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The mucosal immune system consists of T and B lymphocytes and accessory cells that function to protect the human body from pathogens and toxins that enter the host via the mucosal surfaces. One major pathogen is human immunodeficiency virus (HIV), the causative agent of the acquired immunodeficiency syndrome (AIDS). By the year 2000, an estimated 35-40 million people will be infected with HIV worldwide (Chin, 1991). The most common mode of transmission of HIV is via sexual contact where HIV-infected cells or possibly cell-free HIV initiate infection at the mucosal surfaces of the vagina or the rectum (Milman and Sharma, 1994). Based on studies performed in the simian immunodeficiency virus (SIV) model, it appears that HIV infection of newborns may be initiated at the mucosal surfaces of the alimentary canal after swallowing HIV during birth (Baba et al., 1994, 1995). In vitro studies suggest that cells residing in mucosal tissues may be the first cells to become infected with HIV after exposure at the mucosal surface (Batman et al., 1994). The mucosal immune system is therefore in a pivotal position to play a key role in resistance to as well as contribute to the morbidity associated with HIV infection. This chapter will introduce basic concepts of T-helper cell regulation of the mucosal antibody response, followed by a discussion of the effects of HIV infection on the mucosal immune system and conclude with current strategies being employed to prevent HIV infection at mucosal surfaces.

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.

The induction of effective mucosal immunity that also provides systemic immunity is a considerable challenge. Over the past two years, efforts to develop novel mucosal vaccine delivery systems to induce mucosal immunity against bacterial and viral diseases, including HIV, have dramatically increased. Here we cite novel vaccines and delivery systems being used to establish effective mucosal immunity.

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)

Department of Pathology, Duke University Medical Center, Durham, NC 27710, USA Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7571, USA Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA Human Vaccine Institute, Duke University Medical Center, Durham, NC 27710, USA Cistron Biotechnology, 10 Bloomfield Ave., Pine Brook, NJ 07058, USA Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294-2170, USA Department of Immunology, Duke University Medical Center, Durham, NC 27710, USA

To address an existing controversy concerning the presence of HIV-1-specific antibodies of the IgA isotype in the female genital tract secretions of highly-exposed but persistently seronegative (HEPSN) women, 41 samples of plasma and cervicovaginal lavage (CVL) fluid were distributed to six laboratories for their blinded evaluation using ELISA with 10 different HIV-1 antigens, chemiluminescence-enhanced Western blots (ECL-WB), and virus neutralization. HIV-specific IgG or IgA antibodies in plasma samples from HEPSN women were absent or detectable only at low levels. In CVL, 11/41 samples displayed low levels of reactivity in ELISA against certain antigens. However, only one sample was positive in two of five laboratories. All but one CVL sample yielded negative results when analyzed by ECL-WB. Viral neutralizing activity was either absent or inconsistently detected in plasma and CVL. Plasma and CVL samples from 26 HIV-1-infected women were used as positive controls. Irrespective of the assays and antigens used, the results generated in all laboratories displayed remarkable concordance in the detection of HIV-1-specific antibodies of the IgG isotype. In contrast, IgA antibodies to HIV-1 antigens were not detected with consistency, and where present, IgA antibodies were at markedly lower levels than IgG. Although HIV-neutralizing activity was detected in plasma of all HIV-1-infected women, only a few of their CVL samples displayed such activity. In conclusion, frequent HIV-1 sexual exposure does not stimulate uniformly detectable mucosal or systemic HIV-1-specific responses, as convincingly documented in the present blindly performed study using a broad variety of immunological assays. Although HIV-1-infection leads to vigorous IgG responses in plasma and CVL, it does not stimulate sustained IgA responses in either fluid.

