Plasmablast Response Research Articles

Differential regulation of B cell responses to acute and chronic infection by histone-modifying complexes

Abstract Molecular regulation of B cell differentiation is critical for formation of immune memory and becomes dysregulated in B cell lymphomas. Alterations in the genomic landscape that allow or restrict access of transcription factors are critical to enact gene expression profiles that in turn drive cell differentiation. Chromatin accessibility can be modulated by enzymes that modify the N-terminal tail of histones. The Trithorax group protein MLL1 forms histone-modifying complexes to permit transcription, whereas the two Polycomb repressor complexes (PRC) work together to repress gene expression. The dynamic regulation of these complexes can modulate B cell fate and function during immune responses. Yet, little is known about the temporal and spatial requirements of MLL1, canonical PRC1 and PRC2 during acute and chronic immune responses. Deletion of the PRC2 member EED (Eedf/fCd23Cre/+ mice) resulted in a complete absence of GC and B cell memory following immunization or influenza infection. Mll1f/fCd23Cre/+ mice also could not mount an effective GC response to immunization, yet their plasmablast response remained intact. In contrast, the canonical PRC1 member BMI-1 was required for plasmablast but not GC responses. Surprisingly, the requirement for EED and MLL1 in a chronic persisting infection differed from acute responses. In contrast to acute responses, Eedf/fCd23Cre/+ mice infected with LCMV-Docile or P. chabaudi had normal GC formation, whereas Mll1f/fCd23Cre/+ mice formed GCs to the acute LCMV strain but not in chronic infection. Together, our data demonstrate the differential requirements of these complexes in B cell fate decisions, as well as suggest epigenetic regulatory checks are subverted in persisting infections.

Suppressed plasmablast responses in febrile infants, including children with Kawasaki disease

BackgroundKawasaki disease (KD), the leading cause of acquired heart disease in children, primarily affects infants and toddlers. Investigations on immune responses during KD are hampered by a limited understanding of normal immune responses in these ages. It’s well known that Infants have poorer vaccine responses and difficulty with maintaining prolonged serum immunity, but there are few studies on human infants detailing immune deficiencies. Limited studies propose an inability to maintain life-long bone marrow plasma cells. Plasmablasts are a transitional cell form of B cells that lead to long-term Plasma cells. Plasmablasts levels rise in the peripheral blood after exposure to a foreign antigen. In adult studies, these responses are both temporally and functionally well characterized. To date, there have been few studies on plasmablasts in the predominant age range of KD.MethodsChildren presenting to an urban pediatric emergency room undergoing laboratory evaluation, who had concern of KD or had fever and symptoms overlapping those of KD, were recruited. Peripheral blood mononuclear cells were isolated and evaluated utilizing flow cytometry with specific B cell markers from 18 KD subjects and 69 febrile controls.ResultsPlasmablast numbers and temporal formation are similar between infectious disease controls and KD subjects. In both groups, infants have diminished plasmablast responses compared to older children.ConclusionIn this single-time point survey, infants have a blunted peripheral plasmablast response. Overall, similar plasmablast responses in KD and controls support an infectious disease relationship to KD. Future time-course studies of plasmablasts in infants are warranted as this phenomenon may contribute to observed immune responses in this age group.

Plasmablast antibody repertoires in elderly influenza vaccine responders exhibit restricted diversity but increased breadth of binding across influenza strains

Seasonal influenza vaccines elicit antibody responses that can prevent infection, but their efficacy is reduced in the elderly. While a subset of elderly individuals can still mount sufficient vaccine-induced antibody responses, little is known about the properties of the vaccine-induced antibody repertoires in elderly as compared to young responders. To gain insights into the effects of aging on influenza vaccine-induced antibody responses, we used flow cytometry and a cell-barcoding method to sequence antibody heavy and light chain gene pairs expressed by individual blood plasmablasts generated in response to influenza vaccination in elderly (aged 70–89) and young (aged 20–29) responders. We found similar blood plasmablast levels in the elderly and young responders seven days post vaccination. Informatics analysis revealed increased clonality, but similar heavy chain V(D)J gene usage in the elderly as compared to young vaccine responders. Although the elderly responders exhibited decreased antibody sequence diversity and fewer consequential mutations relative to young responders, recombinant antibodies from elderly responders bound a broader range of influenza strain HAs. Thus elderly influenza vaccine responders mount plasmablast responses with restricted diversity but with an increased breadth of binding across influenza strains. Our results suggest that the ability to generate plasmablast responses encoding cross-strain binding antibodies likely represents a mechanism important to vaccine responses in the elderly.

