Development Of Humoral Immune Response Research Articles

Dysfunctional mitochondria, disrupted levels of reactive oxygen species, and autophagy in B cells from common variable immunodeficiency patients.

Common Variable Immunodeficiency (CVID) patients are characterized by hypogammaglobulinemia and poor response to vaccination due to deficient generation of memory and antibody-secreting B cells. B lymphocytes are essential for the development of humoral immune responses, and mitochondrial function, hreactive oxygen species (ROS) production and autophagy are crucial for determining B-cell fate. However, the role of those basic cell functions in the differentiation of human B cells remains poorly investigated. We used flow cytometry to evaluate mitochondrial function, ROS production and autophagy processes in human naïve and memory B-cell subpopulations in unstimulated and stimulated PBMCs cultures. We aimed to determine whether any alterations in these processes could impact B-cell fate and contribute to the lack of B-cell differentiation observed in CVID patients. We described that naïve CD19+CD27- and memory CD19+CD27+ B cells subpopulations from healthy controls differ in terms of their dependence on these processes for their homeostasis, and demonstrated that different stimuli exert a preferential cell type dependent effect. The evaluation of mitochondrial function, ROS production and autophagy in naïve and memory B cells from CVID patients disclosed subpopulation specific alterations. Dysfunctional mitochondria and autophagy were more prominent in unstimulated CVID CD19+CD27- and CD19+CD27+ B cells than in their healthy counterparts. Although naïve CD19+CD27- B cells from CVID patients had higher basal ROS levels than controls, their ROS increase after stimulation was lower, suggesting a disruption in ROS homeostasis. On the other hand, memory CD19+CD27+ B cells from CVID patients had both lower ROS basal levels and a diminished ROS production after stimulation with anti-B cell receptor (BCR) and IL-21. The failure in ROS cell signalling could impair CVID naïve B cell activation and differentiation to memory B cells. Decreased levels of ROS in CVID memory CD19+CD27+ B cells, which negatively correlate with their in vitro cell death and autophagy, could be detrimental and lead to their previously demonstrated premature death. The final consequence would be the failure to generate a functional B cell compartment in CVID patients.

Hemophilia A Research Program (HARP): A Novel Study Design for a Multicenter and Decentralized Prospective Observational Longitudinal Cohort Study in Hemophilia A

Background: Hemophilia A is a rare X-linked bleeding disorder caused by factor VIII (FVIII) deficiency. There is a compelling need in hemophilia to better understand the causes of bleeding in females and the factors underlying FVIII inhibitor development. To meet this need we are establishing a new NHLBI funded Hemophilia A Analytical Cohort Research Program (HARP) as part of a public-private partnership to support an Intergenerational Precision Medicine Program for the study of factor VIII (FVIII) immunogenicity in severe hemophilia A (Harpf8.org). The HARP objectives are: 1) Longitudinal assessment of bleeding in females with a severe hemophilia A genotype beginning in pregnancy; 2) Longitudinal assessment of anti-FVIII antibody formation in severe hemophilia A from birth through at least the first 2 years of life; 3) Studies of causes of bleeding in females with a severe hemophilia A genotype; 4) Studies of the immune response to FVIII in severe hemophilia A; 5) Development of a well-curated annotated data set of genetic, hemophilia, immune, environmental, psychosocial, and quality of life metrics; 6) Development of a multi-omics and immunologic biospecimen resource; and 7) Development of the hemophilia analytical Portal (F8portal.org). Study design: The HARP is a multicenter and decentralized prospective observational longitudinal cohort study beginning in pregnancy. It will follow 50 mother-baby pairs where the babies have severe hemophilia A and the mothers have a severe hemophilia A genotype. The private Clinical Consortium (CC) will oversee the study throughout the U.S., including sites that care for the mothers (up to 200 sites) and sites that care for the child (up to 70 sites). The CC will also conduct decentralized aspects of the study including collection of patient reported outcomes and local sample collection. The primary maternal endpoint is primary postpartum hemorrhage; the primary pediatric endpoint is rate of development of humoral immune response to FVIII and proportion that progress to clinical inhibitors. Mother and child secondary endpoints reflect the diverse research the HARP resource will support. Inclusion criteria for the pregnant person are: Pregnant and prior to 37 weeks gestation, known or at-risk to have a severe hemophilia A genotype, pregnant with a fetus at-risk for severe hemophilia A, ability to give informed consent, and > 18 years old. Inclusion criteria for the child are: severe hemophilia A, born to a person in the study. Exclusion/discontinuation criteria are: Presence of another significant bleeding disorder in mother or child; mother age <18 years; no fetus is at-risk to inherit severe hemophilia A; the child does not have severe hemophilia A; the child has a severe immune disorder; the mother or child participates in another study with blood collection volumes that exceed safety limits; mother's or child's healthcare will be outside the U.S.; the mother is a prisoner; any other reason that in the opinion of the investigator a subject is unsuitable for the study Summary: The HARP innovates in multiple areas of clinical study design to conduct a national prospective longitudinal intergenerational study in a rare disorder beginning in pregnancy. The structure includes a new multidisciplinary CC, systematic inclusion of Lived Experience Experts, strategies for broad community engagement, recruitment, and retention, and decentralized operations. This cohort will enable deep characterization, including genomics and other -omics, on pregnancy-associated bleeding and drivers of FVIII inhibitors in hemophilia A. The HARP Shareable Resource will be comprised of well annotated data and biological samples accessible to researchers through a portal, F8portal.org, and a linked biorepository.

