IgG Ab Titers Research Articles

A novel adjuvant for vaccine development in the aged

A conformationally-biased, response-selective agonist of human C5a 65–74 (EP67) activated antigen presenting cells (APC) from aged C57Bl/6 mice in vitro and the generation of antigen (Ag)-specific antibody (Ab) responses in aged mice in vivo. EP67, induced the release of the pro-inflammatory cytokines IL-6, TNFα, and INFγ from splenic APCs obtained from both aged and young mice. Both aged and young mice produced high Ag-specific IgG Ab titers when immunized with EP67-containing vaccines to ovalbumin (OVA-EP67) and to a protein (rPrp1) from the cell wall of Coccidioides (rPrp1-EP67). Immunization with EP67-containing vaccines resulted in higher IgG titers in both young and aged mice compared to mice immunized with OVA adsorbed to alum (OVA/alum) and Prp1 admixed with CpG (rPrp1 +CpG). Aged and young mice immunized with the EP67-containing vaccines generated higher titers of IgG1 and IgG2b relative to their aged-matched counterparts immunized with OVA/alum or Prp1 +CpG. These results indicate that EP67 induces humoral immunity in aged mice not obtainable with alum and CpG. These results support the use of EP67 as a potential vaccine adjuvant suited to the elderly.

Adenovirus F protein as a delivery vehicle for botulinum B

BackgroundImmunization with recombinant carboxyl-terminal domain of the heavy chain (Hc domain) of botulinum neurotoxin (BoNT) stimulates protective immunity against native BoNT challenge. Most studies developing a botulism vaccine have focused on the whole Hc; however, since the principal protective epitopes are located within β-trefoil domain (Hcβtre), we hypothesize that immunization with the Hcβtre domain is sufficient to confer protective immunity. In addition, enhancing its uptake subsequent to nasal delivery prompted development of an alternative vaccine strategy, and we hypothesize that the addition of targeting moiety adenovirus 2 fiber protein (Ad2F) may enhance such uptake during vaccination.ResultsThe Hcβtre serotype B immunogen was genetically fused to Ad2F (Hcβtre/B-Ad2F), and its immunogenicity was tested in mice. In combination with the mucosal adjuvant, cholera toxin (CT), enhanced mucosal IgA and serum IgG Ab titers were induced by nasal Hcβtre-Ad2F relative to Hcβtre alone; however, similar Ab titers were obtained upon intramuscular immunization. These BoNT/B-specific Abs induced by nasal immunization were generally supported in large part by Th2 cells, as opposed to Hcβtre-immunized mice that showed more mixed Th1 and Th2 cells. Using a mouse neutralization assay, sera from animals immunized with Hcβtre and Hcβtre-Ad2F protected mice against 2.0 LD50.ConclusionThese results demonstrate that Hcβtre-based immunogens are highly immunogenic, especially when genetically fused to Ad2F, and Ad2F can be exploited as a vaccine delivery platform to the mucosa.

A role for macrophage IκB kinase in the mucosal adjuvant activity of edema toxin for sublingual vaccines (46.2)

Abstract It has been demonstrated that nuclear factor κB (NF-κB) activation by IκB kinase (Ikk)β deletion in macrophages increases pro-inflammatory cytokines responses and enhances pathogen clearance. To address whether this pathway also regulates the mucosal adjuvant activity of edema toxin (EdTx), LysMcre x Ikkβf/f (IkkβΔMø) mice with Ikkβ-deleted macrophages were sublingually immunized with 50µg of Yersinia pestis F1V fusion protein with or without 15µg of EdTx as an adjuvant. Both control and IkkβΔMø mice immunized with F1V+EdTx developed higher levels of serum Abs, i.e., IgG, IgG1, IgG2a and IgA, when compared to mice immunized with F1V alone. The IkkβΔMø mice exhibited higher serum IgG2a levels (and IgG2a:IgG1 ratio) than control counterpart immunized with F1V+EdTx, suggesting that immune responses to F1V are biased toward Th1-type. These mice also showed elevated levels of F1V-specific serum IgA Abs than control littermates. The effect of Ikkβ deletion was more pronounced in secretory Abs since EdTx as adjuvant promoted higher fecal IgA Abs, as well as IgG and IgA Ab titers in saliva and vaginal washes of IkkβΔMø mice. These studies reveal a new role for macrophages and NF-κB in shaping mucosal immune responses induced by EdTx derivative adjuvants via the sublingual mucosa.