AS03- and MF59-Adjuvanted Influenza Vaccines in Children.

Influenza is a major cause of respiratory disease leading to hospitalization in young children. However, seasonal trivalent influenza vaccines (TIVs) have been shown to be ineffective and poorly immunogenic in this population. The development of live-attenuated influenza vaccines and adjuvanted vaccines are important advances in the prevention of influenza in young children. The oil-in-water emulsions MF59 and adjuvant systems 03 (AS03) have been used as adjuvants in both seasonal adjuvanted trivalent influenza vaccines (ATIVs) and pandemic monovalent influenza vaccines. Compared with non-adjuvanted vaccine responses, these vaccines induce a more robust and persistent antibody response for both homologous and heterologous influenza strains in infants and young children. Evidence of a significant improvement in vaccine efficacy with these adjuvanted vaccines resulted in the use of the monovalent (A/H1N1) AS03-adjuvanted vaccine in children in the 2009 influenza pandemic and the licensure of the seasonal MF59 ATIV for children aged 6 months to 2 years in Canada. The mechanism of action of MF59 and AS03 remains unclear. Adjuvants such as MF59 induce proinflammatory cytokines and chemokines, including CXCL10, but independently of type-1 interferon. This proinflammatory response is associated with improved recruitment, activation and maturation of antigen presenting cells at the injection site. In young children MF59 ATIV produced more homogenous and robust transcriptional responses, more similar to adult-like patterns, than did TIV. Early gene signatures characteristic of the innate immune response, which correlated with antibody titers were also identified. Differences were detected when comparing child and adult responses including opposite trends in gene set enrichment at day 3 postvaccination and, unlike adult data, a lack of correlation between magnitude of plasmablast response at day 7 and antibody titers at day 28 in children. These insights show the utility of novel approaches in understanding new adjuvants and their importance for developing improved influenza vaccines for children.

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.

Plasmablasts With a Mucosal Phenotype Contribute to Plasmacytosis in Systemic Lupus Erythematosus.

To analyze the composition of known plasmacytosis in systemic lupus erythematosus (SLE) to obtain further insight into the nature of underlying mechanisms. Plasmablasts from patients with active SLE, patients with inactive/treated SLE, and healthy controls were characterized by flow cytometry, enzyme-linked immunospot assay, and Transwell migration assays and compared to vaccination-induced plasmablasts. Serum cytokine levels were analyzed by Luminex assay, and histologic analysis of kidney biopsy specimens was performed. Circulating plasmablasts in SLE expressed markers of mucosal immune reactions. IgA, CCR10, and β7 integrin were expressed by 48%, 40%, and 38% of plasmablasts, respectively, with varying coexpression patterns. Consistent with mucosal homing, some SLE plasmablasts migrated toward the mucosal chemokine CCL28 and secreted polymeric IgA. SLE plasmablasts shared phenotypic characteristics with antigen-specific plasmablasts induced by oral, but not parenteral, vaccinations. Autoreactive antibody-secreting cells of the IgG and IgA isotypes were detectable, but only the emergence of phenotypically mucosal plasmablasts was positively associated with serum interleukin-2 and platelet-derived growth factor BB levels. Our data suggest that distinct plasmablast differentiation pathways jointly contribute to peripheral plasmacytosis in SLE, i.e., a cytokine-amplified mucosal "steady-state" plasmablast response, and an autoreactive plasmablast response, representing conventional autoimmunity. Our results indicate an overly activated mucosal immune system in patients with SLE, with both immunologic and clinical implications.

B Cell-Extrinsic Myd88 and Fcer1g Negatively Regulate Autoreactive and Normal B Cell Immune Responses.

