Mucosal immunity in the female genital tract

Immunoglobulin (Ig)-producing cells in mucosal tissues represent quantitatively the most important humoral immune system of the body. All exocrine tissue sites contain immunocytes (B-cell blasts and plasma cells) that mainly synthesize dimers and larger polymers of IgA (collectively called pIgA) with incorporated J chain. Such pIgA is actively transported to external secretions as secretory IgA (SIgA) by the polymeric Ig receptor (pIgR), a transmembrane epithelial glycoprotein also called the secretory component (SC). The same transport mechanism includes pentameric IgM to generate SIgM. Although the most active SIgA system occurs in the gut, secretory immunity also operates in the female genital tract, with considerable pIgA production in the cervical mucosa and fallopian tubes. The origin of these local IgA immunocytes remains undefined. In mice, both lymphoid tissue in the large bowel (GALT) and nasopharynx (NALT) have been suggested as inductive sites for B cells homing to the urogenital tract. It is well established that integrin alpha 4 beta 7 is used by primed lymphoid cells to enter the intestinal lamina propria through interactions with mucosal addressin cell adhesion molecule (MAdCAM)-1 expressed on venule endothelium. However, alpha 4 beta 7 does not appear to be an important homing molecule in the airways, and the same might be true for the urogenital tract; this could explain that high levels of IgA antibodies occur in cervicovaginal secretions of mice after nasal immunization. The endometrium can likewise perform pIgR-mediated external translocation of pIgA that in this tissue appears to be mainly derived from serum, partly under hormonal regulation. In addition, paracellular diffusion of serum-derived and locally produced IgG through epithelia is an important part of humoral immunity in the female genital tract.     

Intranasal immunization with attenuated live influenza vaccine in asthma

Attenuated live intranasal influenza vaccine ("Alice") was given to 20 asthmatics and 9 control subjects. Pulmonary function were performed before and after, with emphasis on tests of small airways function (using flow volume curves with air and a helium-oxygen mixture). In subjects with a low influenza A antibody titer, there was a 4-fold rise in titer to the vaccine, whereas those subjects with a high titer showed no rise. There were no significant changes in pulmonary function in any parameters measured, and no significant symptoms were reported. We have concluded from this study that "Alice" appears safe for use in asthma and was capable of producing an antibody titer rise in persons with low titers.     

Comparison of the oral, rectal, and vaginal immunization routes for induction of antibodies in rectal and genital tract secretions of women

To determine which mucosal immunization routes may be optimal for induction of antibodies in the rectum and female genital tract, groups of women were immunized a total of three times either orally, rectally, or vaginally with a cholera vaccine containing killed Vibrio cholerae cells and the recombinant cholera toxin B (CTB) subunit. Systemic and mucosal antibody responses were assessed at 2-week intervals by quantitation of CTB-specific antibodies in serum and in secretions collected directly from mucosal surfaces of the oral cavity, rectum, cervix, and vagina with absorbent wicks. The three immunization routes increased levels of specific immunoglobulin G (IgG) in serum and specific IgA in saliva to similar extents. Rectal immunization was superior to other routes for inducing high levels of specific IgA and IgG in rectal secretions but was least effective for generating antibodies in female genital tract secretions. Only vaginal immunization significantly increased both specific IgA and specific IgG in both the cervix and the vagina. In addition, local production of CTB-specific IgG in the genital tract could be demonstrated only in vaginally immunized women. Vaginal immunization did not generate antibodies in the rectum, however. Thus, generation of optimal immune responses to sexually transmitted organisms in both the rectal and the genital mucosae of women may require local immunization at both of these sites.     

Intranasal immunization with a plant virus expressing a peptide from HIV-1 gp41 stimulates better mucosal and systemic HIV-1-specific IgA and IgG than oral immunization

One axiom at the basis of epilepsy research is that there exists an imbalance between excitation and inhibition. This abnormality can be achieved by an increase of excitation on principal cells, a decreased inhibition (i.e. disinhibition) or both. This review focuses on dysfunction of inhibition, and in particular on the 'dormant basket cell hypothesis'. This hypothesis states that, (1) interneurones are functionally disconnected from excitatory afferents, resulting in hyperexcitability of principal neurones and loss of paired pulse inhibition, (2) when properly activated, interneurones can still perform their task, i.e. suppress epileptiform activity and restore paired pulse inhibition. The aim of this review is to discuss the evidence in support of the 'dormant basket cell hypothesis'. We will first discuss the rationale underlying the hypothesis and the criteria needed to validate the hypothesis. We will then show that, (1) the key experimental data offered in support of the hypothesis (Bekenstein and Lothman, 1993. Dormancy of inhibitory interneurones in a model of temporal lobe epilepsy. Science 259, 97-100; Sloviter, 1991. Permanently altered hippocampal structure, excitability, and inhibition after experimental status epilepticus in the rat: the 'dormant basket cell' hypothesis and its relevance to temporal lobe epilepsy. Hippocampus 1, 41-66) are difficult to interpret, and (2) recent recordings from interneurones in epileptic tissue argue against the hypothesis. The 'dormant basket cell hypothesis' is then discussed in the broader context of disinhibition.     

