Mucosal Ab Research Articles

IL-12 Is an Effective Adjuvant for Induction of Mucosal Immunity

We addressed the effects of two cytokines, IL-6 and IL-12, derived from APCs, for the development of mucosal IgA Ab responses following their nasal delivery with the protein vaccine tetanus toxoid (TT). Mice treated nasally with IL-6 and TT showed higher TT-specific serum IgG (mainly IgG1 and IgG2b) Ab responses than did control mice, but exhibited no IgE and negligible secretory IgA (S-IgA) Ab responses. In contrast, IL-12 administered nasally with TT not only induced sharp increases in TT-specific serum IgG (mainly IgG1 and IgG2b) and IgA, but also elevated mucosal S-IgA Ab responses. Coadministration of IL-6 and IL-12 with TT did not enhance the mucosal or serum Ab responses over those seen with IL-12 alone. TT-specific CD4+ T cells from mice given TT with IL-6 or IL-12 produced higher levels of IFN-gamma, IL-6, and IL-10 than did those from control mice, but only negligible levels of IL-4 and IL-5. In summary, both intranasal IL-6 and IL-12 induced serum Abs that protected mice from systemic challenge with TT, whereas only IL-12 induced mucosal S-IgA Ab responses. The significance of IL-12-induced Th1-type responses for regulation of both mucosal and systemic immunity is discussed.

Nasal Immunization of Nonhuman Primates with Simian Immunodeficiency Virus p55gagand Cholera Toxin Adjuvant Induces Th1/Th2 Help for Virus-Specific Immune Responses in Reproductive Tissues

AbstractFemale rhesus macaques were nasally immunized with p55gag (p55) of SIV and cholera toxin as a mucosal adjuvant. Nasal immunization induced Ag-specific IgA and IgG Abs in mucosal secretions (e.g., cervicovaginal secretions, rectal washes, and saliva) and serum. Furthermore, high numbers of p55-specific IgA and IgG Ab-forming cells were induced in mucosal effector sites, i.e., uterine cervix, intestinal lamina propria, and nasal passage. p55-specific CD4+ T cells in both systemic and mucosal compartments expressed IFN-γ and IL-2 (Th1-type)- as well as IL-5, IL-6, and IL-10 (Th2-type)-specific mRNA. Moreover, p55-specific CTL activity was demonstrated in lymphocytes from blood, tonsils, and other lymphoid tissues. These results show that nasal immunization with SIV p55 with cholera toxin elicits both Th1- and selective Th2-type cytokine responses associated with the induction of SIV-specific mucosal and serum Abs, and CTL activity. These results offer a promise for the development of protective mucosal immunity to SIV.

Secretory Component Delays the Conversion of Secretory IgA into Antigen-Binding Competent F(ab′)2: A Possible Implication for Mucosal Defense

Secretory component (SC) represents the soluble ectodomain of the polymeric Ig receptor, a membrane protein that transports mucosal Abs across epithelial cells. In the protease-rich environment of the intestine, SC is thought to stabilize the associated IgA by unestablished molecular mechanisms. To address this question, we reconstituted SC-IgA complexes in vitro by incubating dimeric IgA (IgAd) with either recombinant human SC (rSC) or SC isolated from human colostral milk (SCm). Both complexes exhibited an identical degree of covalency when exposed to redox agents, peptidyl disulfide isomerase, and temperature changes. In cross-competition experiments, 50% inhibition of binding to IgAd was achieved at approximately 10 nM SC competitor. Western blot analysis of IgAd digested with intestinal washes indicated that the alpha-chain in IgAd was primarily split into a 40-kDa species, a phenomenon delayed in rSC- or SCm-IgAd complexes. In the same assay, either of the SCs was resistant to degradation only if complexed with IgAd. In contrast, the kappa light chain was not digested at all, suggesting that the F(ab')2 region was left intact. Accordingly, IgAd and SC-IgAd digestion products retained functionality as indicated by Ag reactivity in ELISA. Size exclusion chromatography under native conditions of digested IgAd and rSC-IgAd demonstrates that SC exerts its protective role in secretory IgA by delaying cleavage in the hinge/Fc region of the alpha-chain, not by holding together degraded fragments. The function of integral secretory IgA and F(ab')2 is discussed in terms of mucosal immune defenses.

A Nontoxic Adjuvant for Mucosal Immunity to Pneumococcal Surface Protein A

In this study, we demonstrated that pneumococcal surface protein A (PspA) nasally administered with a nontoxic A subunit mutant of cholera toxin (mCT) S61F elicited a protective immune response. Immunization with PspA and mCT elicited higher levels of PspA-specific IgG and IgA Abs in serum and of IgG and IgA anti-PspA Ab-forming cells in spleens, cervical lymph nodes (CLN), and lung tissue when compared to nonimmunized mice. Furthermore, significant PspA-specific IgA Abs were induced in saliva and nasal secretions. These responses were dependent on the use of mCT as a mucosal adjuvant. The PspA-specific Ab responses induced by mCT S61F were comparable with those induced by native CT (nCT). Analysis of cytokine responses showed that nasal PspA plus mCT S61F enhanced the induction of PspA-specific CD4+ T cells producing IL-4 but not IFN-gamma in CLN at both the protein and mRNA levels. Importantly, significant numbers of mice intranasally immunized with PspA plus mCT S61F were protected from lethal challenge with capsular serotype 3 Streptococcus pneumoniae A66. These results show that intranasal administration of PspA together with mCT S61F is an effective mucosal vaccine against pneumococcal infection and induces CD4+ Th2-type cells, which provide help for both mucosal and systemic Ab responses.