MyD88 and FcR common γ-chain (Fcer1g, FcRγ) elicit proinflammatory responses to exogenous Ags. Deletion of these receptors in autoimmune models has generally led to reduced overall disease. In B cells, Myd88 is required for anti-DNA and anti-RNA autoantibody responses, whereas Fcer1g is not expressed in these cells. The roles of these receptors in myeloid cells during B cell autoimmune activation remain less clear. To investigate the roles of Myd88 and Fcer1g in non-B cells, we transferred anti-self-IgG (rheumatoid factor) B cells and their physiologic target Ag, anti-chromatin Ab, into mice lacking Fcer1g, Myd88, or both and studied the extrafollicular plasmablast response. Surprisingly, we found a markedly higher and more prolonged response in the absence of either molecule; this effect was accentuated in doubly deficient recipients, with a 40-fold increase compared with wild-type recipients at day 10. This enhancement was dependent on CD40L, indicating that Myd88 and FcRγ, presumably on myeloid APCs, were required to downregulate T cell help for the extrafollicular response. To extend the generality, we then investigated a classic T cell-dependent response to (4-hydroxy-3-nitrophenyl)acetyl conjugated to chicken γ globulin and found a similar effect. Thus, these results reveal novel regulatory roles in the B cell response for receptors that are typically proinflammatory.

T Follicular Helper Cell-Derived IL-4 Is Required for IgE Production during Intestinal Helminth Infection.

IgE production plays a crucial role in protective as well as pathogenic type 2 immune responses. Although the cytokine IL-4 is required for the development of IgE-producing plasma cells, the source of IL-4 and cellular requirements for optimal IgE responses remain unclear. Recent evidence suggests that T follicular helper (Tfh) cells are the primary producer of IL-4 in the reactive lymph node during type 2 immune responses. As Tfh cells are also required for the development of plasmablasts derived from germinal center and extrafollicular sources, we hypothesized that this cell subset is essential for the IgE plasmablast response. In this study, we show that during intestinal helminth infection, IL-4 derived from Tfh cells is required for IgE class switching and plasmablast formation. Notably, early IgE class switching did not require germinal center formation. Additionally, Tfh cell-derived IL-4 was required to maintain the Th2 response in the mesenteric lymph nodes of infected mice. Collectively, our results indicate that IL-4-producing Tfh cells are central orchestrators of the type 2 immune response in the reactive lymph nodes during parasitic helminth infection.

Robust memory responses against influenza vaccination in pemphigus patients previously treated with rituximab.

Rituximab is a therapeutic anti-CD20 monoclonal antibody widely used to treat B cell lymphoma and autoimmune diseases, such as rheumatic arthritis, systemic lupus erythematosus, and autoimmune blistering skin diseases (AIBD). While rituximab fully depletes peripheral blood B cells, it remains unclear whether some preexisting B cell memory to pathogens or vaccines may survive depletion, especially in lymphoid tissues, and if these memory B cells can undergo homeostatic expansion during recovery from depletion. The limited data available on vaccine efficacy in this setting have been derived from rituximab-treated patients receiving concomitant chemotherapy or other potent immunosuppressants. Here, we present an in-depth analysis of seasonal influenza vaccine responses in AIBD patients previously treated with rituximab, who generally did not receive additional therapeutic interventions. We found that, despite a lack of influenza-specific memory B cells in the blood, patients mount robust recall responses to vaccination, comparable to healthy controls, both at a cellular and a serological level. Repertoire analyses of plasmablast responses suggest that they likely derive from a diverse pool of tissue-resident memory cells, refractory to depletion. Overall, these data have important implications for establishing an effective vaccine schedule for AIBD patients and the clinical care of rituximab-treated patients in general and contribute to our basic understanding of maintenance of normal and pathogenic human B cell memory.

Plasmablast Response to Primary Rhesus Cytomegalovirus (CMV) Infection in a Monkey Model of Congenital CMV Transmission.

Human cytomegalovirus (HCMV) is the most common congenital infection worldwide and the leading infectious cause of neurologic deficits and hearing loss in newborns. Development of a maternal HCMV vaccine to prevent vertical virus transmission is a high priority, yet protective maternal immune responses following acute infection are poorly understood. To characterize the maternal humoral immune response to primary CMV infection, we investigated the plasmablast and early antibody repertoire using a nonhuman primate model with two acutely rhesus CMV (RhCMV)-infected animals-a CD4+ T cell-depleted dam that experienced fetal loss shortly after vertical RhCMV transmission and an immunocompetent dam that did not transmit RhCMV to her infant. Compared to the CD4+ T cell-depleted dam that experienced fetal loss, the immunocompetent, nontransmitting dam had a more rapid and robust plasmablast response that produced a high proportion of RhCMV-reactive antibodies, including the first identified monoclonal antibody specific for soluble and membrane-associated RhCMV envelope glycoprotein B (gB). Additionally, we noted that plasmablast RhCMV-specific antibodies had variable gene usage and maturation similar to those observed in a monkey chronically coinfected with simian immunodeficiency virus (SIV) and RhCMV. This study reveals characteristics of the early maternal RhCMV-specific humoral immune responses to primary RhCMV infection in rhesus monkeys and may contribute to a future understanding of what antibody responses should be targeted by a vaccine to eliminate congenital HCMV transmission. Furthermore, the identification of an RhCMV gB-specific monoclonal antibody underscores the possibility of modeling future HCMV vaccine strategies in this nonhuman primate model.