Antibodies and antibody-secreting cells in the female genital tract after vaginal or intranasal immunization with cholera toxin B subunit or conjugates

We studied the antibody response including antibody-secreting cells (ASC) in the female genital tract of mice after mucosal immunizations with the recombinant B subunit of cholera toxin (rCTB) perorally, intraperitoneally, vaginally, and intranasally (i.n.). The strongest genital antibody responses as measured with a novel perfusion-extraction method were induced after vaginal and i.n. immunizations, and these routes also gave rise to specific immunoglobulin A (IgA) and IgG ASC in the genital mucosa. Specific ASC in the iliac lymph nodes, which drain the female genital tract, were seen only after vaginal immunization. Progesterone treatment increased the ASC response in the genital tissue after all mucosal immunizations but most markedly after vaginal immunization. We also tested rCTB as a carrier for human gamma globulin (HGG) and the effect of adding cholera toxin (CT) as an adjuvant for the induction of systemic and genital antibody responses to HGG after vaginal and i.n. immunizations. Vaginal immunizations with HGG conjugated to rCTB resulted in high levels of genital anti-HGG antibodies whether or not CT was added, while after i.n. immunization the strongest antibody response was seen with the conjugate together with CT. In summary, vaginal and i.n. immunization give rise to a specific mucosal immune response including ASC in the genital tissue, and vaginal immunization also elicits ASC in the iliac lymph nodes. We have also shown that rCTB can act as an efficient carrier for a conjugated antigen for induction of a specific antibody response in the genital tract of mice after vaginal or i.n. immunization.     

Influence of mucosal and parenteral immunization with a replication-defective mutant of HSV-2 on immune responses and protection from genital challenge

Platelet microbicidal proteins (PMPs) are hypothesized to exert microbicidal effects via cytoplasmic membrane disruption. Transmission electron microscopy demonstrated a temporal association between PMP exposure, damage of the Staphylococcus aureus cytoplasmic membrane ultrastructure, and subsequent cell death. To investigate the mechanisms of action of PMPs leading to membrane damage, we used flow cytometry to compare the effects of two distinct PMPs (thrombin-induced PMP-1 [tPMP-1] or PMP-2) with human neutrophil defensin-1 (hNP-1) on transmembrane potential (Deltapsi), membrane permeabilization, and killing of S. aureus. Related strains 6850 (Deltapsi -150 mV) and JB-1 (Deltapsi -100 mV; a respiration-deficient menadione auxotroph of 6850) were used to assess the influence of Deltapsi on peptide microbicidal effects. Propidium iodide (PI) uptake was used to detect membrane permeabilization, retention of 3,3'-dipentyloxacarbocyanine (DiOC5) was used to monitor membrane depolarization (Deltapsi), and quantitative culture or acridine orange accumulation was used to measure viability. PMP-2 rapidly depolarized and permeabilized strain 6850, with the extent of permeabilization inversely related to pH. tPMP-1 failed to depolarize strain 6850, but did permeabilize this strain in a manner directly related to pH. Depolarization, permeabilization, and killing of strain JB-1 due to PMPs were significantly less than in strain 6850. Growth in menadione reconstituted Deltapsi of JB-1 to a level equivalent to 6850, and was associated with greater depolarization due to PMP-2, but not tPMP-1. Reconstitution of Deltapsi also enhanced permeabilization and killing of JB-1 due to tPMP-1 or PMP-2. Both PMP-2 and tPMP-1 caused significant reductions in viability of strain 6850. In contrast to tPMP-1 or PMP-2, defensin hNP-1 depolarized, permeabilized, and killed both strains 6850 and JB-1 equally, and in a manner directly related to pH. Collectively, these data indicate that membrane dysfunction and cell death due to tPMP-1, PMP-2, or hNP-1 likely involve different mechanisms. These findings may also reveal new insights into the microbicidal activities versus mammalian cell toxicities of antimicrobial peptides.     