Controlled Lipidation and Encapsulation of Peptides as a Useful Approach to Mucosal Immunizations

To generate a useful strategy for mucosal immunization, we have developed an approach of lipidating a multiple Ag peptide (MAP) containing part of the V3 loop from HIV-1 gp120IIIB. In this work, we compare two delivery systems, lipidated MAP in PBS and encapsulation in poly(DL-lactide-co-glycolide) microparticles. Subcutaneous immunization, followed by intragastric administration of MAP peptide entrapped or not entrapped in microparticles, induced mucosal and systemic immune responses at local and distant sites, including mucosal IgA in saliva, vaginal secretions and feces, and IgG in blood. However, lipidated Ag delivered in microparticles induced higher levels of mucosal Abs, particularly of intestinal IgA, and generated CTL responses. In contrast, lipidated MAP delivered by nasal route microparticles was less effective in inducing CTL responses. These results demonstrate the feasibility of using a lipidated multimeric peptide for mucosal immunization to stimulate both systemic and mucosal immune systems, including the genital tract, irrespective of the route or method of delivery and without requiring the use of a carrier or an extraneous adjuvant.

Role of αβ and γδ T Cells in the Host Response to Salmonella Infection as Demonstrated in T-Cell-Receptor-Deficient Mice of Defined Ity Genotypes

Salmonella spp. are facultative intracellular bacteria which enter the body through the intestinal tract. We studied the roles of T cells expressing either the a and b chains or the g and d chains of the T-cell receptor (ab T cells or gd T cells, respectively) in the host defense against Salmonella using mice genetically deficient in either ab T cells, gd T cells, or both T-cell subsets. These mutant strains of mice were infected orally or intraperitoneally with Salmonella dublin, and the progression of the disease was monitored by determining bacterial numbers in the feces, gut wall, Peyer’s patches, mesenteric lymph nodes, spleen, and liver. Since susceptibility to Salmonella infection in mice is strongly affected by the alleles at the Ity locus, T-cell-mutant mice with either the Ity-sensitive or Ity-resistant phenotype were tested for resistance to S. dublin infection. We found that even though large numbers of intraepithelial and mucosal ab and gd T cells populate the normal intestine, they have no role in controlling the invasion of S. dublin into the intestine or the subsequent bacterial replication in the Peyer’s patches or gut wall. Furthermore, systemic infections were equally severe for the first 6 days in normal, ab T-cell-deficient, and gd T-cell-deficient mice, and ab but not gd T cells were required for clearance of S. dublin, regardless of the Ity phenotype. However, mice that lacked both T-cell subsets had higher bacterial counts in their livers 15 to 18 days after infection than did ab T-cell-deficient mice, suggesting that gd T cells can contribute to acquired immunity to S. dublin.

Oral immunization with simian immunodeficiency virus p55gag and cholera toxin elicits both mucosal IgA and systemic IgG immune responses in nonhuman primates.

Rhesus macaques were orally immunized with a mucosal vaccine consisting of two different concentrations (1 mg vs 250 microg) of recombinant SIV p55gag (p55) with or without cholera toxin (CT, 50 microg) as a mucosal adjuvant. The plasma from macaques receiving the higher dose of p55 (1 mg) and CT had higher p55-specific IgG and IgA Ab titers compared with macaques that received the lower dose of p55 (250 microg) and CT. Further, high levels of p55-specific IgG and IgA Abs were present in external secretions from both groups. The level of p55-induced T cell responses was elevated in PBMCs isolated from the high dose group compared with the low dose group. When culture supernatants from these p55-stimulated PBMCs were examined for Th1 (IFN-gamma) and Th2 (IL-4 and IL-10) cytokines, both IFN-gamma and IL-10 were present, but IL-4 was absent. CD4+ T cells isolated from these p55-stimulated PBMCs contained IFN-gamma spot-forming cells (SFCs) but not IL-4 SFCs. These results were further confirmed by cytokine-specific reverse transcriptase PCR analysis, where p55-specific CD4+ T cells expressed mRNA for IFN-gamma, IL-6, and IL-10, but not IL-4. These findings suggest that oral immunization of nonhuman primates induced both IFN-gamma-secreting Th1 and select Th2 cytokine (e.g., IL-6 and IL-10)-producing CD4+ Th cells, which accounted for the generation of p55-specific systemic and mucosal Ab responses.

Mucosal immunity to HIV-1: systemic and vaginal antibody responses after intranasal immunization with the HIV-1 C4/V3 peptide T1SP10 MN(A).