Massive plasmablast response elicited in the acute phase of hantavirus pulmonary syndrome.

Beside its key diagnostic value, the humoral immune response is thought to play a protective role in hantavirus pulmonary syndrome. However, little is known about the cell source of these antibodies during ongoing human infection. Herein we characterized B-cell subsets circulating in Andes-virus-infected patients. A notable potent plasmablast (PB) response that increased 100-fold over the baseline levels was observed around 1 week after the onset of symptoms. These PB present a CD3neg CD19low CD20neg CD38hi CD27hi CD138+/- IgA+/- surface phenotype together with the presence of cytoplasmic functional immunoglobulins. They are large lymphocytes (lymphoblasts) morphologically coincident with the 'immunoblast-like' cells that have been previously described during blood cytology examinations of hantavirus-infected patients. Immunoreactivity analysis of white blood cell lysates suggests that some circulating PB are virus-specific but we also observed a significant increase of reactivity against virus-unrelated antigens, which suggests a possible bystander effect by polyclonal B-cell activation. The presence of this large and transient PB response raises the question as to whether these cells might have a protective or pathological role during the ongoing hantavirus pulmonary syndrome and suggest their practical application as a diagnostic/prognostic biomarker.

Dendritic Cells Regulate Extrafollicular Autoreactive B Cells via T Cells Expressing Fas and Fas Ligand

The extrafollicular (EF) plasmablast response to self-antigens that contain Toll-like receptor (TLR) ligands is prominent in murine lupus models and some bacterial infections, but the inhibitors and activators involved have not been fully delineated. Here, we used two conventional dendritic cell (cDC) depletion systems to investigate the role of cDCs on a classical TLR-dependent autoreactive EF response elicited inrheumatoid-factor B cells by DNA-containing immune complexes. Contrary to our hypothesis, cDC depletion amplified rather than dampened the EF response in Fas-intact but not Fas-deficient mice. Further, we demonstrated that cDC-dependent regulation requires Fas and Fas ligand (FasL) expression by Tcells, but not Fas expression by B cells. Thus, cDCs activate FasL-expressing Tcells that regulate Fas-expressing extrafollicular helper T (Tefh) cells. These studies reveal a regulatory role for cDCs in Bcell plasmablast responses and provide a mechanistic explanation for the excess autoantibody production observed in Fas deficiency.

Peripheral VH4+plasmablasts demonstrate autoreactive B cell expansion toward brain antigens in early multiple sclerosis patients.

Plasmablasts are a highly differentiated, antibody secreting B cell subset whose prevalence correlates with disease activity in Multiple Sclerosis (MS). For most patients experiencing partial transverse myelitis (PTM), plasmablasts are elevated in the blood at the first clinical presentation of disease (known as a clinically isolated syndrome or CIS). In this study we found that many of these peripheral plasmablasts are autoreactive and recognize primarily gray matter targets in brain tissue. These plasmablasts express antibodies that over-utilize immunoglobulin heavy chain V-region subgroup 4 (VH4) genes, and the highly mutated VH4+ plasmablast antibodies recognize intracellular antigens of neurons and astrocytes. Most of the autoreactive, highly mutated VH4+ plasmablast antibodies recognize only a portion of cortical neurons, indicating that the response may be specific to neuronal subgroups or layers. Furthermore, CIS-PTM patients with this plasmablast response also exhibit modest reactivity toward neuroantigens in the plasma IgG antibody pool. Taken together, these data indicate that expanded VH4+ peripheral plasmablasts in early MS patients recognize brain gray matter antigens. Peripheral plasmablasts may be participating in the autoimmune response associated with MS, and provide an interesting avenue for investigating the expansion of autoreactive B cells at the time of the first documented clinical event.