Persistent humoral immune response in youth throughout the COVID-19 pandemic: prospective school-based cohort study

Understanding the development of humoral immune responses of children and adolescents to SARS-CoV-2 is essential for designing effective public health measures. Here we examine the changes of humoral immune response in school-aged children and adolescents during the COVID-19 pandemic (June 2020 to July 2022), with a specific interest in the Omicron variant (beginning of 2022). In our study “Ciao Corona”, we assess in each of the five testing rounds between 1874 and 2500 children and adolescents from 55 schools in the canton of Zurich with a particular focus on a longitudinal cohort (n=751). By July 2022, 96.9% (95% credible interval 95.3–98.1%) of children and adolescents have SARS-CoV-2 anti-spike IgG (S-IgG) antibodies. Those with hybrid immunity or vaccination have higher S-IgG titres and stronger neutralising responses against Wildtype, Delta and Omicron BA.1 variants compared to those infected but unvaccinated. S-IgG persist over 18 months in 93% of children and adolescents. During the study period one adolescent was hospitalised for less than 24 hours possibly related to an acute SARS-CoV-2 infection. These findings show that the Omicron wave and the rollout of vaccines boosted S-IgG titres and neutralising capacity. Trial registration number: NCT04448717. https://clinicaltrials.gov/ct2/show/NCT04448717.

Effects of the complement system on antibody formation and function: implications for transplantation.

In antibody-mediated allograft rejection, donor-reactive antibodies cause transplant injury in part via complement activation. New mechanistic insights indicate complement also modulates development of humoral immune responses. Herein we review recent data that describes how complement affects antibody formation and we discuss therapeutic implications. Extravasating T cells interacting with integrins express and activate intracellular complement that drives immune-metabolic adaptations vital for CD4 + helper cells. Marginal zone B cells can acquire intact major histocompatibility complexes from dendritic cells via complement-dependent trogocytosis for presentation to T cells. Activated B cells in germinal centers receive co-stimulatory signals from T-helper cells. These germinal center B cells undergo coordinate shifts in surface complement regulator expression that permit complement receptor signaling on the germinal center B cells required for affinity maturation. The positively selected, high-affinity B cells can differentiate into plasma cells that produce donor-HLA-reactive antibodies capable of ligating endothelial, among other, graft cells. Subsequent sublytic complement attack can stimulate endothelial cells to activate CD4 + and CD8 + T cells, promoting cellular and humoral rejection. Newly developed complement inhibitors are being tested to prevent/treat transplant rejection. The complement system influences T-cell, B-cell and endothelial-cell activation, and thereby contributes allograft injury. Emerging therapeutic strategies targeting complement activation have the potential to prevent or abrogate transplant injury and improve transplant outcomes.

Antibody responses after documented COVID-19 disease in patients with autoimmune rheumatic disease.