Chlamydia trachomatis Native Major Outer Membrane Protein Induces Partial Protection in Nonhuman Primates: Implication for a Trachoma Transmission-Blocking Vaccine

A vaccine is likely the most effective strategy for controlling human chlamydial infections. Recent studies have shown immunization with Chlamydia muridarum major outer membrane protein (MOMP) can induce significant protection against infection and disease in mice if its native trimeric structure is preserved (nMOMP). The objective of this study was to investigate the immunogenicity and vaccine efficacy of Chlamydia trachomatis nMOMP in a nonhuman primate trachoma model. Cynomolgus monkeys (Macaca fascicularis) were immunized systemically with nMOMP, and monkeys were challenged ocularly. Immunization induced high serum IgG and IgA ELISA Ab titers, with Abs displaying high strain-specific neutralizing activity. The PBMCs of immunized monkeys produced a broadly cross-reactive, Ag-specific IFN-gamma response equivalent to that induced by experimental infection. Immunized monkeys exhibited a significant decrease in infectious burden during the early peak shedding periods (days 3-14). However, at later time points, they exhibited no difference from control animals in either burden or duration of infection. Immunization had no effect on the progression of ocular disease. These results show that systemically administered nMOMP is highly immunogenic in nonhuman primates and elicits partially protective immunity against ocular chlamydial challenge. This is the first time a subunit vaccine has shown a significant reduction in ocular shedding in nonhuman primates. A partially protective vaccine, particularly one that reduces infectious burden after primary infection of children, could interrupt the natural trachoma reinfection cycle. This would have a beneficial effect on the transmission between children and sensitized adults which drives blinding inflammatory disease.

Intrarectal Vaccination with Recombinant Vaccinia Virus Expressing Carcinoembronic Antigen Induces Mucosal and Systemic Immunity and Prevents Progression of Colorectal Cancer

The gastrointestinal mucosa contains an intact immune system that protects the host from pathogens and communicates with the systemic immune system. Absorptive epithelial cells in the mucosa give rise to malignant tumors although the interaction between tumor cells and the mucosal immune system is not well defined. The pathophysiology of colorectal cancer has been elucidated through studies of hereditary syndromes, such as familial adenomatous polyposis, a cancer predisposition syndrome caused by germline mutations in the adenomatous polyposis coli tumor suppressor gene. Patients with FAP develop adenomas and inevitably progress to invasive carcinomas by the age of 40. To better delineate the role of mucosal immunity in colorectal cancer, we evaluated the efficacy of intrarectal recombinant vaccinia virus expressing the human carcinoembryonic Ag (CEA) in a murine FAP model in which mice are predisposed to colorectal cancer and also express human CEA in the gut. Mucosal vaccination reduced the incidence of spontaneous adenomas and completely prevented progression to invasive carcinoma. The therapeutic effects were associated with induction of mucosal CEA-specific IgA Ab titers and CD8(+) CTLs. Mucosal vaccination was also associated with an increase in systemic CEA-specific IgG Ab titers, CD4(+) and CD8(+) T cell responses and resulted in growth inhibition of s.c. implanted CEA-expressing tumors suggesting communication between mucosal and systemic immune compartments. Thus, intrarectal vaccination induces mucosal and systemic antitumor immunity and prevents progression of spontaneous colorectal cancer. These results have implications for the prevention of colorectal cancer in high-risk individuals.