Intranasal immunization with CTL epitope peptides from HIV-1 or ovalbumin and the mucosal adjuvant cholera toxin induces peptide-specific CTLs and protection against tumor development in vivo

To evaluate the ability of mucosal immunization protocols using peptide immunogens to induce CTL responses, BALB/c and C57BL/6 mice were immunized intranasally (i.n.) with peptides corresponding to a known CTL epitope in HIV-1 glycoprotein 120 or OVA, respectively, and the mucosal adjuvant cholera toxin (CT). Intranasal immunization of BALB/c mice with a 10- or 15-amino acid peptide corresponding to a CTL determinant in HIV-1 glycoprotein 120 and CT induced peptide-specific CTLs in spleen cells that persisted through 35 days after the last immunization. Intranasal immunization of C57BL/6 mice with the octameric OVA peptide and CT produced similar results with detectable peptide-specific CTL in both the cervical lymph node and spleen. To test whether CTL induced by i.n. immunization with OVA peptide and CT were functional in vivo, groups of C57BL/6 mice were injected with E.G7-OVA tumor cells that express the OVA protein and monitored for tumor growth. Animals immunized i.n. with OVA and CT were protected against tumor development as efficiently as animals immunized by the potent CTL induction protocol of i.v. injection with OVA-pulsed dendritic cells. Intranasal immunization with peptides corresponding to known CTL epitopes and CT provides a noninvasive route of immunization for the induction of CTL responses in vivo.     

Novel mucosal immunization with polysaccharide-protein conjugates entrapped in alginate microspheres

A novel mucosal immunization was examined using biocompatible and biodegradable alginate microspheres containing a conjugate of polysaccharide antigen and cholera toxin B subunit (CTB). In order to prepare the alginate microspheres with diameters of less than 5 microm, a new diffusion-controlled interfacial gelation technique was developed. Also, in order to improve the mucosal immune response, a pneumococcal capsular polysaccharide type 19 (PS19) was conjugated to the CTB (PS19-CTB). This conjugate was subsequently encapsulated into the alginate microspheres. The loading content of PS19-CTB to the alginate microspheres was 60%. An in vitro sustained release pattern was observed with the antigen-loaded microspheres, showing 80% antigen release within one day. Mucosal and systemic immunities following oral immunization with the alginate microspheres were studied. Balb/c mice were immunized perorally three times at intervals of two weeks. Peroral immunization with 25 microg of PS19-CTB entrapped in the alginate microspheres evoked both the mucosal IgA and systemic IgM responses to PS19 in small intestine and in sera, respectively. The results suggest that both the mucosal and systemic antibody responses could be induced by oral administration of the PS19-CTB antigen entrapped in alginate microspheres.     

Induction of protective immunity against Japanese encephalitis in mice by immunization with a plasmid encoding Japanese encephalitis virus premembrane and envelope genes

A DNA vaccine plasmid containing the Japanese encephalitis (JE) virus premembrane (prM) and envelope (E) genes (designated pcDNA3JEME) was evaluated for immunogenicity and protective efficacy in mice. Two immunizations of 4-week-old female ICR mice with pcDNA3JEME by intramuscular or intradermal injections at a dose of 10 or 100 microg per mouse elicited neutralizing (NEUT) antibodies at titers of 1:10 to 1:20 (90% plaque reduction), and all immunized mice survived a challenge with 10,000 50% lethal doses of the P3 strain of JE virus. A single immunization with 100 microg of pcDNA3JEME did not elicit detectable NEUT antibodies but induced protective immunity. Spleen cells obtained from BALB/c mice immunized once with 10 or 100 microg of pcDNA3JEME contained JE virus-specific memory cytotoxic T lymphocytes (CTLs). BALB/c mice maintained detectable levels of memory B cells and CTLs for at least 6 months after one immunization with pcDNA3JEME at a dose of 100 microg. The CTLs induced in BALB/c mice immunized twice with 100 microg of pcDNA3JEME were CD8 positive and recognized mainly the envelope protein. These results indicate that pcDNA3JEME has the ability to induce a protective immune response which includes JE virus-specific antibodies and CTLs.     

