Specific and Cross-reactive Plasmablast Response in Humans after Primary and Secondary Immunization with Vi Capsular Polysaccharide Typhoid Vaccine.

Secondary immunization with polysaccharide vaccines may imply a risk of hyporesponsiveness. Despite the wide use of typhoid Vi capsular polysaccharide vaccine, its potential tendency to hyporesponsiveness has been inadequately addressed. While previous studies have explored serum antibody responses, we applied a more sensitive approach, a single-cell assay for circulating plasmablasts, to compare primary and secondary responses. Twelve subjects received primary and booster doses of the Vi vaccine (Typherix® ) at 30- to 37-month intervals. Plasmablasts specific to the Vi or typhoidal O antigens or cross-reactive with paratyphoid and non-typhoidal Salmonella strains were identified as antibody-secreting cells (ASC) with ELISPOT. Before vaccinations, none had plasmablasts specific to the antigens tested. Twelve of 12 subjects showed a Vi-specific response after primary, but only eight of 12 after booster vaccination. All responded to typhoidal O-9,12 antigen after both immunizations. The geometric mean of plasmablasts specific to the Vi antigen was 59 (95% CI 24-119) and 1 (0-54) IgA + IgG + IgM-ASC/106 peripheral blood mononuclear cell (PBMC) after primary and booster immunizations, respectively, and 20 (9-49) and 56 (29-103) to the O-9,12 antigen. We detected 1 (0-28) and 17 (6-36) ASC/106 PBMC cross-reactive with Salmonella Paratyphi A; 3 (0-30) and 22 (8-48) with S. Paratyphi B; 3 (0-29) and 18 (7-47) with S. Paratyphi C; 19 (10-34) and 51 (26-94) with Salmonella Enteritidis; and 1 (0-35) and 23 (9-52) with Salmonella Typhimurium, respectively. One-third of the vaccinees, although responding to the O-9,12 antigen, failed to respond to the Vi antigen after booster immunization, suggesting hyporesponsiveness in part of the vaccinees. The findings warrant further investigation.

Cross-reactive immune response induced by the Vi capsular polysaccharide typhoid vaccine against Salmonella Paratyphi strains.

There are no vaccines in clinical use against paratyphoid fever, caused by Salmonella Paratyphi A and B or, rarely, C. Oral Salmonella Typhi Ty21a typhoid vaccine elicits a significant cross-reactive immune response against S. Paratyphi A and B, and some reports suggest cross-protective efficacy against the disease. These findings are ascribed to the O-12 antigen shared between the strains. The Vi capsular polysaccharide vaccine has been shown to elicit antibodies reactive with O-9,12. Twenty-five volunteers immunized with the parenteral Vi vaccine (Typherix(®) ) were explored for plasmablasts cross-reactive with paratyphoid strains; the responses were compared to those in 25 age- and gender-matched volunteers immunized with Ty21a (Vivotif(®) ). Before vaccination, 48/50 vaccinees had no plasmablasts reactive with the antigens. Seven days after vaccination, 15/25 and 22/25 Vi- and Ty21a-vaccinated volunteers had circulating plasmablasts producing antibodies cross-reactive with S. Paratyphi A, 18/25 and 23/25 with S. Paratyphi B and 16/25 and 9/25 with Paratyphi C, respectively. Compared to the Ty21a group, the Vi group showed significantly lower responses to S. Paratyphi A and B and higher to S. Paratyphi C. To conclude, the Vi vaccine elicited a cross-reactive plasmablast response to S. Paratyphi C (Vi antigen in common) and less marked responses to S. Paratyphi A and B than the Ty21a preparation. S. Paratyphi A and B both being Vi-negative, the result can be explained by trace amounts of bacterial cell wall O-12 antigen in the Vi preparation, despite purification. The clinical significance of this finding remains to be determined.

Humoral immune response to keyhole limpet haemocyanin, the protein carrier in cancer vaccines.

Keyhole limpet haemocyanin (KLH) appears to be a promising protein carrier for tumor antigens in numerous cancer vaccine candidates. The humoral immune response to KLH was characterized at the single-cell level with ELISPOT combined with separations of cell populations according to their expression of homing receptors (HRs). The analysis of HR expressions is expected to reveal the targeting of the immune response in the body. Eight orally primed and four nonprimed volunteers received KLH-vaccine subcutaneously. Circulating KLH-specific plasmablasts were found in all volunteers, 60 KLH-specific plasmablasts/10(6) PBMC in the nonprimed and 136/10(6) in the primed group. The proportion of L-selectin(+) plasmablasts proved high and integrin α(4)ß(7) (+) low. KLH serving as protein carrier in several vaccines, the homing profile of KLH-specific response may be applicable to the cancer antigen parts in the same vaccines. The present data reflect a systemic homing profile, which appears advantageous for the targeting of immune response to cancer vaccines.