Passive transfer of a monoclonal antibody (MAb) specific for glycoprotein D (gD) is highly effective in preventing the development of herpes simplex virus type 1-induced stromal keratitis. In the present study, we investigated whether animals which had been functionally depleted of T-cell subsets or asialo GM1+ cells would continue to be responsive to MAb therapy. BALB/c mice were depleted of CD4+, CD8+, or asialo GM1+ cells by treatment with anti-L3T4, anti-Lyt 2.2, or anti-asialo GM1 antibodies, respectively. Functional depletion of CD4+ cells was documented by the loss of delayed-type hypersensitivity responsiveness, while CD8+ cell depletion was accompanied by abrogation of cytotoxic lymphocyte activity. Anti-asialo GM1 treatment led to the loss of natural killer cell lytic activity. Mice depleted of the desired cell population and infected on the scarified cornea with herpes simplex virus type 1 uniformly developed necrotizing stromal keratitis by 3 weeks postinfection. A single inoculation of anti-gD MAb (55 micrograms) given intraperitoneally 24 h postinfection strongly protected hosts depleted of CD4+ cells against stromal keratitis. Likewise, antibody treatment in CD8+ or asialo GM1+ cell-depleted hosts was as therapeutically effective as that seen in non-cell-depleted mice. We also observed that in cell-depleted mice, the virus spread into the central nervous system and caused encephalitis. The CD4+ cell-depleted mice were the most severely affected, as 100% developed fatal disease. Anti-gD MAb treatment successfully protected all (32 of 32) CD4+-, CD8+-, or asialo GM1(+)-depleted hosts against encephalitis. We therefore conclude that antibody-mediated prevention of stromal keratitis and encephalitis does not require the obligatory participation of CD4+, CD8+, or asialo GM1+ cells. However, when mice were simultaneously depleted of both CD4+ and CD8+ T-cell subsets, antibody treatment could not prevent fatal encephalitis. Thus, antibody can compensate for the functional loss of one but not two T-lymphocyte subpopulations.

Dendritic cell (DC) accumulation in T-cell zones of secondary lymph organs is a critical event in the maximization of the primary immune response. Here, we demonstrate that mast cell (MC) control of elevated DC trafficking during infection enables the primary adaptive humoral immune response to proceed with heightened intensity. Elevated DC accumulation in draining lymph nodes (DLNs) was found to be the product of continual and incremental recruitment of DCs into the infected tissue site prior to their egress to DLNs. MCs contribute to this pattern of DC trafficking by the release of TNF, which was found to coordinately activate local blood vessel endothelium and DLNs to increase their expression of CD62E and CCL21, respectively. Blockade of either protein interfered with DC accumulation in DLNs, as well as the intensity of the humoral response to bacterial challenge. Thus, MCs enable the humoral immune response to proceed with enhanced intensity, which may be an important consideration in rationale vaccine design. Supported by grants DK50814 and AI50021 from the National Institutes of Health.

Recent studies have reported that mast cells play a role in the trafficking of dendritic cells and lymphocytes to lymph nodes draining sites of infection. To test the hypothesis that mast cell activators could be used as vaccine adjuvants, C3H/HeN mice were intradermally vaccinated in the ear on days 0 & 21 with 0.5 μg recombinant anthrax protective antigen (rPA) alone or combined with 30 μg compound 48/80 (C 48/80), a mast cell activator. CpG oligodeoxynucleotide (CpG ODN, 1 μg) was used as a control. Ear swelling was monitored 24 hours after immunization to measure injection site inflammation. Mice immunized with rPA alone had an average ear thickness (mm) of 0.0059 while mice immunized with rPA + C 48/80 had an ear thickness of 0.0426 (p < 0.001 vs rPA alone). Mice immunized with rPA + CpG ODN had an ear thickness of 0.1519 (p < 0.001 vs rPA), significantly greater than swelling induced by C 48/80 (p < 0.001). Day 42 serum from mice immunized with PA alone had serum anti-PA IgG titers of 1:27,238 while mice immunized with PA + C 48/80 had anti-PA titers of 1:10,568,984 (p < .001 vs PA alone). Similar titers were detected in mice vaccinated with rPA plus CpG ODN. Antibodies induced with rPA + C 48/80 neutralized anthrax lethal toxin. Our findings suggest that mast cell activators provide effective vaccine adjuvant activity when delivered intradermally while inducing less injection site inflammation than CpG ODN.

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.

