Antibody-secreting Cells In Peripheral Blood Research Articles

Exploring Prime-Boost Vaccination Regimens with Different H1N1 Swine Influenza A Virus Strains and Vaccine Platforms.

In a previous vaccination study in pigs, heterologous prime-boost vaccination with whole-inactivated H1N1 virus vaccines (WIV) induced superior antibody responses and protection compared to homologous prime-boost vaccination. However, no pan-H1 antibody response was induced. Therefore, to stimulate both local and systemic immune responses, we first vaccinated pigs intranasally with a pseudorabies vector vaccine expressing the pH1N1 hemagglutinin (prvCA09) followed by a homologous or heterologous WIV booster vaccine. Homologous and heterologous WIV-WIV vaccinated groups and mock-vaccinated or prvCA09 single-vaccinated pigs served as control groups. Five weeks after the second vaccination, pigs were challenged with a homologous pH1N1 or one of two heterologous H1N2 swine influenza A virus strains. A single prvCA09 vaccination resulted in complete protection against homologous challenge, and vector-WIV vaccinated groups were significantly better protected against heterologous challenge compared to the challenge control group or WIV-WIV vaccinated groups. Furthermore, vector-WIV vaccination resulted in broader hemagglutination inhibition antibody responses compared to WIV-WIV vaccination and higher numbers of antibody-secreting cells in peripheral blood, draining lymph nodes and nasal mucosa. However, even though vector-WIV vaccination induced stronger antibody responses and protection, we still failed to induce a pan-H1 antibody response.

Augmented ILT3/LILRB4 Expression of Peripheral Blood Antibody Secreting Cells in the Acute Phase of Kawasaki Disease.

Kawasaki disease (KD) is an acute, systemic vasculitis syndrome that occurs in children. The clinical symptoms and epidemiologic features of KD strongly suggest that KD is triggered by unidentified infectious agents in genetically predisposed patients. In addition, a number of studies have described the role of B cells in the development of KD. To obtain a mechanistic insight into the humoral immune response of B-lineage cells in KD patients, we examined peripheral blood antibody secreting cells (ASCs) and inhibitory immunoreceptors, immunoglobulin-like transcript (ILT)/leukocyte immunoglobulin-like receptor (LILR), on each B cell subpopulation. Eighteen Japanese KD patients and thirteen healthy control subjects were recruited for this study. Their peripheral blood mononuclear cells were examined by flow cytometry for the number of CD19 B cells, the size of each B cell subset and the expression of the inhibitory isoforms of ILT/LILR on the B cell subset. The frequency of CD19CD27 ASCs was significantly increased in the acute phase of KD and reduced after high-dose intravenous immunoglobulin (IVIG) treatment. Interestingly, while ILT2/LILRB1 expression was ubiquitously observed on every B cell/ASCs subset and the level was not significantly different after IVIG, ILT3/LILRB4 (B4) was uniquely expressed on only ASCs, and its expression was significantly decreased after IVIG. In the acute phase of KD, the frequency of ASCs is high with augmented B4 expression, whereas it is lower with decreased B4 expression after IVIG. Further studies of B4 expression on ASCs in autoimmune and infectious diseases will be needed to confirm the significance of our findings.

Affinity maturation occurs in channel catfish (Ictalurus punctaus) following immunization with a T-cell dependent antigen