Patients with autoimmune rheumatic diseases (AIRD) are suspected to have less robust immune responses during COVID-19 due to underlying immune dysfunction and the use of immune-suppressive drugs. Fifty consecutive patients with a diagnosis of AIRD on disease-modifying drugs were included at around 30 days after a confirmatory test for COVID-19. Fifty controls matched one to one for age, sex, and severity of COVID-19 were also included at around 30 days after testing positive for COVID-19. Antibody titers for anti-spike protein IgG and anti-nucleocapsid protein IgG were estimated. Cases (mean age 45.9 ± 13; 76% females) and controls (mean age 45.9 ± 13; 76% females) had similar proportion of comorbidities. Of the cases, 4 had moderate and 1 had severe COVID-19, while 3 and 1 of controls had moderate and severe COVID-19 respectively. Positivity of anti-N IgG was similar between patients (80%) and controls (90%) (p = 0.26). Similarly, anti-S IgG was positive in 82% of patients and 86% of controls (p = 0.79). Both the antibodies were negative in seven (14%) patients and five (10%) of controls (p = 0.76, Fischer exact test). Only anti-N IgG titers were lower in patients as compared to controls. In four patients with rheumatoid arthritis, two with spondyloarthritis and one with eosinophilic fasciitis both antibodies were not detectable. They did not differ from the rest of the cohort in clinical characteristics. The patients with AIRD had adequate protective antibody responses to COVID-19 at a median of 30 days post-infection. Thus, the presence of AIRD or the use of immunosuppressants does not seem to influence the development of humoral immune response against COVID-19. Key Points • Patients with autoimmune rheumatic diseases (AIRD) are suspected to have less robust immune responses. • In our cohort of 50 patients with AIRD with confirmed COVID-19, only seven did not have detectable protective antibodies at 30 days post infection. • Patients with AIRD on immunosuppressants have adequate protective antibodies post COVID-19 disease, at rates similar to that in health controls.

Development of Humoral Immune Response to Thermostable Newcastle Disease Vaccine Strain I-2 in Ring-Necked Pheasant (Phasianus colchicus)

Development of Humoral Immune Response to Thermostable Newcastle Disease Vaccine Strain I-2 in Ring-Necked Pheasant (Phasianus colchicus)

VANGUARD®crLyme: A next generation Lyme disease vaccine that prevents B. burgdorferi infection in dogs

Lyme disease, a public health threat of significance to both veterinary and human medicine, is caused by the tick (Ixodes) transmitted spirochete, Borreliella burgdorferi. Here we report on the immunogenicity and efficacy of VANGUARD®crLyme (Zoetis), the most recent canine Lyme disease vaccine to be approved by the United States Department of Agriculture. VANGUARD®crLyme is a subunit vaccine consisting of outer surface protein A (OspA) and a recombinant outer surface protein C (OspC) based-chimeric epitope protein (chimeritope) that consists of at least 14 different linear epitopes derived from diverse OspC proteins. The combination of OspA and the OspC chimeritope (Ch14) in the vaccine formulation allows for the development of humoral immune responses that work synergistically to target spirochetes in both ticks and in mammals. Immunogenicity was assessed in purpose-bred dogs. A two-dose vaccination protocol resulted in high antibody titers to OspA and Ch14 and vaccinal antibody reacted with 25 different recombinant OspC variants. Efficacy was demonstrated using an Ixodes scapularis -purpose bred dog challenge model. Vaccination with VANGUARD®crLyme provided protection against infection and prevented the development of clinical manifestations and histopathological changes associated with Lyme disease.

The Risk G Allele of the Single-Nucleotide Polymorphism rs928413 Creates a CREB1-Binding Site That Activates IL33 Promoter in Lung Epithelial Cells.

Cytokine interleukin 33 (IL-33) is constitutively expressed by epithelial barrier cells, and promotes the development of humoral immune responses. Along with other proinflammatory mediators released by the epithelium of airways and lungs, it plays an important role in a number of respiratory pathologies. In particular, IL-33 significantly contributes to pathogenesis of allergy and asthma; genetic variations in the IL33 locus are associated with increased susceptibility to asthma. Large-scale genome-wide association studies have identified minor “G” allele of the single-nucleotide polymorphism rs928413, located in the IL33 promoter area, as a susceptible variant for early childhood and atopic asthma development. Here, we demonstrate that the rs928413(G) allele creates a binding site for the cAMP response element-binding protein 1 (CREB1) transcription factor. In a pulmonary epithelial cell line, activation of CREB1, presumably via the p38 mitogen-activated protein kinases (MAPK) cascade, activates the IL33 promoter containing the rs928413(G) allele specifically and in a CREB1-dependent manner. This mechanism may explain the negative effect of the rs928413 minor “G” allele on asthma development.