Gliadin activates mucosal cell mediated immunity in cultured rectal mucosa from coeliac patients and a subset of their siblings

BACKGROUND: CD3 and gamma delta cells in the rectal mucosa increase after local instillation of gluten in children with coeliac disease and in half of their siblings. Aim- To establish an in vitro system for assessing immunological changes induced by gluten in the rectum. PATIENTS AND METHODS: Rectal biopsy specimens obtained from 13 treated coeliac children, nine of their siblings, and nine controls were cultured in vitro with a peptic-tryptic digest of gliadin or ovalbumin. CD3 and CD25 cells were counted, and the expression of adhesion molecules evaluated. RESULTS: In the lamina propria of coeliac biopsy samples cultured with gliadin, but not in those from controls, the expression of vascular cell adhesion molecule 1 (VCAM-1) was enhanced, and the number of CD25 cells was significantly higher than in those cultured in medium alone; the density of intraepithelial CD3 cells was also significantly higher. No differences were noted in coeliac biopsy specimens cultured with ovalbumin. A discriminant analysis allowed correct classification of all controls and all coeliacs but one, but three of nine siblings were allocated to the coeliac group. CONCLUSIONS: Our data confirm that gliadin is able to activate cell mediated immunity in the rectal mucosa in coeliac patients and in a subset of their first degree relatives.     

Oral immunization with a Salmonella typhimurium vaccine vector expressing recombinant enterotoxigenic Escherichia coli K99 fimbriae elicits elevated antibody titers for protective immunity

Bovine enterotoxigenic Escherichia coli (ETEC) continues to cause mortality in piglets and newborn calves. In an effort to develop a safe and effective vaccine for the prevention of F5(+) ETEC infections, a balanced lethal asd+ plasmid carrying the complete K99 operon was constructed and designated pMAK99-asd+. Introduction of this plasmid into an attenuated Salmonella typhimurium Deltaaro Deltaasd strain, H683, resulted in strain AP112, which stably expresses E. coli K99 fimbriae. A single oral immunization of BALB/c and CD-1 mice with strain AP112 elicited significant mucosal immunoglobulin A (IgA) titers that remained elevated for >11 weeks. IgA and IgG responses in serum specific for K99 fimbriae were also induced, with a prominent IgG1, as well as IgG2a and IgG2b, titer. To assess the derivation of these antibodies, a K99 isotype-specific B-cell ELISPOT analysis was conducted by using mononuclear cells from the lamina propria of the small intestines (LP), Peyer's patches (PP), and spleens of vaccinated and control BALB/c mice. This analysis revealed elevated numbers of K99 fimbria-specific IgA-producing cells in the LP, PP, and spleen, whereas elevated K99 fimbria-specific IgG-producing cells were detected only in the PP and spleen. These antibodies were important for protective immunity. One-day-old neonates from dams orally immunized with AP112 were provided passive protection against oral challenge with wild-type ETEC, in contrast to challenged neonates from unvaccinated dams or from dams vaccinated with a control Salmonella vector. These results confirm that oral Salmonella vaccine vectors effectively deliver K99 fimbriae to mucosal inductive sites for sustained elevation of IgA and IgG antibodies and for eliciting protective immunity.     

Tumor immunity and autoimmunity induced by immunization with homologous DNA

The immune system can recognize self antigens expressed by cancer cells. Differentiation antigens are prototypes of these self antigens, being expressed by cancer cells and their normal cell counterparts. The tyrosinase family proteins are well characterized differentiation antigens recognized by antibodies and T cells of patients with melanoma. However, immune tolerance may prevent immunity directed against these antigens. Immunity to the brown locus protein, gp75/ tyrosinase-related protein-1, was investigated in a syngeneic mouse model. C57BL/6 mice, which are tolerant to gp75, generated autoantibodies against gp75 after immunization with DNA encoding human gp75 but not syngeneic mouse gp75. Priming with human gp75 DNA broke tolerance to mouse gp75. Immunity against mouse gp75 provided significant tumor protection. Manifestations of autoimmunity were observed, characterized by coat depigmentation. Rejection of tumor challenge required CD4(+) and NK1.1(+) cells and Fc receptor gamma-chain, but depigmentation did not require these components. Thus, immunization with homologous DNA broke tolerance against mouse gp75, possibly by providing help from CD4(+) T cells. Mechanisms required for tumor protection were not necessary for autoimmunity, demonstrating that tumor immunity can be uncoupled from autoimmune manifestations.     

Baculovirus-like particles in the reproductive tracts of female parasitoid wasps

Virus-like particles have been found in specific regions of the reproductive tracts of three different braconid wasps, all parasitoids of the tobacco budworm, Heliothis virescens. The particles are nuclear in origin, and Feulgen cytochemistry of particulate fluid in the calyx and oviducts of one species has revealed the presence of DNA. On the basis of apparent structural homologies, it is suggested that the parasitoid particles are related to baculoviruses.     