Affinity maturation of the antibody response, a process of antibody affinity increasing over response, is one of the key features of the mammalian immune system. However, the process is incompletely understood in teleost, including channel catfish (Ictalurus punctaus). In this study, IgM affinity maturation in channel catfish was investigated by estimating the kinetics of antibody affinity using ELISA and ELISPOT assays. Fish were immunized with a T-cell dependent antigen (TNP-KLH), and individual serum IgM antibody titers and affinities, and IgM+ antibody-secreting cells (ASCs) in peripheral blood were analyzed over a period of 14 weeks. A detectable serum anti-TNP response developed by 2-weeks post-immunization, and the maximal antibody production was observed by 6-weeks post-immunization. The average affinity of anti-TNP serum antibody increased consistently and reached the maximum by 10-weeks post-immunization. The increase of antibody affinity beyond the point of optimal antibody titer revealed that the affinity maturation of IgM antibody response occurred in channel catfish. Dissection of dynamics of individual affinity subpopulations indicated that a significant proportion of low affinity subpopulations appeared at early response, and high affinity subpopulations appeared predominantly at later, resulting in a 100-fold increase in affinity over response. Additional, TNP+ IgM+ ASCs was detected by 2-weeks post-immunization and achieved the maximal number by 6-weeks post-immunization. Using an inhibition ELISPOT assay, the findings of a consistent increase in the average affinity of secreted IgM antibody by peripheral blood ASCs, as the immune response progressed, confirmed the occurrence of the affinity maturation. Taken together, the results of this study indicated that affinity maturation occurred in channel catfish following immunization with a TD antigen TNP-KLH.

Analysis of peripheral blood B and Tfh cells as predictors of antibody responses in individuals receiving candidate blood-stage malaria vaccines in a Phase Ia clinical trial

Background Here we report on a Phase Ia clinical vaccine trial using the AMA1 antigen from the blood-stage Plasmodium falciparum malaria parasite. A variety of promising ‘mixed-modality’ regimens were tested, with all volunteers primed with chimpanzee adenovirus-ChAd63, and then subsequently boosted with the orthopoxvirus MVA and/or protein-in-adjuvant (Alhydrogel ± CPG 7909) using 8 or 16 week prime-boost intervals. The induction of high titre, neutralizing antibodies is deemed essential for protection against blood-stage malaria. Peripheral blood antibody secreting cells (ASC) and memory B cells (mBC) play important roles in antibody production and maintenance. Lymph node (LN) CD4+ follicular helper T cells (Tfh cells) mediate cognate control of antigen-specific antibody responses. Recently Tfh-like cells have been found to be present in the CXCR5+ subset of memory CD4+ T cells in human peripheral blood. This facilitates analysis of the role of Tfh responses in regulation of vaccine-elicited antibody responses, although there is much debate over the relationship between LN and circulating populations. Phenotypic definition of the latter has also proved complex, with recent studies indicating that phenotypically-distinct Tfh populations with different functional profiles are elicited by different antigenic stimuli. Characterizing T cell and B cell populations across a variety of immunisation regimes may lead to greater understanding of their value as quantitative and qualitative predictors of antibody responses. Materials and methods ASC and mBC responses were measured using a standardized B cell ELISPOT method, using fresh and frozen PBMC respectively, whilst antibody titre was measured using a standardized ELISA for AMA1. Multiparameter flow cytometry was also subsequently run using frozen PBMC to characterise total peripheral CD4+ Tfh populations and the AMA1-specific CD4+ T cell response.

Chronic differentiation of human mucosal IgA-secreting cells during B cell depletion therapy with rituximab. (161.3)

Abstract During mucosal immune responses, IgA-secreting plasmablasts are generated from mucosal B cells. We previously demonstrated that at steady-state, most human peripheral blood antibody-secreting cells produce IgA and express mucosal homing receptors, and suggested their generation in mucosal immune responses. In the current study, we analyzed the blood of patients who received B cell depletion therapy with rituximab (anti-CD20) for treatment of rheumatoid arthritis before and at 2-9months after rituximab infusion by flow cytometry. While CD20+ B cells were reduced to <0.02% of their intial numbers before therapy, CD19+/CD27hi/CD20- antibody-secreting cells remained detectable in the blood at 26-119% of their initial numbers. These circulating antibody-secreting cells expressed IgA that bound to bacterial antigens, beta7 integrin and CCR10 before and during B cell depletion, suggesting their mucosal origin. High expression of HLA-DR and Ki-67 and in vitro migration towards CCL28 and CXCL12 qualified these cells as recently activated plasmablasts, reflecting the survival of functional B cells during rituximab treatment. Consistently, IgA+ plasmablasts and plasma cells were identified in the lamina propria during B cell depletion. Our data implicate that a population of mucosal B cells is resistant to rituximab and self-sufficient in adult humans and not replenished by CD20+ B cells immigrating from blood, lymphoid tissue, or bone marrow, that is, B cells depleted by rituximab.