Human isotype-dependent inhibitory antibody responses against Mycobacterium tuberculosis.

Accumulating evidence from experimental animal models suggests that antibodies play a protective role against tuberculosis (TB). However, little is known about the antibodies generated upon Mycobacterium tuberculosis (MTB) exposure in humans. Here, we performed a molecular and functional characterization of the human B‐cell response to MTB by generating recombinant monoclonal antibodies from single isolated B cells of untreated adult patients with acute pulmonary TB and from MTB‐exposed healthcare workers. The data suggest that the acute plasmablast response to MTB originates from reactivated memory B cells and indicates a mucosal origin. Through functional analyses, we identified MTB inhibitory antibodies against mycobacterial antigens including virulence factors that play important roles in host cell infection. The inhibitory activity of anti‐MTB antibodies was directly linked to their isotype. Monoclonal as well as purified serum IgA antibodies showed MTB blocking activity independently of Fc alpha receptor expression, whereas IgG antibodies promoted the host cell infection. Together, the data provide molecular insights into the human antibody response to MTB and may thereby facilitate the design of protective vaccination strategies.

Plasmablasts During Acute Dengue Infection Represent a Small Subset of a Broader Virus-specific Memory B Cell Pool

Dengue is endemic in tropical countries worldwide and the four dengue virus serotypes often co-circulate. Infection with one serotype results in high titers of cross-reactive antibodies produced by plasmablasts, protecting temporarily against all serotypes, but impairing protective immunity in subsequent infections. To understand the development of these plasmablasts, we analyzed virus-specific B cell properties in patients during acute disease and at convalescence. Plasmablasts were unrelated to classical memory cells expanding in the blood during early recovery. We propose that only a small subset of memory B cells is activated as plasmablasts during repeat infection and that plasmablast responses are not representative of the memory B cell repertoire after dengue infection.

Cell-Mediated Immunity Against Antigenically Drifted Influenza A(H3N2) Viruses in Children During a Vaccine Mismatch Season.

Emergence of antigenically drifted influenza A(H3N2) viruses resulted in reduced vaccine effectiveness in all age groups during the 2014-2015 influenza season. In children, inactivated influenza vaccine (IIV) elicited neutralizing antibodies (Abs) against drifted strains at significantly lower levels than against the vaccine strain. Little is known about the cross-reactivity of cell-mediated immunity against drifted strains in children. Children aged 3-17 years (n = 48) received IIV during the 2014-2015 influenza season. Peripheral blood mononuclear cells, collected before (on day 0) and after (on days 7 and 21) vaccination were evaluated for induction of cross-reactive plasmablasts, memory B cells, and cytokine-secreting CD4(+) and CD8(+) T cells against the vaccine and drifted A(H3N2) viruses by an enzyme-linked immunospot assay and flow cytometry. IIV increased frequencies of plasmablasts and memory B cells. The overall induction of the T-cell response was not significant. Both B-cell and T-cell responses showed significant cross-reactivity against A(H3N2) viruses. Age and preexisting immunity affected virus-specific plasmablast responses and fold-change of T-cell responses, respectively. The proportion of T-helper type 1-prone (ie, interferon γ- or tumor necrosis factor α-secreting) CD4(+) T cell responses also increased with age. In children aged 3-17 years, B- and T-cell responses following IIV receipt showed significant cross-reactivity against A(H3N2) viruses during a vaccine mismatch season.

High-dose influenza vaccine favors acute plasmablast responses rather than long-term cellular responses

High-dose (HD) influenza vaccine shows improved relative efficacy against influenza disease compared to standard-dose (SD) vaccine in individuals ⩾65years. This has been partially credited to superior serological responses, but a comprehensive understanding of cell-mediated immunity (CMI) of HD vaccine remains lacking. In the current study, a total of 105 participants were randomly administered HD or SD vaccine and were evaluated for serological responses. Subsets of the group (n=12–26 per group) were evaluated for B and T cell responses at days 0, 7, 14 and 28 post-vaccination by flow cytometry or ELISPOT assay. HD vaccine elicited significantly higher hemagglutination inhibition (HI) titers than SD vaccine at d28, but comparable titers at d365 post-vaccination. HD vaccine also elicited higher vaccine-specific plasmablast responses at d7 post-vaccination than SD vaccine. However, long-lived memory B cell induction, cytokine-secreting T cell responses and persistence of serological memory were comparable regardless of vaccine dose. More strategies other than increased Ag amount may be needed to improve CMI in older adults.Trial Registration: ClinicalTrials.gov NCT 01189123