Abstract 2425: Absence of Auto-Antibodies against Cardiac Troponin I Predicts Improvement of Left Ventricular Function after Acute Myocardial Infarction

Application of antibodies against cardiac troponin I (cTnI-Ab) can induce dilation and dysfunction of the heart in mice. Recently, we demonstrated that immunization with cTnI induces inflammation and fibrosis in myocardium of mice. Others have shown that autoanti-bodies to cTnI are present in patients with acute coronary syndrome. But little is known about the clinical relevance of detected cTnI-Ab. First, anti-cTnI and anti-cTnT antibody titers were measured in sera from 272 patients with dilated- (DCM) and 185 with ischemic- (ICM) cardiomyopathy. Secondly, 108 patients with acute myocardial infarction (AMI) were included for a follow-up study. Heart characteristics were determined by magnetic resonance imaging 4 days and 6 –9 months after AMI. Altogether, in 7,0% of patients with DCM and in 9,2% with ICM an anti-cTnI IgG antibody titer ≥1:160 was measured. In contrast, only in 1,7% of patients with DCM and in 0,5% with ICM an anti-cTnT IgG antibody titer ≥1:160 was detected. Ten out of 108 patients included in the follow-up study were tested positive for cTnI-Ab with IgG Ab titers ≥1:160. TnI-Ab negative patients showed a significant increase in LVEF and stroke volume 6 –9 months after AMI. In contrast, there was no significant increase in LVEF and stroke volume in TnI-Ab positive patients. We demonstrate for the first time that the prevalence of cTnI-Abs in patients with AMI has an impact on the improvement of the LVEF over a study period of 6 –9 months.

Absence of Auto‐Antibodies against Cardiac Troponin I Predicts Improvement of Left Ventricular Function after Acute Myocardial Infarction

Background: Application of antibodies (Ab) against cardiac troponin I (cTnI) can induce dilation and dysfunction of the heart in mice. But little is known about the clinical relevance of detected cTnI‐Ab.Methods: First, anti‐cTnI and anti‐cTnT Ab titers have been measured in sera from 272 patients with dilated‐ (DCM) and 185 with ischemic‐ (ICM) cardiomyopathy. Secondly, 108 patients with acute myocardial infarction (AMI) were included for a follow‐up study. All 108 patients were treated according to current guidelines. Heart characteristics were determined by magnetic resonance imaging 3–4 days and 6–9 months after AMI.Results: Altogether, in 14,3% of patients with DCM and in 17,3% with ICM an anti‐cTnI IgG‐Ab titer ≥1:80 was measured. In contrast, only in 1,8% of patients with DCM and in 0,5% with ICM an anti‐cTnT IgG‐Ab titer ≥1:80 was detected. Ten out of 108 patients included in the follow‐up study were tested positive for cTnI‐Ab with IgG Ab titers ≥1:160. TnI‐Ab negative patients showed a significant increase in LVF and stroke volume 6–9 months after AMI. In contrast, there was no significant increase in LVF and stroke volume in TnI‐Ab positive patients.Conclusion: We demonstrate for the first time that the prevalence of cTnI‐Abs in patients with AMI has an impact on the improvement of the LVF.

αE Integrin Compensates for β7 Deficiency in the Nasal Passages (NP) Following Nasal Immunization with Cholera Toxin (CT) (41.10)

Abstract Nasal immunization stimulates robust Ab responses in the head and neck lymph nodes (HNLN) and nasal-associated lymphoid tissue (NALT). Studies showed that B lymphocyte trafficking in the HNLN and NALT is both L-selectin- and β7-dependent. β7 integrin generally associates with αE or α4 integrins to mediate lymphocyte retention in the epithelium or the mucosa, respectively. To study the contribution of β7 integrin to upper respiratory immunity, β7−/− mice were nasally dosed with CT on days 0, 7, and 14. Mucosal IgA or serum IgG Ab titers were not different from B6 β7+/+ mice at any of the time points examined til day 42. Vaginal IgG responses were slightly enhanced in β7−/− mice, and absent in L-selectin−/− mice. However, the absence of β7 did alter the types of supporting B cells found in the NP. Increased expression of αE was observed on day 42 despite the absence of its co-receptor, β7. This increased αE expression was not observed in B6 β7+/+ mice. αE+ B cells continued to be found in the β7−/− small intestinal lamina propria. Minimal αE+ B cells were found in the HNLN. From these studies, although mucosal Ab responses remain intact in β7−/− mice, it does appear that different homing receptor-dependent mechanisms are employed. This work is supported by NIH AI-55563.