Mucosal immunology of the genital and gastrointestinal tracts and HIV-1 infection

The male and female genital tracts are protected by a local immune system that displays features distinguishing them from other mucosal sites. In contrast to the intestinal tract, where locally produced IgA is the dominant Ig, secretions of the male and female genital tract contain predominantly IgG of both local and systemic origin. Genital tract tissues also lack mucosal lymphoepithelial inductive sites analogous to intestinal Peyer's patches; consequently, local immunization or infections with sexually transmitted pathogens induce low immune responses. Human immunodeficiency virus 1 (HIV-1) infection must be primarily considered as a mucosal disease with extensive involvement of the systemic immune compartment. Although the majority of infections is acquired through the genital mucosa, a high rate of virus replication and profound CD4(+) T cell depletion occurs in the intestinal mucosa and other mucosal tissues shortly after infection. Evaluation of HIV-specific antibodies in sera and external secretions, including vaginal washes and semen, unexpectedly revealed a selective lack of IgA responses. Moreover, specific antibody-secreting cells in peripheral blood were of the IgG isotype, even in mucosally infected individuals. Whether humoral responses to previously or newly encountered antigens are compromised in HIV-1-infected persons is under current investigation.

Diagnostic Approach to Acute Diarrheal Illness in a Military Population on Training Exercises in Thailand, a Region of Campylobacter Hyperendemicity

High rates of Campylobacter fluoroquinolone resistance highlight the need to evaluate diagnostic strategies that can be used to assist with clinical management. Diagnostic tests were evaluated with U.S. soldiers presenting with acute diarrhea during deployment in Thailand. The results of bedside and field laboratory diagnostic tests were compared to stool microbiology findings for 182 enrolled patients. Campylobacter jejuni was isolated from 62% of the cases. Clinical and laboratory findings at the time of presentation were evaluated to determine their impact on the posttest probability, defined as the likelihood of a diagnosis of Campylobacter infection. Clinical findings, the results of tests for inflammation (stool occult blood testing [Hemoccult], fecal leukocytes, fecal lactoferrin, plasma C-reactive protein), and the numbers of Campylobacter-specific antibody-secreting cells in peripheral blood failed to increase the posttest probability above 90% in this setting of Campylobacter hyperendemicity when these findings were present. Positive results by a Campylobacter-specific commercial enzyme immunoassay (EIA) and, less so, a research PCR were strong positive predictors. The negative predictive value for ruling out Campylobacter infection, defined as a posttest probability of less than 10%, was similarly observed with these Campylobacter-specific stool-based tests as well the fecal leukocyte test. Compared to the other tests evaluated, the Campylobacter EIA is a sensitive and specific rapid diagnostic test that may assist with diagnostic evaluation, with consideration of the epidemiological setting, logistics, and cost.

Paucity of Antigen-Specific IgA Responses in Sera and External Secretions of HIV-Type 1-Infected Individuals

This study was undertaken to resolve existing controversies with respect to the detection of IgA HIV-1-specific mucosal antibodies in infected individuals. External secretions, including tears, nasal, rectal, and vaginal washes, saliva, semen, urine, and sera were obtained from 50 HIV-1-infected individuals and 20 controls using collection procedures that minimize the irritation of mucosal surfaces. Levels of total and antigen (gp120 and gp160)-specific antibodies of the IgG and IgA isotypes were measured by assays that proved reliable in a large multicenter study: quantitative ELISA and chemiluminescence-enhanced Western blot analyses. Although the levels of total IgG and IgA were increased or remained unchanged in body fluids of HIV-1-infected individuals as compared to the controls, HIV-1-specific IgA antibodies were either absent or present at low levels even in secretions with characteristically high relative contents of total IgA vs. IgG (saliva, tears, and rectal and nasal washes). In these secretions, HIV-1-specific IgG antibodies dominated. In assessing levels and frequency of detection of IgG antibodies, both female and male genital tract secretions, urine, and nasal wash were preferable to parotid saliva and especially to rectal wash. External secretions contained IgG antibodies to gp160 > gp120 > gp41 and p24; when present, IgA antibodies were predominantly directed at gp160. Analyses of peripheral blood antibody-secreting cells (ASC) isolated from the same individuals paralleled these serological findings: gp160-specific IgG-secreting ASC were dominant. Therefore, in striking contrast to other mucosally encountered microbial infections, HIV-1 does not induce vigorous specific IgA responses in any body fluid examined or in ASC in peripheral blood.