Plasma cell differentiation is controlled by multiple cell division-coupled epigenetic programs

The genomic loci associated with B cell differentiation that are subject to transcriptional and epigenetic regulation in vivo are not well defined, leaving a gap in our understanding of the development of humoral immune responses. Here, using an in vivo T cell independent B cell differentiation model, we define a cellular division-dependent cis-regulatory element road map using ATAC-seq. Chromatin accessibility changes correlate with gene expression and reveal the reprogramming of transcriptional networks and the genes they regulate at specific cell divisions. A subset of genes in naive B cells display accessible promoters in the absence of transcription and are marked by H3K27me3, an EZH2 catalyzed repressive modification. Such genes encode regulators of cell division and metabolism and include the essential plasma cell transcription factor Blimp-1. Chemical inhibition of EZH2 results in enhanced plasma cell formation, increased expression of the above gene set, and premature expression of Blimp-1 ex vivo. These data provide insights into cell-division coupled epigenetic and transcriptional processes that program plasma cells.

Time- and dose-dependent development of humoral immune responses to Ascaridia galli in experimentally and naturally infected chickens

Factors affecting the development of Ascaridia galli–specific humoral responses and their protective roles are largely unknown. We investigated the effects of time and infection dose on A. galli–specific IgY antibody levels following experimental infection. The acquisition and development of new infections and reinfections were also monitored by using tracer birds. Relationships between the retrospective IgY and the final worm burden of the birds were investigated to determine whether humoral immune responses generated during infection provide protection to the host animal.Young chickens were infected (+) with either 100 or 1000 embryonated eggs of A. galli (100+: n = 45; 1000+: n = 45) or kept as uninfected controls (CON: n = 10). Uninfected birds were also added to each infection group as tracer (T) birds (T100+; n = 5 and T1000+; n = 5). Faecal egg counts and IgY antibody concentrations in plasma and egg yolk were determined at selected intervals. Final worm burdens were quantified at 28 weeks post infection (wpi).The plasma antibody (PAB) and egg yolk antibody (EAB) levels of experimentally infected birds were compared to those of control and tracer birds throughout the study period, and PAB levels were found to depend initially on the infection dose but thereafter mainly on reinfections. Starting at wpi 2, 1000+ had consistently higher PAB levels than CON did (P < 0.05). With exceptions at wpi 0, 2 and 12, PAB levels were also higher (P < 0.05) or tended to be higher (P < 0.10) in 100+ than in CON. An earlier and higher increase was observed in the PAB levels of T1000+ than in those of T100+, implying that (re-)infection occurrence depended on the infection dose. Although 1000+ showed higher (P < 0.05) EAB levels than CON did at both wpi 14 and 18, EAB levels were higher in 100+ than in CON only at wpi 28 (P < 0.05). The total worm burdens in the initial experimentally infected birds were similar (P = 0.257); they were also highly comparable between experimentally and naturally infected birds, indicating that final worm burden was mainly determined by the naturally occurring infections resulting from continuous exposure. When all available information on the retrospective plasma and egg yolk IgY levels was collectively evaluated to estimate the larval or total worm burdens of the experimentally infected birds, both PAB and EAB levels at particular wpi were significantly associated with worm burden, especially with larval count. In conclusion, our data support the hypothesis that the number of larvae, rather than the number of mature worms, affects the antibody levels in both plasma and egg yolk. Despite the increased levels of A. galli–specific antibodies in plasma and egg yolk throughout the study period, only a weak indication was found that antibodies might be directly associated with protection.

Humoral immune responses to infection: common mechanisms and unique strategies to combat pathogen immune evasion tactics.