The impact of early viral infections and graft-versus-host disease on immune reconstitution following paediatric stem cell transplantation.

Viral infections and graft-versus-host disease (GVHD) render an impact on both the clinical and immunological recovery following allogeneic hematopoietic stem cell transplantation (HSCT). We studied the recuperation of the immune defence after transplant in the paediatric setting and assessed the impact of early (<100 days post-HSCT) viral [cytomegalovirus (CMV), Ebstein-Barr virus (EBV) and adenovirus] reactivations/infections and GVHD. Fifty-one paediatric recipients of HSCT were enrolled. T cell recovery was evaluated on lymphocyte subpopulations using flow cytometry and functionally by measuring T cell excision circles (TRECs) and through the analysis of T lymphocyte responses to mitogens. B cell recovery was studied by flow cytometry and functionally by ELISPOT. Acute and mild chronic GVHD allowed for a brisk recovery of both cellular and humoral immunity while moderate to severe chronic graft-versus-host disease (cGVHD) associated with a significant, tampering effect on the immunological recovery after transplant. In the former group, the early viral reactivations/infections seemingly linked with a delayed recovery of T lymphocytes and low TRECs values. Moderate to severe cGVHD appears to associate with an impaired immunological recovery after HSCT. Early viral infections linked with prolonged T cell immunodeficiency and thymic dysfunction may be indicative of the presence of subclinical GVHD.

Interaction of orally administered Lactobacillus rhamnosus GG with skin and gut microbiota and humoral immunity in infants with atopic dermatitis.

BACKGROUND: The intestinal mucosa functions as a defence barrier against gut intraluminar antigens. The maturational events in the gut parallel its step-wise colonization. Atopic dermatitis (AD) is associated with aberrant barrier functions of the skin epithelium and, in a subgroup of patients, of the gut mucosa. OBJECTIVE: To investigate the interaction of Lactobacillus rhamnosus GG (LGG) with skin and gut microbiota and humoral immunity in infants with AD. METHODS: Thirty-nine infants with AD were randomized for a 3-month period in a double-blind design to receive extensively hydrolysed casein formula supplemented with (n=19) or without (n=20) LGG (ATCC 53103) 5.0 × 107 CFU/g to achieve a daily intake of 3.4 × 109 CFU. Sampling (blood and fecal samples, cotton swab from the skin) was carried out at entry, 1 and 3 months thereafter. Ig-secreting cells were determined by enzyme-linked immunospot and the proportions of CD19(+)CD27(+) B cells among peripheral blood leucocytes by flow cytometry. The major groups of gut and skin bacteria were characterized using PCR. RESULTS: The proportions of IgA- and IgM-secreting cells decreased significantly in the treated group; the baseline-adjusted ratios for treated vs. untreated at 1 month were 0.59 (95%CI 0.36-0.99, P=0.044) for IgA- and 0.53 (95%CI 0.29-0.96, P=0.036) for IgM-secreting cells. The proportions of CD19(+)CD27(+) B cells increased in the probiotic-treated infants but not in the untreated. There were no significant differences in bifidobacterial species composition of the gut between the study groups. On the skin, the bacterial counts of Bifidobacterium genus vs. Clostridium coccoides in the treated and untreated infants were similar. CONCLUSION AND CLINICAL RELEVANCE: Specific probiotics may enhance gut barrier function and aid in the development of immune responses. Thus, specific probiotics may afford protection against offending macromolecules in the gut and provide control for future infections by accelerated immunological maturation (ClinicalTrials.gov ID NCT01148667).

Decreased numbers of circulating plasmablasts and differences in IgA1-plasmablast homing to skin in coeliac disease and dermatitis herpetiformis.

The two clinical phenotypes of gluten enteropathy, coeliac disease (CD) and dermatitis herpetiformis (DH), were characterized for numbers and homing profiles of circulating final effector B cells, plasmablasts, identified as immunoglobulin (Ig)-secreting cells (ISC). In CD, the numbers of ISC were approximately 50% lower than in DH or controls. ISC expressed peripheral lymph node homing receptor (HR), L-selectin, less frequently in CD (54%) and DH (52%) patients than in controls (70%). The expression of gut mucosal HR, alpha(4)beta(7), was less frequent in CD (42%) than in DH (65%) or controls (60%). In DH, but not in CD or controls, a higher proportion of IgA1-ISC (40%) than IgA2-ISC (25%) expressed the skin HR, cutaneous lymphocyte-associated antigen. In gluten enteropathy circulating plasmablasts are more mature, but decreased in number, and have distorted homing profiles. Differential IgA1-plasmablast homing could be associated with the development of skin rash with IgA1-deposits in DH but not in CD.