Paucity of Antigen-Specific IgA Responses in Sera and External Secretions of HIV-Type 1-Infected Individuals

This study was undertaken to resolve existing controversies with respect to the detection of IgA HIV-1-specific mucosal antibodies in infected individuals. External secretions, including tears, nasal, rectal, and vaginal washes, saliva, semen, urine, and sera were obtained from 50 HIV-1-infected individuals and 20 controls using collection procedures that minimize the irritation of mucosal surfaces. Levels of total and antigen (gp120 and gp160)-specific antibodies of the IgG and IgA isotypes were measured by assays that proved reliable in a large multicenter study: quantitative ELISA and chemiluminescence-enhanced Western blot analyses. Although the levels of total IgG and IgA were increased or remained unchanged in body fluids of HIV-1-infected individuals as compared to the controls, HIV-1-specific IgA antibodies were either absent or present at low levels even in secretions with characteristically high relative contents of total IgA vs. IgG (saliva, tears, and rectal and nasal washes). In these secretions, HIV-1-specific IgG antibodies dominated. In assessing levels and frequency of detection of IgG antibodies, both female and male genital tract secretions, urine, and nasal wash were preferable to parotid saliva and especially to rectal wash. External secretions contained IgG antibodies to gp160> gp120> gp41 and p24; when present, IgA antibodies were predominantly directed at gp160. Analyses of peripheral blood antibody-secreting cells (ASC) isolated from the same individuals paralleled these serological findings: gp160-specific IgG-secreting ASC were dominant. Therefore, in striking contrast to other mucosally encountered microbial infections, HIV-1 does not induce vigorous specific IgA responses in any body fluid examined or in ASC in peripheral blood.

Optimization of an ovine antibody-secreting cell assay for detection of antigen-specific immunoglobulin production in peripheral blood leukocytes.

Enumeration of antibody-secreting cells in peripheral blood by enzyme-linked immunospot (ELISPOT) has been used in human studies to detect antigen-specific antibody production at mucosal tissue sites. An alternative assay for detecting and quantitating antigen-specific antibody responses involves culturing circulating peripheral blood antibody-secreting cells and quantitating specific antibody production in culture supernatant by ELISA. In the present study, antigen-specific peripheral blood lymphocytes were isolated from subcutaneously immunized sheep and the parameters for maximizing in vitro antibody production by in vivo-induced antibody-secreting cells optimized for this species. Maximum antibody-secreting cell responses were observed in peripheral blood collected four days after antigen challenge. The addition of lipopolysaccharide and antisheep immunoglobulin had no effect on in vitro antibody secretion by blood antibody-secreting cells, while the effects of pokeweed mitogen were highly variable. However, the combination of anti-Ig and recombinant ovine interleukin-6 to peripheral blood lymphocyte cultures was found to markedly and consistently enhance specific antibody production. In unstimulated cultures, the optimal peripheral blood lymphocyte concentration for generating the greatest antibody responses was 5.0 x 107 cells per mL, but in cultures stimulated with recombinant ovine interleukin-6/antisheep immunoglobulin, the optimal cell concentration was lowered to approximately 1.0 x 107 cells per mL. In vitro, peak immunoglobulin production was usually achieved by day one in unstimulated cultures. In recombinant ovine interleukin-6/antisheep immunoglobulin-stimulated cultures, antibody levels were similar to unstimulated cultures by day one, however, the levels continued to rise during incubation to reach a maximum between days four and five of incubation. This optimized antibody-secreting cell culture assay is amenable for increasing the sensitivity and reducing the cell numbers required for quantitating antigen-specific antibody induction in large-scale immunization trials in sheep and other large animal species.