To optimize mucosal immune responses to the HIV-1 peptide vaccine candidate T1SP10 MN(A), we intranasally immunized BALB/c and C57BL/6 mice with C4/V3 HIV-1 peptide together with the mucosal adjuvant cholera toxin (CT). Four doses over a 4-wk period resulted in peak serum anti-peptide IgG titers of > 1:160,000 in BALB/c mice and > 1:520,000 in C57BL/6 mice, and significant levels (>1:30,000) persisted in both strains of mice for longer than 6 mo. Furthermore, intranasal immunization with peptide and CT induced serum IgG reactivity to HIV-1 gp120 and HIV-1(MN) neutralizing responses. The primary anti-peptide IgG subclass was IgG1, suggesting a predominant Th2-type response. In addition to elevated serum anti-peptide A responses, intranasal immunization with T1SP10 MN(A) and CT induced both vaginal anti-peptide IgG and IgA responses, which persisted for 91 days in both strains of mice. Vaginal anti-HIV IgA was frequently associated with secretory component, suggesting transepithelial transport of IgA into vaginal secretions. Cervical lymph nodes contained the highest relative concentration of anti-T1SP10 MN(A) IgG-producing cells, while the spleen was the next major site of anti-T1SP10 MN(A) IgG-producing cells. Ag-specific proliferative responses were also detected in cervical lymph node and spleen cell populations after intranasal immunization with T1SP10 MN(A) and CT. In addition, intranasal immunization with T1SP10 MN(A) and CT was able to induce anti-HIV cell-mediated immunity in vivo as indicated by the induction of delayed-type hypersensitivity. Therefore, intranasal immunization with hybrid HIV peptides provides a noninvasive route of immunization that induces both long-lived systemic and mucosal Ab responses as well as cell-mediated immunity to HIV.

MUCOSAL AND SERUM ANTIBODY (Ab) RESPONSES TO PARENTERAL IMMUNIZATION OF 1-YR-OLD INFANTS WITH H. INFLUENZAE B CAPSULAR (PRP) ANTIGEN CONJUGATED TO A DIPH IHERIA PROTEIN CARRIER

Seventeen healthy infants age 9-15 mos received a 2-injection sequence of a PRP oligosaccharide conjugate vaccine. Sera and samples of nasal mucus obtained using saline washes (NW) were taken before the 1° and 2° injections and 1 mo and 6 mos after the 2°. A rise in total serum anti-PRP Ab [determined by radio-antigen binding (RAB)] occurred in 15 of 17. NW anti-PRP Ab developed in 6 of 17 as detected by RAB and in 8 of 17 as detected by an Ig class-specific immunosorbent assay (ELISA); these ELIS.A responses occurred as IgG (7 of 17), IgM (5 of 17), and IgA (4 of 17). Ab responses in NW were related to the magnitude of the serum Ab response: in 8 infants with post-2° serum Ab of <2 μg/ml, NW responses were detected in 0 of 8 by RAB and 1 of 8 by ELISA; in 9 infants with serum Ab >2 μg/ml, NW responses were detected in 6 of 9 by RAB and 7 of 9 by ELISA. At 6 mos after 2° injection, anti-PRP Ab was still detectable in NW in 6 of 16 subjects. Thus, a mucosal Ab component may contribute to the protective potential of conjugate vaccines.

958 PRODUCTION OF MUCOSAL ANTIBODY FOLLOWING PARENTERAL VACCINATION WITH HAEMOPHILUS INFLUENZAE TYPE B CAPSULAR POLYSACCHARIDE (PRP)

Anti-PRP antibody (ab) in sera and mucosal secretions from 5 children (>18 mo of age) and 4 adults was quantified by radioantigen binding, before and 3 weeks after immunization with 15 μg of PRP. Total IgA in secretions was quantified by laser-nephelometry. 8 of 9 subjects had detectable serum ab prior to vaccination and all produced an increase (children 4.5-640X, adults 3.3-48X) in serum ab following immunization. Prior to vaccination 3 of 5 children had detectable ab in nasal secretions (519-1828 ng ab/mg IgA). 2 of the 3 children with preimmunization nasal ab had an increase of ab following vaccination (2.3 & 7.7X) and 1 had no response. Both children with nondetectable preimmunization nasal ab levels had detectable levels following vaccination (1026 & 2093 ng ab/mg IgA). All adults had ab in nasal mucus (120-1917 ng ab/mg IgA) and 3 of 4 adults had ab in parotid saliva (127-241 ng ab/mg IgA) prior to vaccination. 3 of 4 adults had an increase (1.6-6.2X) and 1 had a transient decrease of nasal ab and all adults had an increase in salivary ab (3-8.5X in those with detectable preimmunization levels and to 1419 ng ab/mg IgA in the other subject) following vaccination. The subjects who did not have an increase in mucosal ab had the highest preimmunization mucosal ab titers. We conclude that, unlike vaccination with many other non-replicating antigens, PRP immunization often increases mucosal as well as serum ab.