Effects of continuous exposure to stromal cell-derived factor-1 alpha on T cell rolling and tight adhesion to monolayers of activated endothelial cells.

Immobilized stromal cell-derived factor-1 alpha (SDF-1 alpha) has been shown to induce tight adhesion of T cells to purified ICAM-1 in assays done under flow conditions. In this study, we show that soluble SDF-1 alpha induced a rapid (within 20 s) cessation of rolling and tight adhesion of >90% of the rolling T cells on monolayers of activated endothelial cells under similar flow. Within 4 min, the T cells had either started to migrate between the endothelial cells or re-entered the rolling and circulating lymphocyte pool. This deadherence of the firmly bound cells, with either ensuing transmigration or continued rolling, was most likely due to desensitization of lymphocytes to the continuously present SDF-1 alpha. The released rolling lymphocytes could still respond to other activating signals by a second round of tight adhesion. Pretreating the lymphocytes with pertussis toxin almost completely blocked the effect of the chemokine, confirming that the induction of firm adhesion was due to the function of the chemokine on the lymphocytes and not the endothelial cells. Pretreating the endothelium with SDF-1 alpha did not lead to firm adhesion of subsequently added lymphocytes, also indicating that the effect was due to soluble, not endothelially bound, chemokine. Blocking experiments showed that the same molecules mediated rolling before and after SDF-1 alpha-induced tight adhesion. This is the first study to demonstrate the effect of soluble SDF-1 alpha on T cell rolling on an endothelial cell monolayer. The data broaden our understanding of the stimulatory factors directing the firm adhesion and ensuing transmigration of leukocytes into tissues through activated endothelium.

Isolation of bovine neutrophils with biomagnetic beads: comparison with standard Percoll density gradient isolation methods.

A prerequisite for studies on bovine neutrophils is a reliable method of neutrophil isolation from blood to obtain highly purified cell populations that are functionally active. Since current techniques of neutrophil isolation fall short of these requirements, we have developed a newer and more effective technique for isolation of bovine neutrophils that utilizes biomagnetic beads coated with a monoclonal antibody that recognizes an abundant surface antigen on bovine neutrophils to purify these cells. Comparison of the purity and viability of bovine neutrophils isolated by a conventional method (continuous Percoll density gradient) with this new method showed that neutrophils isolated with biomagnetic beads were higher in purity and had an increased yield. In addition, cells isolated with biomagnetic beads demonstrated normal or even improved function in assays of chemotaxis, phagocytosis, degranulation, and respiratory burst activity. Finally, bovine neutrophils isolated using this method showed an overall lower level of spontaneous apoptosis, which correlates well with the high level of viability observed in the purified cell preparations. Thus, this method represents a significant advance over current methods for isolating bovine neutrophils and would be widely applicable to labs studying the biochemistry and signal transduction pathways in these cells.

Jejuna of patients with insulin-dependent diabetes mellitus (IDDM) show signs of immune activation.

The roles of enteric viruses and food antigens as possible triggers in human insulin-dependent diabetes mellitus and the evidence that mucosal-associated homing receptors are important in both human and experimental diabetes prompted us to undertake an immunohistochemical study of intestinal specimens from patients with IDDM. We studied jejunal morphology and immunohistochemistry in 26 patients with IDDM, 13 of whom had the HLA-DQB1*0201 gene and therefore a higher risk of coeliac disease. The findings were compared with those in specimens from age-matched controls. Villous structure and the density of the intraepithelial lymphocytes were normal in every biopsy specimen. The extent of positivity with anti-DR and -DP antibodies in the villous epithelium was significantly greater in the specimens from patients than in those from controls (P = 0.0002 in both comparisons). The crypts were also more positive: for DR P = 0.0001, and for DP P = 0.002. The densities of T cells, CD4+, CD8+, and T cell receptor alpha/beta+ and gamma/delta+ cells in the epithelium and lamina propria were similar in patients and controls, but the patients had significantly more alpha 4/beta 7 integrin+ cells in the lamina propria (P = 0.006). No difference was seen between HLA-DQB1*0201-positive and -negative patients. These findings reflect a stage of inflammation in the structurally normal intestines of patients with IDDM and suggest secretion of inflammatory Th1-type cytokines in the intestine.