Humoral immune responses are crucial for protection against invading pathogens and are the underlying mechanism of protection for most successful vaccines. Our understanding of how humoral immunity develops is largely based on animal models utilizing experimental immunization systems. While these studies have made enormous progress for the field and have defined many of the fundamental principles of B cell differentiation and function, we are only now beginning to appreciate the complexities of humoral immune responses induced by infection. Co-evolution of the adaptive immune system and the pathogenic world has created a diverse array of B cell responses to infections, with both shared and unique strategies. In this review, we consider the common mechanisms that regulate the development of humoral immune responses during infection and highlight recent findings demonstrating the evolution of unique strategies used by either host or pathogen for survival.

Sex bias in mouse humoral immune response to influenza vaccine depends on the vaccine type

The study explored influence of biological sex on development of humoral immune response to seasonal trivalent whole inactivated virus (WIV) and split virus (SV) influenza vaccines in outbred Swiss mouse model. To this end, mice of both sexes were immunized with WIV (WIV mice) and SV vaccines (SV mice) and examined for specific antibody response. Irrespective of sex, total IgG and neutralizing antibody responses to distinct virus strains were weaker in SV than in WIV mice. In WIV mice of both sexes, irrespective of strain specificity, IgG isotype response was dominated by IgG2a antibodies, while in SV mice nearly equal representation of IgG2a and IgG1 antibodies was found. The analyses of sex differences showed higher titers of H1N1-specific and both H1N1- and H3N2-specific total IgG and neutralizing antibodies in female WIV and SV mice, respectively. Additionally, sexual dimorphism in IgG subclass profile depended on vaccine type. Specifically, compared with males, in females WIV shifted IgG2a/IgG1 antibody ratio towards IgG2a isotype on the account of weaker IgG1 response, whereas in SV mice, irrespective of virus strain, IgG2a and IgG1 isotypes were equally represented in both sexes. These findings indicate the vaccine type-dependent sex bias in antibody response to inactivated influenza vaccines.

Investigation of three outbreaks of Porcine Epidemic Diarrhea in Germany in 2016 demonstrates age dependent differences in the development of humoral immune response

Porcine epidemic diarrhea (PED) has reemerged in Europe since 2014. Characterized by a rapid onset of diarrhea in pigs of all ages, morbidity can reach up to 100% whereas mortality is variable. The virus strains involved in the recent European outbreaks all cluster together with US strains (S INDEL) that lead to less severe clinical signs. In this study, fattening pigs and suckling piglets (n = 105) on farms with no prior PED history were monitored after an acute outbreak of the disease, caused by an S INDEL strain of PED virus (PEDV). For diagnostic investigations in the affected farms, real time RT-PCR was performed to detect PEDV RNA in individually taken fecal samples, and two commercial ELISA kits, both based on the N protein of PEDV, were used to detect IgG in serum samples of pigs experiencing acute signs of the disease. PEDV RNA could be detected in fecal samples up to 14 days after initial sampling. Comparing both ELISAs by Cohens Kappa showed substantial agreement (κ = 0,771). Antibodies were detectable in all fattening pigs (100%) within 10 days after the occurrence of first clinical signs and remained detectable for about two months at least in 20.6% (farm 1) and 45.7% (farm 2) of the animals, respectively. In contrast, only 18 of 34 (52.9%) suckling piglets seroconverted. Although, PEDV RNA was found in fecal samples of all piglets, 13 piglets did not demonstrate antibodies at any sampling day. PCR to detect PEDV RNA in fecal samples seems to be a reliable diagnostic tool during and after the acute outbreak. In the present study, IgG ELISA kits proved to be a feasible diagnostic tool, but age dependent differences in detection rate and persistence of antibodies need to be considered.

Follicular CD8+ T Cells: Origin, Function and Importance during HIV Infection.

The lymphoid follicle is critical for the development of humoral immune responses. Cell circulation to this site is highly regulated by the differential expression of chemokine receptors. This feature contributes to the establishment of viral reservoirs in lymphoid follicles and the development of some types of malignancies that are able to evade immune surveillance, especially conventional CD8+ T cells. Interestingly, a subtype of CD8+ T cells located within the lymphoid follicle (follicular CD8+ T cells) was recently described; these cells have been proposed to play an important role in viral and tumor control, as well as to modulate humoral and T follicular helper cell responses. In this review, we summarize the knowledge on this novel CD8+ T cell population, its origin, function, and potential role in health and disease, in particular, in the context of the infection by the human immunodeficiency virus.

Identification of Interleukin 40, a novel B cell-associated cytokine.