Deficient IgA1 immune response to nasal cholera toxin subunit B in primary IgA nephropathy

Twelve IgA nephropathy (IgAN) patients and 18 controls were immunized with novel protein antigens, cholera toxin subunit B (CTB) via the nasal route and keyhole limpet hemocyanin (KLH) subcutaneously. Antibody secreting cells and antibody response in body fluids were determined by ELISPOT assay and ELISA, respectively. Analysis of variance showed, in contrast to controls (P < 0.001), no CTB-specific IgA response in the nasal washes of patients with IgAN. Significantly lower numbers of CTB-specific antibody-secreting cells in peripheral blood (P < 0.001) and CTB-specific antibodies in plasma (P < 0.005) were found in IgAN, both restricted to the IgA1 subclass. The proportions of CTB-specific IgA1-secreting cells in bone marrow aspirates correlated significantly with the corresponding ratios in plasma, with significantly lower values (P < 0.005) in IgAN as compared to controls. These results support the existence of a "mucosa-bone marrow axis" in humans, but no dysregulation of this axis was found in IgAN. The deficient mucosal IgA immune response to CTB observed in this study after primary mucosal immunization indicates that patients with IgAN have a defective immune response when challenged intranasally. These patients may depend on more frequent and/or prolonged antigen encounter at mucosal sites before efficient mucosal immunity is established. Repeated seeding of antigen-specific cells to secondary lympoid organs could result secondarily in the relative hyperresponsiveness found in IgAN upon reactivation by parenteral immunization.

Peripheral blood antibody-secreting cells in the evaluation of the immune response to an oral vaccine

Specific antibody-secreting cells (ASC) appear in the blood as a response to oral vaccination in humans. Based on information from animal experiments, these cells are believed to be migrating to the mucosa. This review summarizes a series of studies aimed at a detailed characterization of the ASC response to a prototype oral vaccine Salmonella typhi Ty21a, with respect to its kinetics, Ig-class distribution, antigen specificity, influence of the administration route and nature of the antigen, and the corresponding antibody responses in serum. Different vaccine formulations as well as dosage schedules are compared, and the response to booster immunization is described. The response manifested by ASC in blood is shown to be independent from serum antibody responses. Moreover, it is shown to parallel with the results obtained for protection in field trials. Finally, some data on the homing receptor expression of these cells are presented, giving further evidence for the mucosal homing of these cells. The ASC assay offers a practical means for assessing immune response to oral vaccines in humans. It can be used as a laboratory parameter correlated with protection conferred by an oral typhoid vaccine. It can even be applied to measure active mucosal immunity, i.e., protective immunity by showing the relative reduction of the ASC response to an oral dose of live vaccine.

Anti-myelin basic protein and anti-proteolipid protein antibody-secreting cells in the cerebrospinal fluid of patients with acute optic neuritis.

To study the intrathecal synthesis of anti-myelin basic protein (MBP) and anti-proteolipid protein (PLP) antibodies in patients in the early stages of multiple sclerosis. A study of consecutive patients with acute optic neuritis (ON) who were undergoing lumbar punctures in an ambulatory unit. Eleven patients with acute idiopathic ON and 14 patients with acute ON as a symptom of definite multiple sclerosis (the diagnosis of which was supported by clinical or laboratory findings). Nineteen patients with other neurological diseases (10 with inflammatory diseases) served as controls. Numbers of anti-MBP and anti-PLP antibody-secreting cells in peripheral blood and cerebrospinal fluid samples that were enumerated with an immunospot assay. Cerebrospinal fluid cells that secreted anti-MBP or anti-PLP antibodies were detected in 10 of 15 and in 21 of 23 patients with acute ON, while they were detected in nine of 18 and in six of 18 patients with other neurological diseases, respectively. Patients with ON had significantly more anti-PLP-secreting cells than did patients with other neurological diseases (P < .01). No difference was observed for anti-MBP-secreting cells. A significant correlation between the time from onset and the number of anti-PLP-secreting cells was found in patients with idiopathic ON (P < .02). These data suggest that anti-PLP antibodies are a more specific finding in demyelinating disease than anti-MBP antibodies. Furthermore, they suggest that anti-PLP antibodies may arise as a consequence of the demyelinating process.