Homing potentials of circulating antibody-secreting cells after administration of oral or parenteral protein or polysaccharide vaccine in humans.

The site of antigen encounter influences the Ig-distribution and homing potentials of circulating antibody-secreting cells (ASC) induced. After oral antigen administration, the majority ASC secrete the mucosal Ig-isotype, IgA, and all of them express the gut homing receptor (HR), alpha 4 beta 7, thus implying mucosal homing of these cells. Parenteral protein vaccine induces an IgG-dominated response with a low proportion of alpha 4 beta 7 expressing cells. However, a polysaccharide vaccine, even if administered parenterally, elicits an IgA-dominated response, hence suggesting homing to the mucosa. In order to study the influence of the nature of the antigen on the targeting of the ASC response, the present work compares the homing potentials of circulating ASC in humans after administration of an oral Salmonella Typhi Ty21a vaccine (antigen studied: O-9,12 polysaccharide), an oral recombinant cholera vaccine (antigen studied: cholera toxin B-subunit, CTB protein), a parenteral pneumococcal vaccine (antigen studied: Pnc capsular polysaccharide 19F) or a parenteral tetanus toxoid vaccine (antigen studied: TT protein). alpha 4 beta 7 was expressed on a higher proportion of ASC induced by oral O-9,12 (99%) and CTB (99%) than by parenteral Pnc (70%) or TT (63%). L-selectin, the peripheral lymph node HR, was expressed on a smaller proportion of ASC induced by O-9,12 (37%) or CTB (43%) than of those induced by Pnc (78%) or TT (81%). The results imply that even if the nature of the antigen has a profound effect on the Ig-distribution of the ASC response, it does not seem to influence the targeting of the response.

Enteric infections in an endemic area induce a circulating antibody-secreting cell response with homing potentials to both mucosal and systemic tissues.

Enteric infections induce a response of circulating pathogen-specific antibody-secreting cells (ASC). The expression of homing receptors (HRs) on these cells was studied in patients with diarrhea caused by Vibrio cholerae in Bangladesh, an area in which cholera is endemic. The gut HR, alpha4beta7, was expressed by approximately 80% of the ASC, indicating mucosal homing of these cells. However, the peripheral lymph node HR, L-selectin, was also expressed by approximately 80% of the ASC specific to either cholera toxin or O antigen. In earlier findings after oral immunization in nonendemic areas, alpha4beta7 has been expressed by approximately 100% and L-selectin by approximately 50% of the ASC. In comparison, the present data speak for a more systemic targeting of the immune response associated with long-lasting immunity in an endemic area. The results thus provide insight for the continued development and evaluation of vaccines.

Glutamate decarboxylase-reactive peripheral blood lymphocytes from patients with IDDM express gut-specific homing receptor alpha4beta7-integrin.

Migration of lymphocytes to the pancreas is a prerequisite for insulitis in IDDM. Mucosal vascular addressin (MAdCAM-1), involved in the recirculation of lymphocytes to the gut, has been found in the inflamed islets in NOD mice. In humans, triggers of the gut immune system (e.g., early exposure to cow's milk proteins in infancy, exposure to enteroviral infections) have been associated with IDDM. To study the possible link between the gut immune system and IDDM, we tested the expression of the alpha4beta7-integrin, a homing receptor for MAdCAM-1, on GAD65-reactive lymphocytes. Using immunomagnetic cell sorting, we depleted the lymphocytes with high expression of alpha4beta7-integrin in the peripheral blood mononuclear cell population from IDDM patients and patients with autoimmune polyendocrine disease type 1 (APD-I). The depletion led to a marked decrease (mean 70%) in the cellular response against GAD65 in three of six IDDM patients and in one subject at high risk for IDDM. A decrease of 37% in the GAD response was observed after depletion in the case of one APD-I patient who also had IDDM. Cellular response to tetanus toxoid increased in the majority of patients as well as in three control subjects studied. We demonstrated that a remarkable population of islet cell antigen-reactive lymphocytes express the gut-specific homing receptor, which emphasizes the role of gut immunity in IDDM. The manipulation of the gut immune system is therefore proposed as a tool for modulation of the autoimmunity against pancreatic beta-cells in IDDM.

Homing potentials of circulating lymphocytes in humans depend on the site of activation: oral, but not parenteral, typhoid vaccination induces circulating antibody-secreting cells that all bear homing receptors directing them to the gut.