The generation of recombinant single-chain antibodies from either non-immune or immune phage display antibody libraries is an effective means to obtain high affinity antibodies against a specific target. Non-immune libraries contain a wide variety of antibodies but these are often low affinity. Immune libraries contain a high frequency of high affinity antibodies, but are typically limited to a single antigen. Due to the V(H) and V(L) recombination that occurs during antibody library construction, we hypothesized that an immune antibody library produced against one member of a protein family would contain antibodies specific for other members of the same protein family. Here, we tested this hypothesis by mining an existing anti-human Toll-like receptor-2 (hTLR2) antibody library for antibodies specific for other members of the TLR family. This procedure, referred to as homolog mining, proved to be effective. Using a cell-based system to pan and screen the anti-TLR2 library, we identified single chain antibodies specific for three of the four hTLR2 homologs we targeted. The antibodies identified, anti-murine TLR2, anti-hTLR5, and anti-hTLR6, bind specifically to their target, with no cross-reactivity to hTLR2 or other TLRs tested. These results demonstrate that combinatorial re-assortment of V(H) and V(L) fragments from multiple sources during Ab library construction increases Ab repertoire complexity, allowing antibody libraries produced by immunization with one antigen to be used to obtain antibodies specific to related antigens. The principle of homolog mining may be extended to other protein families and will facilitate and accelerate antibody production processes.

Apolipoprotein E (apoE) is a 34 kDa glycosylated protein with multiple biological properties. In addition to its role in cholesterol transport, apoE has in vitro immunomodulatory properties. Recent data suggest that these immunomodulatory effects of apoE may be biologically relevant, and apoE-deficient mice have altered immune responses after bacterial inoculation and increased susceptibility to endotoxemia induced by lipopolysaccharide (LPS). To better understand the mechanism by which apoE-modulates immune responses, we tested the role of human apoE isoforms in assays of human T cell proliferation, and analyzed the immune responses of apoE-deficient mice. Both the E3 and E4 isoforms of apoE induced similar suppression of human lymphocyte function in assays of T cell proliferation, including mitogenic responses to phytohaemagglutin (PHA), stimulation of the T cell receptor with αCD3, and antigen-specific response to tetanus toxoid. ApoE-deficient mice showed no quantitative differences in thymic, splenic, or bone marrow lymphocyte populations, nor were there in vitro abnormalities in splenocyte proliferation after stimulation with αCD3 to suggest an inherent T cell defect in apoE-deficient mice. ApoE deficient animals, however, had significantly higher levels of antigen-specific IgM after immunization with tetanus toxoid, and impaired delayed type hypersensitivity responses as compared to control C57-BL/6 mice. These results support a growing body of evidence demonstrating an interplay between lipid metabolism and immune responses, and suggest that apoE plays a biologically relevant role in regulating humoral and cell-mediated immunity.—Laskowitz, D. T., D. M. Lee, D. Schmechel, and H. F. Staats. Altered immune responses in apolipoprotein E-deficient mice. J. Lipid Res. 2000. 41: 613–620.

Cholera is endemic to many countries. Recent major outbreaks of cholera have prompted World Health Organization to recommend oral cholera vaccination as a public-health strategy. Variation in percentage of seroconversion upon cholera vaccination has been recorded across populations. Vaccine-induced responses are influenced by host genetic differences. We have investigated association between single-nucleotide polymorphic (SNP) loci in and around 296 immunologically relevant genes and total anti-lipopolysaccharide (LPS) antibody response to a killed whole-cell vaccine, comprising LPS from multiple strains of Vibrio cholerae. Titers derived from standard vibriocidal assays were also analyzed to gain further insights on validated SNP associations. Vaccination was administered to 1000 individuals drawn from India. Data on two independent random subsets, each comprising ∼500 vaccinees, were used for discovery of genomic associations and validation, respectively. Significant associations of four SNPs and haplotypes in three genes (MARCO, TNFAIP3 and CXCL12) with AR were discovered and validated, of which two in TNFAIP3 and CXCL12 were also significantly associated with immunity (fourfold increase in vibriocidal titers). CXCL12 is a neutrophil and lymphocyte chemoattractant that is upregulated in response to V. cholerae infection. LPS in the vaccine possibly provides signals that mimic those of the live bacterium. TNFAIP3 promotes intestinal epithelial barrier integrity and provides tight junction protein regulation; possible requirements for adequate response to the vaccine. LPS is a potent activator of innate immune responses and a ligand of MARCO. Variants in this gene have been found to be associated with LPS response, but not with high vibriocidal titer level.