a. All tested adjuvants elicited comparable levels of E7-specific IgG₁. The longest duration and greatest magnitude of CTL response was seen following two immunizations with the highest dose of E7 and Quil-A. Simultaneous immunization with a Th1 and a T helper 2 (Th2)-promoting adjuvant gave a Th1-type response. However, E7 and Quil-A were unable to induce a Th1-type response (as measured by the inability to generate anti-E7 IgG_2a antibody) in animals with a pre-existing Th2-type response to E7. These results suggest that saponin adjuvants may be suitable for immunotherapy in humans where a Th1-type response is sought, provided that there is no pre-existing Th2-type response to the antigen.     

Low-dose oral tolerance due to antigen in the diet suppresses differentially the cholera toxin-adjuvantized IgE, IgA and IgG response

BACKGROUND: Cholera toxin (CT) is used as a mucosal adjuvant amongst other applications for studying food allergy because oral administration of antigen with CT induces an antigen-specific type 2 response, including IgE and IgA production. Previously established oral tolerance due to antigen in the diet may radically impact on the CT-adjuvantized immune response. The present study served to evaluate the effect of previously established low-dose oral tolerance on the CT-adjuvantized immune response towards a food antigen. METHODS: Mice fed a diet containing microgram levels of the soy protein Kunitz soy-trypsin inhibitor (KSTI) (F0 mice) and mice fed a soy-free diet (F2 mice) were orally immunized with KSTI and CT. KSTI-specific serum IgG1, IgG2a, IgA and IgE and fecal IgA were monitored. KSTI-stimulated cell proliferation and interleukin (IL)-6 production were determined. RESULTS: The anti-KSTI IgE and IgA responses in the F0 mice were substantially suppressed, while the IgG1 and IgG2a responses were not suppressed after five oral immunizations. The response suppression tended to decline with increasing numbers of immunizations suggesting that the suppression could be overcome by multiple immunizations. However, cell proliferation and IL-6 production were clearly suppressed even after five immunizations. CONCLUSIONS: Priorly established low-dose oral tolerance considerably suppressed the CT-adjuvantized KSTI-specific IgE, IgA and cellular immune response but only weakly and transiently the IgG response. The results revealed that low-dose oral tolerance includes the mucosal IgA response and that CT, albeit mediating an antigen-specific response, does not fully abrogate previously established oral tolerance.     

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.     

Mucosal immunization of CD4+ T cell-deficient mice with an inactivated virus induces IgG and IgA responses in serum and mucosal secretions

Immunoglobulin (Ig) class switching can occur in the absence of alphabeta+ or gammadelta+ T cells when mice are infected with certain live viruses, although CD4 T helper cells are believed to be essential for induction of a high-affinity antibody response and for efficient isotype switching from IgM to IgG and IgA production. However, little information is available about the immune responses after mucosal immunization of CD4+ T cell-deficient mice with inactivated virus. In this study, we show that intranasal immunization with formalin-inactivated influenza A/PR8/34 virus induces IgG and IgA responses in serum and IgA responses in mucosal secretions in CD4+ T cell-deficient mice. All four subclasses of IgG were produced. IgG1/IgG2a ratios were found to be from 1 to 1.75, indicating that both Th1 and Th2 immune responses are induced by the inactivated influenza virus. The sera and mucosal secretions were found to have neutralizing activity against influenza virus in vitro. In addition, the mucosally immunized CD4+ T cell-deficient mice were protected completely from challenge with a lethal dose of live, pathogenic influenza virus. To our knowledge, this is the first demonstration that mucosal immunization with an inactivated virus induces immune responses in serum and mucosal secretions in CD4+ T cell-deficient mice.     

Calcium phosphate nanoparticles induce mucosal immunity and protection against herpes simplex virus type 2

Previously we reported that calcium phosphate nanoparticles (CAP) represented a superior alternative to alum adjuvants in mice immunized with viral protein. Additionally, we showed that CAP was safe and elicited no detectable immunoglobulin E (IgE) response. In this study, we demonstrated that following mucosal delivery of herpes simplex virus type 2 (HSV-2) antigen with CAP, CAP adjuvant enhanced protective systemic and mucosal immunity versus live virus. Mice were immunized intravaginally and intranasally with HSV-2 protein plus CAP adjuvant (HSV-2+CAP), CAP alone, phosphate-buffered saline, or HSV-2 alone. HSV-2+CAP induced HSV-specific mucosal IgA and IgG and concurrently enhanced systemic IgG responses. Our results demonstrate the potency of CAP as a mucosal adjuvant. Furthermore, we show that systemic immunity could be induced via the mucosal route following inoculation with CAP-based vaccine. Moreover, neutralizing antibodies were found in the sera of mice immunized intranasally or intravaginally with HSV-2+CAP. Also, the results of our in vivo experiments indicated that mice vaccinated with HSV-2+CAP were protected against live HSV-2 infection. In conclusion, these preclinical data support the hypothesis that CAP may be an effective mucosal adjuvant that protects against viral infection.     