Bacterial c-di-GMP Is an Immunostimulatory Molecule

Cyclic diguanylate (c-di-GMP) is a bacterial intracellular signaling molecule. We have shown that treatment with exogenous c-di-GMP inhibits Staphylococcus aureus infection in a mouse model. We now report that c-di-GMP is an immodulator and immunostimulatory molecule. Intramammary treatment of mice with c-di-GMP 12 and 6 h before S. aureus challenge gave a protective effect and a 10,000-fold reduction in CFUs in tissues (p < 0.001). Intramuscular vaccination of mice with c-di-GMP coinjected with S. aureus clumping factor A (ClfA) Ag produced serum with significantly higher anti-ClfA IgG Ab titers (p < 0.001) compared with ClfA alone. Intraperitoneal injection of mice with c-di-GMP activated monocyte and granulocyte recruitment. Human immature dendritic cells (DCs) cultured in the presence of c-di-GMP showed increased expression of costimulatory molecules CD80/CD86 and maturation marker CD83, increased MHC class II and cytokines and chemokines such as IL-12, IFN-gamma, IL-8, MCP-1, IFN-gamma-inducible protein 10, and RANTES, and altered expression of chemokine receptors including CCR1, CCR7, and CXCR4. c-di-GMP-matured DCs demonstrated enhanced T cell stimulatory activity. c-di-GMP activated p38 MAPK in human DCs and ERK phosphorylation in human macrophages. c-di-GMP is stable in human serum. We propose that cyclic dinucleotides like c-di-GMP can be used clinically in humans and animals as an immunomodulator, immune enhancer, immunotherapeutic, immunoprophylactic, or vaccine adjuvant.

Oral vaccination with salmonella simultaneously expressing Yersinia pestis F1 and V antigens protects against bubonic and pneumonic plague.

The gut provides a large area for immunization enabling the development of mucosal and systemic Ab responses. To test whether the protective Ags to Yersinia pestis can be orally delivered, the Y. pestis caf1 operon, encoding the F1-Ag and virulence Ag (V-Ag) were cloned into attenuated Salmonella vaccine vectors. F1-Ag expression was controlled under a promoter from the caf1 operon; two different promoters (P), PtetA in pV3, PphoP in pV4, as well as a chimera of the two in pV55 were tested. F1-Ag was amply expressed; the chimera in the pV55 showed the best V-Ag expression. Oral immunization with Salmonella-F1 elicited elevated secretory (S)-IgA and serum IgG titers, and Salmonella-V-Ag(pV55) elicited much greater S-IgA and serum IgG Ab titers than Salmonella-V-Ag(pV3) or Salmonella-V-Ag(pV4). Hence, a new Salmonella vaccine, Salmonella-(F1+V)Ags, made with a single plasmid containing the caf1 operon and the chimeric promoter for V-Ag allowed the simultaneous expression of F1 capsule and V-Ag. Salmonella-(F1+V)Ags elicited elevated Ab titers similar to their monotypic derivatives. For bubonic plague, mice dosed with Salmonella-(F1+V)Ags and Salmonella-F1-Ag showed similar efficacy (>83% survival) against approximately 1000 LD(50) Y. pestis. For pneumonic plague, immunized mice required immunity to both F1- and V-Ags because the mice vaccinated with Salmonella-(F1+V)Ags protected against 100 LD(50) Y. pestis. These results show that a single Salmonella vaccine can deliver both F1- and V-Ags to effect both systemic and mucosal immune protection against Y. pestis.

Is there any relationship between toxoplasma infection and reactive arthritis?