B cells naturally induced during dengue virus infection release soluble CD27, the plasma level of which is associated with severe forms of pediatric dengue

The CD27 and CD38 antigens are highly expressed on the plasmablast surface, and a massive plasmablast response has been described for dengue virus infection. Soluble CD27 and CD38 forms (sCD27 and sCD38, respectively) increase after immune activation. Here, we show increased sCD27 release in cultures of purified polyclonally stimulated B cells. T and B cells isolated from children with dengue spontaneously produced higher levels of sCD27 but not sCD38, compared with healthy children (P=0.03), and sCD27 levels positively correlated with plasmablast frequency in the cultures (rho=0.58, P=0.01). Children with dengue had higher plasma levels of sCD27 and sCD38 than healthy children, which decreased during convalescence. Plasma sCD27 was higher in severe than with mild dengue, but the opposite was observed for sCD38. These findings support a potential new role for B cells in dengue pathogenesis, and sCD27 and sCD38 are novel biomarkers associated with clinical outcome during dengue virus infection.

A Single Human Papillomavirus Vaccine Dose Improves B Cell Memory in Previously Infected Subjects

Although licensed human papillomavirus (HPV) vaccines are most efficacious in persons never infected with HPV, they also reduce infection and disease in previously infected subjects, indicating natural immunity is not entirely protective against HPV re-infection. The aim of this exploratory study was to examine the B cell memory elicited by HPV infection and evaluate whether vaccination merely boosts antibody (Ab) levels in previously infected subjects or also improves the quality of B cell memory. Toward this end, the memory B cells (Bmem) of five unvaccinated, HPV-seropositive subjects were isolated and characterized, and subject recall responses to a single HPV vaccine dose were analyzed. Vaccination boosted Ab levels 24- to 930-fold (median 77-fold) and Bmem numbers 3- to 27-fold (median 6-fold). In addition, Abs cloned from naturally elicited Bmem were generally non-neutralizing, whereas all those isolated following vaccination were neutralizing. Moreover, Ab and plasmablast responses indicative of memory recall responses were only observed in two subjects. These results suggest HPV vaccination augments both the magnitude and quality of natural immunity and demonstrate that sexually active persons could also benefit from HPV vaccination. This study may have important public policy implications, especially for the older ‘catch-up’ group within the vaccine's target population.

Circulating Gut-Homing (α 4 β 7 + ) Plasmablast Responses against Shigella Surface Protein Antigens among Hospitalized Patients with Diarrhea

Developing countries are burdened with Shigella diarrhea. Understanding mucosal immune responses associated with natural Shigella infection is important to identify potential correlates of protection and, as such, to design effective vaccines. We performed a comparative analysis of circulating mucosal plasmablasts producing specific antibodies against highly conserved invasive plasmid antigens (IpaC, IpaD20, and IpaD120) and two recently identified surface protein antigens, pan-Shigella surface protein antigen 1 (PSSP1) and PSSP2, common to all virulent Shigella strains. We examined blood and stool specimens from 37 diarrheal patients admitted to the Infectious Diseases & Beliaghata General Hospital, Kolkata, India. The etiological agent of diarrhea was investigated in stool specimens by microbiological methods and real-time PCR. Gut-homing (α4β7 (+)) antibody-secreting cells (ASCs) were isolated from patient blood by means of combined magnetic cell sorting and two-color enzyme-linked immunosorbent spot (ELISPOT) assay. Overall, 57% (21 of 37) and 65% (24 of 37) of the patients were positive for Shigella infection by microbiological and real-time PCR assays, respectively. The frequency of α4β7 (+) IgG ASC responders against Ipas was higher than that observed against PSSP1 or PSSP2, regardless of the Shigella serotype isolated from these patients. Thus, α4β7 (+) ASC responses to Ipas may be considered an indirect marker of Shigella infection. The apparent weakness of ASC responses to PSSP1 is consistent with the lack of cross-protection induced by natural Shigella infection. The finding that ASC responses to IpaD develop in patients with recent-onset shigellosis indicates that such responses may not be protective or may wane too rapidly and/or be of insufficient magnitude.