Induction of Gut Mucosal Immune Responses: Importance of Genetic Background and Th1/Th2 Cross-Regulation

The reciprocal regulation of T-helper cell (Th) subsets is widely documented in various animal models of infectious diseases. In this study IFN-γ/IL-4 double knockout (DKO) mice were used to analyse the role of Th subsets in mucosal immune responses. We found that the DKO mice had normal IgA differentiation but impaired induction of specific gut mucosal antibody responses after oral immunization using cholera toxin adjuvant. Both Th1 and Th2 responses were reduced compared with wild-type mice. Despite the absence of both IFN-γ and IL-4 in the DKO mice the overall results were similar to previous observations in IFN-γ receptor-knockout (IFN-γR^−/−) mice and did not suggest a strict cross-regulation of the two Th subsets in the gut mucosa. To further examine the role of IFN-γ in mucosal immunity we compared two different mouse strains lacking IFN-γ, i.e. IFN-γ^−/− (C57BL/6) and IFN-γR^−/− mice (129/Sv). We found that IFN-γR^−/− mice exhibited reduced mucosal antibody responses and decreased Th1 and Th2 activity after oral immunization, while IFN-γ^−/− mice had intact antibody responses and increased Th2 responses. Thus, genetic differences were found to critically affect the development of a specific gut mucosal immune response. An enhanced Th2 activity in the Peyer's patches following oral immunization was associated with an ability to mount strong intestinal IgA immunity.     

Induction of antigen-specific antibodies in vaginal secretions by using a nontoxic mutant of heat-labile enterotoxin as a mucosal adjuvant.

Immunization of the female reproductive tract is important for protection against sexually transmitted diseases and other pathogens of the reproductive tract. However, intravaginal immunization with soluble antigens generally does not induce high levels of secretory immunoglobulin A (IgA). We recently developed safe mucosal adjuvants by genetically detoxifying Escherichia coli heat-labile enterotoxin, a molecule with a strong mucosal adjuvant activity, and here we describe the use of the nontoxic mutant LTK63 to induce a response in the mouse vagina against ovalbumin (Ova). We compared intravaginal and intranasal routes of immunization for induction of systemic and vaginal responses against LTK63 and Ova. We found that LTK63 is a potent mucosal immunogen when given by either the intravaginal or intranasal route. It induces a strong systemic antibody response and IgG and long-lasting IgA in the vagina. The appearance of vaginal IgA is delayed in the intranasally immunized mice, but the levels of vaginal anti-LTK63 IgA after repeated immunizations are higher in the intranasally immunized mice than in the intravaginally immunized mice. LTK63 also acts as a mucosal adjuvant, inducing a serum response against Ova, when given by both the intravaginal and intranasal routes. However, vaginal IgA against Ova is stimulated more efficiently when LTK63 and antigen are given intranasally. In conclusion, our results demonstrate that LTK63 can be used as a mucosal adjuvant to induce antigen-specific antibodies in vaginal secretions and show that the intranasal route of immunization is the most effective for this purpose.     

Mistletoe lectins enhance immune responses to intranasally co-administered herpes simplex virus glycoprotein D2

The mucosal adjuvant properties of the three type 2 ribosome-inactivating proteins (RIPs) from the European mistletoe, Viscum album L., were investigated. Mistletoe lectins were compared with cholera toxin (CT) as adjuvants when delivered nasotracheally together with herpes simplex virus glycoprotein D2 (gD2). All three mistletoe lectins (MLI, MLII, MLIII) were potent mucosal adjuvants. Co-administration of MLI, MLII or MLIII with gD2 led to significantly higher levels of gD2-specific mucosal immunoglobulin A (IgA) and systemic immunoglobulin G (IgG) antibody than when the antigen was delivered alone. The levels of antibodies induced were similar to those generated in mice immunized with gD2 and the potent mucosal adjuvant CT. Administration of ML1 with gD2 enhanced the antigen-specific splenic T-cell proliferative response. Interleukin-5 (IL-5), but not interferon-gamma (IFN-gamma), was detected in supernatants from splenocytes stimulated in vitro with gD2. This indicates that MLI enhanced type 2 T-helper cell (Th2) responses to the bystander antigen, gD2. Analysis of the gD2- and lectin-specific IgG subclass titres in mice immunized with gD2 and MLI, MLII or MLIII revealed a high ratio of IgG1 : IgG2a, which is compatible with the selective induction of Th2-type immune 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.     