Immunization of the female genital tract with a DNA-based vaccine

Vaccines are being sought for contraception and the prevention of sexually transmitted diseases. However, progress is slow in this area largely because of lack of information on induction of protective immune responses in genital tract mucosa. In this study, we investigated whether in vivo transfection with a model DNA-based antigen delivered by gene gun technology would induce an antibody response detectable in vaginal secretions. Female rats were immunized with plasmids encoding human growth hormone (HGH) under the control of a cytomegalovirus promoter (pCMV/HGH) via vaginal mucosa (V), Peyer's patch (PP), and/or abdominal skin (S) routes. Localization of HGH in the target tissues demonstrated that all three sites can be transfected in vivo with pCMV/HGH. Vaginal tissues expressed roughly the same level of plasmid as skin. Antibodies to HGH were detectable in serum and vaginal secretions in rats immunized with pCMV/HGH. In the rats primed and boosted vaginally, vaginal immunoglobulin A (IgA) and IgG antibody titers to HGH were sustained for at least 14 weeks, whereas rats immunized via other routes and protocols (S/V, S/S, PP/PP, or PP/V) did not consistently sustain significant vaginal antibody titers beyond week 6. DNA-based immunizations administered by the gene gun may be an effective method of inducing local immunity in the female genital tract.     

Antigen-releasing polymer rings and microspheres stimulate mucosal immunity in the vagina

Several recent studies suggest that direct application of antigen to the vaginal surface may enhance local IgA secretion, but the most effective methods for stimulating immunity at the vaginal surface have not been identified. We used antigen-loaded, biocompatible, vaginal rings to provide controlled and sustained antigen delivery directly to the vaginal mucosal surface. Mice were primed with ferritin, either subcutaneously or orally by ferritin-loaded polymer microspheres, and vaginally boosted by insertion of a ferritin-loaded polymer ring. We found that the vaginal rings were a convenient method for providing controlled antigen delivery to the vagina. Subcutaneously primed mice receiving ferritin-loaded vaginal rings had ferritin-specific IgA in their mucus secretions, while mice receiving blank rings did not. Oral priming with ferritin-loaded poly(lactic acid) microspheres also produced significant levels of ferritin-specific IgA in the vaginal secretions, but required the presence of cholera toxin. Controlled ferritin delivery to mucosal surfaces, either by oral, biodegradable microspheres or vaginal rings, provides a convenient and reliable method for enhancing vaginal IgA production in mice.     

Cytokine- and T helper-dependent lung mucosal immunity in mice with invasive pulmonary aspergillosis

The role of cytokine- and T helper (Th)-dependent lung mucosal antifungal immunity in murine invasive pulmonary aspergillosis (IPA) was investigated. Intact or leukopenic DBA/2 mice were resistant or highly susceptible, respectively, to infection caused by multiple intranasal injections of viable Aspergillus fumigatus conidia. Resistance was associated with unimpaired innate antifungal activity of pulmonary phagocytic cells, concomitant with high-level production of tumor necrosis factor (TNF)-alpha and interleukin (IL)-12 and the presence of interstitial lymphocytes producing interferon-gamma and IL-2. Conversely, production of TNF-alpha and IL-12 was down-regulated in highly susceptible mice, which also had defective innate antifungal immunity and high-level production of IL-4 and IL-10 by lung lymphocytes. Resistance was increased in susceptible mice upon local IL-4 or IL-10 neutralization or IL-12 administration. These results indicate that, similar to observations in mice with disseminated aspergillosis, innate and Th1-dependent immunity play an essential role in host defense against IPA.     

Oral immunization with recombinant Norwalk virus-like particles induces a systemic and mucosal immune response in mice

Recombinant Norwalk virus-like particles (rNV VLPs) produced in insect cells were evaluated as an oral immunogen in CD1 and BALB/c mice by monitoring rNV-specific serum total and subclass immunoglobulin G (IgG) and intestinal IgA responses. Dose and kinetics of response were evaluated in the presence and absence of the mucosal adjuvant cholera toxin (CT). rNV-specific serum IgG and intestinal IgA were detected in the absence of CT, and the number of responders was not significantly different from that of mice administered VLPs with CT at most doses. The use of CT was associated with induction of higher levels of IgG in serum; this effect was greater at higher doses of VLPs. IgG in serum was detected in the majority of animals by 9 days postimmunization (dpi), and intestinal IgA responses were detected by 24 dpi. In the absence of CT, IgG2b was the dominant IgG subclass response in both mouse strains. Thus, nonreplicating rNV VLPs are immunogenic when administered orally in the absence of any delivery system or mucosal adjuvant. These studies demonstrate that rNV VLPs are an excellent model to study the oral delivery of antigen, and they are a potential mucosal vaccine for NV infections.