The diagnosis of reactive arthritis is a challenging clinical problem in daily practice. Although there are many triggering infectious agents for reactive arthritis, Toxoplasmosis, a worldwide parasitic infection has not been reported. We investigated the serologic evidence of Toxoplasma gondii ( T. gondii ) infection in patients with newly diagnosed reactive arthritis after six weeks of the onset of the first symptom but no demonstrable triggering agent for reactive arthritis. Clinical controlled study. We screened serologically the serum toxoplasma IgM and IgG antibody (Ab) titers which revealed toxoplasma infection in 50 patients with reactive arthritis (40 female, 10 men) and no demonstrable triggering agent and control subjects (32 female, 8 male). SPSS 10.0 software package program was used. The mean age of the patients and controls was similar (41.3+/- 12.0 vs. 39.6+/-11.8 years) respectively. The prevalence of IgG Ab titers of T. gondii in patients and controls were found to be 52% and 47.5%, respectively. Mean serum Toxoplasma IgG Ab levels were found to be 16.5+/-14.5 IU/ml, and 16.9+/-13.8 IU/ml in patients and control subjects respectively ( P> 0.05). We did not find any Toxoplasma IgM Ab titer demonstrating the acute or sub-acute infection in the serum of patients or controls. Although past Toxoplasma infection was prevalent in both groups, we did not find any subject with acute Toxoplasma infection in patients with newly diagnosed reactive arthritis and healthy controls. Despite the fact that our study group was small, we suggest that T. gondii does not seem to be a triggering agent for reactive arthritis and past infection may be a coincidental finding.

T Cell Epitope-Specific Defects in the Immune Response to Cat Allergen in Patients with Atopic Dermatitis

Atopic dermatitis (AD) is often associated with high titer IgE antibodies (ab) to allergens, and IL-10-mediated regulation of IFN-gamma has been proposed to contribute to this IgE ab production. However, the relevance of IL-10 and IFN-gamma to IgE associated with AD has not been examined in the context of an allergen-specific system. Analysis of PBMC responses in vitro showed deficient T cell proliferation to overlapping IL-10- (peptide (P) 2:1) and IFN-gamma- (P2:2) inducing chain 2 major epitopes of cat allergen (Fel d 1) in cultures from sensitized AD patients (mean IgE to cat=20.9 IU/ml). Diminished IFN-gamma induction by Fel d 1 and P2:2, along with elevated peptide-induced IL-10 (except for P2:1) was observed in PBMC cultures from AD subjects compared with non-AD (sensitized and non-sensitized) subjects. Neither T cell proliferation nor IFN-gamma production to chain 2 epitopes could be restored by anti-IL-10 mAb in cultures from sensitized AD subjects. Moreover, allergen avoidance was associated with a paradoxical decrease in both IL-10 and IFN-gamma in peptide-stimulated PBMC from these subjects. Control of IFN-gamma production to chain 2 epitopes by IL-10 may be relevant to sensitization status. Development of high titer IgE ab in AD could reflect a failure of this mechanism.

Pinellic acid from the tuber of Pinellia ternata Breitenbach as an effective oral adjuvant for nasal influenza vaccine

This study describes the isolation, purification, characterization, and adjuvant activity of an orally active adjuvant substance from the tuber of Pinellia ternata, as an active herbal component of the traditional Japanese herbal (Kampo) medicine, Sho-seiryu-to (SST, Chinese name: Xiao-Qing-Long-Tang), which has been reported to show oral adjuvant activity for nasally administered influenza HA vaccine [Int. J. Immunopharmacol. 16 (1994) 605]. The active compound was identified as 9 S, 12 S, 13 S-trihydroxy-10 E-octadecenoic acid using infrared spectra, proton magnetic resonance, mass spectrometry, and circular dichroism, and named pinellic acid. Oral administration of pinellic acid (1 μg) to BALB/c mice given primary and secondary intranasal inoculations of influenza HA vaccine (1 μg) enhanced antiviral IgA antibody (Ab) titers 5.2- and 2.5-fold in nasal and bronchoalveolar washes, respectively, and antiviral IgG Ab titers 3-fold in bronchoalveolar wash and serum. Intranasal administration of pinellic acid (1 μg) with influenza HA vaccine (1 μg) slightly enhanced antiviral IgG Ab titers in bronchoalveolar wash and serum but not antiviral IgA Ab titers in nasal and bronchoalveolar washes. Pinellic acid showed no hemolytic activity. The results of this study suggest that pinellic acid may provide a safe and potent oral adjuvant for nasal influenza HA vaccine.