Clostridium difficile toxin A carboxyl-terminus peptide lacking ADP-ribosyltransferase activity acts as a mucosal adjuvant

The receptor binding domains of the most potent mucosal adjuvants, bacterial toxins and plant lectins, are organized in repeat units to recognize specific sugar residues. The lectin-like structure of the C-terminal region of Clostridium difficile toxin A prompted us to investigate the mucosal adjuvant properties of a nontoxigenic peptide corresponding to amino acids 2394 to 2706 (TxA(C314)). We compared TxA(C314) adjuvant activity to those of cholera toxin (CT) and Escherichia coli heat-labile enterotoxin subunit B (EtxB) coadministered orally or nasotracheally with poor peptide antigens (keyhole limpet hemocyanin [KLH] and hen egg lysozyme [HEL]). Levels of anti-KLH-specific serum immunoglobulin G (IgG) and IgA as well as that of mucosal IgA were significantly higher in animals immunized orally with TxA(C314) plus KLH than with KLH alone, CT plus KLH, or EtxB plus KLH. Following intranasal immunization with TxA(C314) plus HEL, levels of serum- and mucosa-specific antibodies were comparable to those induced by coadministering HEL with CT or EtxB. The TxA(C314) adjuvant effect following oral, but not intranasal, immunization was dose dependent. The analysis of the subclasses of anti-KLH-specific IgG isotypes and the cytokines released from splenocytes of immunized mice challenged in vitro with KLH indicates the induction of a mixed Th1/Th2-type immune response, with prevalence of the Th1 branch. We conclude that TxA(C314) enhances immune responses against mucosa-coadministered foreign antigens and represents a promising mucosal adjuvant, especially because its ability to stimulate mixed Th1/Th2 responses with a strong a Th1 component is extremely worthwhile against intracellular pathogens.     

Calcium Phosphate Nanoparticles Induce Mucosal Immunity and Protection against Herpes Simplex Virus Type 2

Previously we reported that calcium phosphate nanoparticles (CAP) represented a superior alternative to alum adjuvants in mice immunized with viral protein. Additionally, we showed that CAP was safe and elicited no detectable immunoglobulin E (IgE) response. In this study, we demonstrated that following mucosal delivery of herpes simplex virus type 2 (HSV-2) antigen with CAP, CAP adjuvant enhanced protective systemic and mucosal immunity versus live virus. Mice were immunized intravaginally and intranasally with HSV-2 protein plus CAP adjuvant (HSV-2+CAP), CAP alone, phosphate-buffered saline, or HSV-2 alone. HSV-2+CAP induced HSV-specific mucosal IgA and IgG and concurrently enhanced systemic IgG responses. Our results demonstrate the potency of CAP as a mucosal adjuvant. Furthermore, we show that systemic immunity could be induced via the mucosal route following inoculation with CAP-based vaccine. Moreover, neutralizing antibodies were found in the sera of mice immunized intranasally or intravaginally with HSV-2+CAP. Also, the results of our in vivo experiments indicated that mice vaccinated with HSV-2+CAP were protected against live HSV-2 infection. In conclusion, these preclinical data support the hypothesis that CAP may be an effective mucosal adjuvant that protects against viral infection.     

HIV-1CN54 DNA疫苗滴鼻免疫小鼠诱发系统和黏膜免疫应答

目的：探讨重组痘苗病毒rVVsyngp120或rVVmCN54gp120候选疫苗是否增强HIV-1CN54合成gp120基因(syngp120)DNA疫苗的免疫原性。方法：第0、7、14、21天用DNA疫苗滴鼻免疫小鼠,第28、35、42天再滴鼻接种rVVsyngp120或rVVmCN54gp120。体外测脾和肠系膜淋巴结(MLN)淋巴细胞增殖应答与CD8^ CTL应答。测血清和黏膜洗液特异的IgG和IgA,并测其是否中和实验室适应株HIV-1SF33。结果：单纯DNA免疫后,脾和MLN淋巴细胞在体外发生增殖应答和CTL应答,且测出血清特异的IgG和黏膜洗液特异的IgA。重组痘苗病毒末次免疫后第2周(第56天),发现rVVmCN54gp120增强MLN淋巴细胞增殖应答和CTL应答,脾CTL应答也增强。rVVsyngp120则增强MLN CTL应答。同时发现：2组重组痘苗病毒免疫的动物其血清中特异IgG抗体滴度均有所增高,但黏膜(粪便和阴道)洗液特异IgA抗体滴度却未增高,未测出血清特异IgA和黏膜洗液特异IgG。免疫血清可中和HIV-1SF33,而阴道洗液却不能。结论：单纯DNA疫苗滴鼻免疫可诱发较弱的系统和黏膜体液免疫与细胞免疫,但维持时间短。重组痘苗病毒主要增强局部黏膜的细胞免疫应答,且稍增强系统体液免疫应答,未增强黏膜的IgA应答。免疫血清有中和作用。     