Antibody-secreting cells in acute urinary tract infection as indicators of local immune response.

Mucosal immune mechanisms are believed to be important in host defense against urinary tract infection. To study the human immune response in the urinary tract, peripheral blood antibody-secreting cells (ASC), believed to originate from the mucosal surfaces, were investigated with the enzyme-linked immunospot assay. Pathogen-specific ASC were found in 17 (89%) of 19 patients with pyelonephritis and in 12 (70%) of 17 with LUTI (lower urinary tract infection); for infections caused by Escherichia coli, the respective figures were 17 (100%) and 10 (71%). The responses in pyelonephritis were stronger (P < .001) and lasted longer than those in LUTI: 15 patients with pyelonephritis (74%) but only 1 with LUTI (6%) had > 100 ASC/10(6) cells. A similar difference was seen in the number of all immunoglobulin-secreting cells. The ASC assay offers a new means for assessing the human immune response in urinary tract infection and may be useful in localizing the infection. It might prove valuable in predicting harmful postinfection processes.

Increased in vitro intestinal permeability in suckling rats exposed to cow milk during lactation.

Specific mucosal barrier functions of the gut develop in the newborn to combat the constant challenge of foreign antigens. To determine whether exposure to cow milk antigens interferes with this maturational process, jejunal permeability to macromolecules and the activation of immune mechanisms were studied in preweaning rats. At the age of 14 days, rat pups were divided into three feeding groups. Controls (n = 18) remained on normal maternal milk; group CM (n = 27) additionally received a daily gavage feed of cow milk; and in group D (n = 23), cow milk was given to dams. At 21 days, when "gut closure" normally occurs, intestinal in vitro absorption of horseradish peroxidase in its intact form was significantly higher in group CM and in group D than in controls (F = 5.6; p = 0.006): group CM: mean, 37.8 ng/h/cm2; 95% confidence interval (CI), 19.4-73.6; group D: mean, 26.9 ng/h/cm2; 95% CI, 8.2-88.2; controls: mean, 4.0 ng/h/cm2; 95% CI, 1.2-13.9. In association with increased jejunal permeability, there was enhanced jejunal eosinophilic infiltration in group CM. In group D, the number of specific antibody-secreting cells in peripheral blood against beta-lactoglobulin was significantly higher than in group CM and controls. These data indicate that there is a critical period in development when feeding cow milk antigens delays gut closure. They further suggest that mucosal barrier function is impaired due to a local hypersensitivity reaction to cow milk antigens, irrespective of the protection of maternal milk or maternal antigen processing.

Immune Response to Prolonged Intestinal Exposure to Antigen

The influence of prolonged intestinal antigen exposure on the immune response in humans was investigated. Two groups of volunteers were vaccinated orally with live Salmonella typhi Ty21a given in enteric-coated capsules three (group 3 x E) or six (group 6 x E) at 2-day intervals. The immune response was followed for 1 month by enumerating the antibody-secreting cells (ASC) in the peripheral blood. Soon after vaccination specific ASC were observed in the blood of all volunteers. In group 3 x E, the response peaked on day 5 or 7 and had faded away by day 14. In group 6 x E, by contrast, there was no clear single peak of the response, and the ASC numbers were high in several consecutive measurements. On day 14, considerable numbers of ASC were still detected in all cases; the responses had mostly faded away by day 22. These results show that prolonged exposure to antigen in the intestine induces a prolonged response of specific ASC in peripheral blood. This is an important prerequisite for the application of the ASC assay to study mucosal infections, in which the actual time of onset is often unknown. In addition, the findings suggest that the ASC assay might offer new possibilities for assessing the persistence of microbial antigens in infections.