Schistosome-Infected IL-4 Receptor Knockout (KO) Mice, in Contrast to IL-4 KO Mice, Fail to Develop Granulomatous Pathology While Maintaining the Same Lymphokine Expression Profile

Th2 lymphocytes have been postulated to play a major role in the immunopathology induced by Schistosoma mansoni infection. Nevertheless, infected IL-4 knockout (KO) and wild-type (wt) mice develop egg granulomas comparable in size. To further investigate the function of the Th2 response in egg pathology we studied IL-4Ralpha-deficient mice, which are nonresponsive to both IL-4 and IL-13. In striking contrast to IL-4 KO animals, infected IL-4Ralpha KO mice developed only minimal hepatic granulomas and fibrosis despite the presence of CD3+ T cells in the residual egg lesions. Moreover, liver lymphokine mRNA levels in these animals and IL-4 KO mice were equivalent. In addition, infected IL-4Ralpha-deficient, IL-4-deficient, and wt animals developed similar egg Ag-specific IgG Ab titers, arguing that CD4-dependent Th activity is intact in KO mice. As expected, IFN-gamma secretion was strongly up-regulated in mesenteric lymph node cultures from both groups of deficient animals, a change reflected in increased serum IgG2a and IgG2b Ab levels. Surprisingly, Th2 cytokine production in infected IL-4Ralpha KO mice was not abolished but was only reduced and resembled that previously documented in IL-4 KO animals. This residual Th2 response is likely to explain the ability of IL-4 KO mice to generate egg granulomas, which cannot be formed in IL-4Ralpha-deficient animals because of their lack of responsiveness to the same cytokine ligands. Taken together, these findings argue that tissue pathology in schistosomiasis requires, in addition to egg-specific CD4+ lymphocytes, a previously unrecognized IL-4Ralpha+ non-T cell effector population.

Targeting of natural killer cells to mammary carcinoma via naturally occurring tumor cell-bound iC3b and beta-glucan-primed CR3 (CD11b/CD18).

Previous reports have suggested that malignant cells frequently generate a humoral immune response that is ineffective in tumor destruction. Despite coating tumors with IgM and IgG that activate the C system via the classical pathway, normal membrane regulators of C (e.g., membrane cofactor protein and CD59) prevent cytotoxicity. Moreover, C3 deposition on tumors does not result in cytotoxic recognition by phagocytes or NK cells bearing C3 receptors capable of mediating destruction of C3-opsonized bacteria or yeast. The current investigation showed that freshly excised mammary tumors bore IgM, IgG, and C3 detectable by flow cytometry. Normal sera contained natural IgM and IgG Abs reactive with breast tumor cell lines, and IgG Ab titers were increased in patients with breast cancer. Breast tumor cell lines incubated in normal serum from AB+ individuals activated the classical, but not the alternative, pathway of C and became coated with C3. Despite exhibiting membrane-bound C3, serum-opsonized breast tumor cell lines were not killed by CR3 (CD11b/CD18)-bearing NK cells. Priming of NK cell CR3 with small soluble yeast beta-glucan polysaccharides enabled CR3-dependent killing of these same C3-bearing tumor cell lines. Tests of mammary carcinoma cells from freshly excised tumors demonstrated that they also bore sufficient amounts of opsonic C3 for cytotoxic recognition by NK cells bearing polysaccharide-primed CR3, whereas they were largely resistant to NK cells bearing unprimed CR3. This study demonstrates the potential utility of using naturally occurring opsonic C3 on tumor cells for specific immunotherapeutic targeting by NK cells and phagocytes bearing polysaccharide-primed CR3.

Correlation of tolerogenicity of a viral antigen with its immunogenicity.

Induction of B cell tolerance or activation was analyzed with vesicular stomatitis virus (VSV) glycoprotein (G) expressed as a neo-self Ag. A membrane form of VSV-G expressed in all tissues, including the bone marrow, induced unresponsiveness at both the Th and B cell level, whereas a soluble form of VSV-G expressed peripherally in liver and kidney did not tolerize B cells and only reversibly anergized Th cells. Interestingly, a similar correlation was found for activation of mature lymphocytes. When mature normal spleen cells were transferred into the two transgenic mouse lines, the membrane form of VSV-G was strongly immunogenic for both Th and B cells, and high VSV-G-specific IgG Ab titers were induced in these transgenic mice. In contrast, spleen cells transferred into mice expressing the soluble form of VSV-G were not activated, and no VSV-G specific Abs were induced. These results indicate that highly immunogenic Ags are strongly tolerogenic for both immature B and T cells.

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.