Specific Ab-secreting cells (ASC) appear in the human blood as a response to oral and parenteral vaccination. The actual contribution of these cells to the defense of the body depends on their final effector site. The homing potentials of mucosally and parenterally induced ASC were compared by examining the homing receptor (HR) expression of circulating specific ASC in the blood of volunteers vaccinated orally or parenterally with the same Ag, Salmonella typhi Ty21a. Circulating lymphocytes were separated into receptor-positive and -negative populations, and the numbers of specific ASC were assayed. The alpha4 beta7 integrin, which acts as a gut HR, was expressed on all (99%) of the mucosally activated ASC, but on only 58% of the parenterally induced ASC or 58% of all Ig-secreting cells of the unvaccinated controls. L-selectin, the peripheral lymph node HR, showed an inverse distribution; it was found on 42% of mucosally activated ASC and on 86% of parenterally induced ASC. These results reveal that all of the circulating ASC after oral vaccination are committed to migrate to the mucosal compartment of the immune system, strongly arguing for a recirculation of activated mucosal cells in humans. By contrast, ASC induced by parenteral vaccination with the same Ag are mostly directed to the systemic compartment, yet a part of them has mucosal homing attitudes as well. These differences indicate that the site of Ag encounter determines the homing potential of the cell.

Circulating immunoglobulin-secreting cells are heterogeneous in their expression of maturation markers and homing receptors.

Immunoglobulin-secreting cells (ISC) in the peripheral blood are active effectors of the human immune defence system on their way to their site of action. We combined immunomagnetic cell separation and ELISPOT to study the expression of maturation markers and homing receptors (HR) on these cells in healthy volunteers. The results revealed that although highly differentiated, peripheral blood ISC are remarkably heterogeneous both with respect to their expression of maturation markers and HR. Moreover, significant differences were demonstrated between the various isotypes. Fewer IgA-secreting cells expressed both markers of early maturation (HLA-DR, HLA-DQ, CD20, and CD21) and of more mature B cells or plasma cells (CD28, CD38, and alpha-syndecan) compared with IgG- and IgM-secreting cells. IgA-secreting cells also showed the lowest proportion of cells positive for the peripheral lymph node HR, L-selectin, or the skin HR, cutaneous lymphocyte antigen (CLA). By contrast, the expression of mucosal HR on IgA-secreting cells did not reveal a more pronounced homing attitude to mucosal tissues than IgG- or IgM-secreting cells. We conclude that peripheral blood ISC are a heterogeneous cell population and that IgA-secreting cells seem to differ from the other isotypes both in respect of expression of HR and the various maturational markers studied.

Mucosally activated circulating human B cells in diarrhea express homing receptors directing them back to the gut.

BACKGROUND & AIMS: Recirculation of mucosal lymphocytes has been established in animals but not in humans. Specific antibody-secreting cells in the blood of patients with diarrhea, initially activated in gut mucosa, are potential recirculating cells. The aim of this study was to determine whether these cells circulate back to gut by analyzing their homing receptors. METHODS: Blood mononuclear cells, separated with immunomagnetic cell sorting into receptor-positive and receptor-negative populations, were assayed for pathogen-specific antibody-secreting cells and all immunoglobulin-secreting cells using enzyme-linked immunospot assay. RESULTS: The gut mucosa homing receptor alpha4beta7 was expressed more frequently on pathogen-specific antibody-secreting cells than on immunoglobulin-secreting cells of healthy controls (P<0.001). Conversely, L-selectin, a homing receptor for peripheral lymph nodes, was found on remarkably fewer antibody-secreting cells of the patients compared with immunoglobulin-secreting cells of controls (32.9% and 70.3%, respectively; P<0.001). Three to 6 months after the disease, specific antibody-secreting cells had disappeared and frequency of L-selectin-and alpha4beta7-expressing cells had returned to control levels. CONCLUSIONS: Circulating mucosally activated antibody-secreting cells express a set of homing receptors guiding them back to the gut. This provides evidence for recirculation of mucosal lymphocytes in humans.

Active immunity is seen as a reduction in the cell response to oral live vaccine.

Oral immunization elicits a response of antibody-secreting cells (ASC) in the peripheral blood; these cells are believed to originate in the mucosa and hence give information on the mucosal immune response. We have shown earlier that oral booster immunization is followed by an elevated ASC response reflecting an immunologic memory. In the present study we show that a booster dose of a live bacterial vaccine given at a time of active mucosal immunity elicits a low ASC response only. This is probably because the multiplication of the live vaccine is inhibited in the gut, resulting in a low actual dose of the antigen. This situation may be an example of the protective immunity manifested when an orally immunized person encounters the pathogen in nature, and could be used to assess the protective immunity.

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.