Comparison of the human immune response to live oral, killed oral or killed parenteral Salmonella typhi TY21A vaccines

The live oral typhoid vaccine Ty21a has proved to confer protection against the disease at least as effectively as killed parenteral vaccines, whereas killed oral vaccines have not been protective in field trials. This prompted us to compare the immune response of subjects vaccinated either with live oral, killed oral or killed parenteral Salmonella typhi Ty21a vaccine. The immune responses were studied by analysis of peripheral blood antibody-secreting cells (ASC), believed to reflect the mucosal immune response. Live and killed bacteria administered by the oral route elicited immune responses of similar specificity and Ig class profile (IgA dominating), but the response to the live vaccine was significantly stronger and lasted longer The administration route, on the other hand, influenced the antigenic specificity of the ASC response suggesting different processing of the antigen by the systemic and local immune systems. Thus, the response after oral vaccination was almost exclusively directed to the surface O-antigen, whereas after parenteral vaccination an equally strong response was seen to the O-antigen, to lipopolysaccharide core and to flagella.

Antibody-secreting peripheral blood lymphocytes induced by immunization with a conjugate consisting of Streptococcus pneumoniae type 12F polysaccharide and diphtheria toxoid

Healthy adult volunteers were injected either with one of two conjugates composed of Streptococcus pneumoniae type 12F polysaccharide (Pn12F) covalently coupled to diphtheria toxoid or with Pn12F alone (as a component of Pnu-Imune, a 23-valent pneumococcus vaccine). The conjugates induced Pn12F-specific antibody-secreting cells in peripheral blood with numbers and isotype distribution similar to those induced by Pnu-Imune, with immunoglobulin A (IgA) as the predominant isotype. The conjugates also elicited high numbers of diphtheria toxoid-specific antibody-secreting cells of the IgG class. There was no distinct booster effect, since a second dose of the conjugates induced antibody-secreting cells at significantly lower numbers than after the first dose. In contrast to the cell numbers, the conjugate vaccines induced higher increases of IgA1 Pn12F antibodies in serum than did Pnu-Imune. However, neither the conjugates nor Pnu-Imune induced a secretory antibody response. Antibody levels in serum and saliva correlated poorly with the frequency of antigen-specific antibody-secreting cells. Circulating antibody-secreting cells present 7 days postimmunization were probably not responsible for the high increase of antibodies in serum but rather represented a population of in vivo-activated B cells with the ability to disseminate the humoral response from the antigen recognition site to distant locations of antibody production.

Immunization of humans with polysaccharide vaccines induces systemic, predominantly polymeric IgA2-subclass antibody responses.

Ig class- and IgA subclass-specific immune responses to protein and polysaccharide Ag were studied in serum, external secretions, and at the single cell level in peripheral blood of systemically immunized adults. Immunization with tetanus toxoid induced predominantly IgG antibody responses in serum and in the PBMC. The IgA antibody response was low, and was mostly of the IgA1 subclass. In contrast, immunization with polysaccharide Ag (Haemophilus influenzae type b, Neisseria meningitidis serogroup A, C, Y, W-135, and Streptococcus pneumoniae capsular polysaccharides) elicited a major IgA response predominantly of the IgA2 isotype. Analysis of the molecular forms of secreted IgA antibodies indicated that polymers were produced early after immunization, irrespective of the nature of the Ag. When compared with serum antibody and to PBMC cell responses, systemic immunization with polysaccharides induced a minor salivary response dominated by IgG and IgM antibodies. In contrast, the presence of antipolysaccharide antibodies in bile, irrespective of their isotype, paralleled the serum response 14 days after the immunization with polysaccharide Ag. These results suggest that biliary Ig were mostly derived from serum. Different patterns of the expression of MHC class II Ag on T cells, B cells, and monocytes during the course of immunization with protein or polysaccharide Ag were observed: whereas protein Ag induced a high frequency of HLA-DP- and HLA-DR-expressing cells early in the course of immunization, polysaccharide vaccines elicited low and protracted increases of HLA-DP+ T cells. Polysaccharide vaccine covalently coupled to a protein carrier induced a higher frequency of antipolysaccharide antibody-secreting cells in peripheral blood and increased the IgG to IgA ratio among polysaccharide-specific antibody-secreting cells.