Immune response induced by recombinant Mycobacterium bovis BCG producing the cholera toxin B subunit

The pentameric form of the cholera toxin B subunit (CTB) is known to be a strong mucosal adjuvant and stimulates antigen-specific secretory immunoglobulin A (IgA) and systemic antibody responses to antigens when given by mucosal routes. To deliver CTB for prolonged periods of time to the respiratory mucosa, we constructed a Mycobacterium bovis bacillus Calmette-Guérin (BCG) strain that produces and secretes assembled pentameric CTB. Mice immunized intranasally (i.n.) with recombinant BCG (rBCG) developed a stronger anti-BCG IgA response in bronchoalveolar lavage fluids (BALF) than mice immunized with nonrecombinant BCG. The total IgA response in the BALF of mice immunized with rBCG was also stronger than that in BALF of mice immunized with the nonrecombinant strain. The induction of IgA was well correlated with an increased production of transforming growth factor beta1. Simultaneous administration of intraperitoneally delivered ovalbumin and of i.n. delivered CTB-producing BCG induced a long-lasting ovalbumin-specific mucosal IgA response as well as a systemic IgG response, both of which were significantly higher than those in mice immunized with nonrecombinant BCG together with ovalbumin. These results suggest that the CTB-producing BCG may be a powerful adjuvant to be considered for future mucosal vaccine development.     

Mucosal Immunity Induced by Pneumococcal Glycoconjugate

Host defenses against Streptococcus pneumoniae involve opsonophagocytosis mediated by antibodies and complement. Because the pneumococcus is a respiratory pathogen, mucosal immunity may play an important role in the defense against infection. The mechanism for protection in mucosal immunity consists of induction of immunity by the activation of lymphocytes within the mucosal-associated lymphoid tissues, transport of antigen-specific B and T cells from inductive sites through bloodstream and distribute to distant mucosal effector sites. Secretory IgA is primarily involved in protection of mucosal surfaces. Mucosal immunization is an effective way of inducing immune responses at mucosal surfaces. Several mucosal vaccines are in various stages of development. A number of mucosal adjuvants have been proposed. CpG oligodeoxynucleotide (ODN) has been shown to be an effective mucosal adjuvant for various antigens. Mucosal immunity induced by intranasal immunization was studied with a pneumococcal glycoconjugate, using CpG ODN as adjuvant. Mice immunized with type 9V polysaccharide (PS) conjugated to inactivated pneumolysin (Ply) plus CpG produced high levels of 9V PS IgG and IgA antibodies compared to the group that received the conjugate alone. High levels of subclasses of IgG1, IgG2 and IgG3 antibodies were also observed in sera of mice immunized with 9V PS-Ply plus CpG. In addition, high IgG and IgA antibody responses were observed in sera of young mice immunized with 9V PS-Ply plus CpG or the conjugate plus non-CpG compared with the group received the conjugate alone. These results reveal that mucosal immunization with pneumococcal glycoconjugate using CpG as adjuvant can confer protective immunity against pneumococcal infection.     

甲型肝炎减毒活疫苗及灭活疫苗灌胃免疫小鼠后的抗体应答

目的 :测定甲肝减毒活疫苗及灭活疫苗灌胃免疫小鼠后的抗体应答效应。方法 :将活或灭活的甲肝疫苗加或不加明胶 ,经胃免疫小鼠 ,于末次免疫后 2w取血清及肠液 ,用间接ELISA法分别检测其中的特异性IgG和IgA抗体水平 ,并与空白及肌注组比较。结果 :实验组特异抗体水平明显高于肌注组 (P <0 0 1) ;加明胶组的免疫效果较不加者好 (IgG :P <0 0 0 1,IgA :P <0 0 5 )。结论 :甲肝活疫苗及灭活疫苗经消化道免疫小鼠后 ,均可诱导全身及局部的抗体应答 ;明胶有增强抗体产生的作用 ,可作为一种安全、廉价的粘膜佐剂被进一步开发与利用。