Abstract We have identified a novel mouse and human cytokine. IL-40 is normally produced in the bone marrow and fetal liver, and by activated B cells. Its sequence predicts a small (~27kDa) secreted protein unrelated to other cytokine gene families, that we have called Interleukin 40 (IL-40). IL40 is only present in mammalian genomes, suggesting a role in mammalian immune responses. Accordingly, IL-40 expression is induced in the mammary gland upon the onset of lactation, and an IL-40−/− mouse exhibits reduced levels of IgA in the serum, gut, feces, and milk. Furthermore, the IL-40−/− mouse has smaller and fewer Peyer’s patches and IgA secreting cells. The gut microbiome of IL-40−/− mice also exhibits altered composition, reflecting the reduced levels of IgA in the gut. We have also determined that B cell precursor populations are altered in the IL-40−/− mouse. Taken together, these observations indicate that IL-40 represents a novel B cell associated cytokine, that plays an important role in the development of humoral immune responses. IL-40 is also expressed by human activated B cells and by several human B cell lymphomas. The latter observation suggests that it may play a role in the pathogenesis of these diseases.

Antibody responses to Sarcoptes scabiei apolipoprotein in a porcine model: relevance to immunodiagnosis of recent infection.

No commercial immunodiagnostic tests for human scabies are currently available, and existing animal tests are not sufficiently sensitive. The recombinant Sarcoptes scabiei apolipoprotein antigen Sar s 14.3 is a promising immunodiagnostic, eliciting high levels of IgE and IgG in infected people. Limited data are available regarding the temporal development of antibodies to Sar s 14.3, an issue of relevance in terms of immunodiagnosis. We utilised a porcine model to prospectively compare specific antibody responses to a primary infestation by ELISA, to Sar s 14.3 and to S. scabiei whole mite antigen extract (WMA). Differences in the antibody profile between antigens were apparent, with Sar s 14.3 responses detected earlier, and declining significantly after peak infestation compared to WMA. Both antigens resulted in >90% diagnostic sensitivity from weeks 8–16 post infestation. These data provide important information on the temporal development of humoral immune responses in scabies and further supports the development of recombinant antigen based immunodiagnostic tests for recent scabies infestations.

Temporal development of the humoral immune response to surface antigens of Moraxella catarrhalis in young infants

The primary Moraxella catarrhalis-specific humoral immune response, and its association with nasopharyngeal colonization, was studied in a cohort of infants from birth to 2 years of age. Results indicated that the levels of antigen-specific IgG, IgA and IgM showed extensive inter-individual variability over time, with IgM and IgA levels to all 9 recombinant domains, from 7 different OMPs, being relatively low throughout the study period. In contrast, the level of antigen-specific IgG was significantly higher for the recombinant domains Hag 385–863, MID 764–913, MID 962–1200, UspA1 557–704 and UspA2 165–318 in cord blood compared to 6 months of age ( P ≤ 0.001). This was a most likely a consequence of maternal transmission of antigen-specific IgG to newborn babies, possibly indicating a future role for these 3 surface antigens in the development of an effective humoral immune response to M. catarrhalis. Finally, at 2 years of age, the levels of antigen-specific IgG still remained far below that obtained from cord blood samples, indicating that the immune response to M. catarrhalis has not matured at 2 years of age. We provide evidence that a humoral antibody response to OMPs UspA1, UspA2 and Hag/MID may play a role in the immune response to community acquired M. catarrhalis colonization events.

Synthetic glycosylphosphatidylinositol microarray reveals differential antibody levels and fine specificities in children with mild and severe malaria

Glycosylphosphatidylinositol (GPI) glycolipids abound on the cell surface at the merozoite stage of Plasmodium falciparum life cycle are a central toxin in malaria. The contribution of GPI specific humoral immune responses to protection against malaria pathology is not clear, since studies on the correlation between anti-GPI antibody titers and disease severity have yielded contradictory results. Here, we present the application of a carbohydrate microarray based on synthetic PfGPI glycans to assess levels and fine specificities of anti-GPI antibody responses in healthy and malaria diseased individuals. Furthermore, the age dependent development of humoral immune responses against GPI in malaria-exposed children was investigated. Anti-GPI antibodies were only rarely found in children under the age of 18 months. Sera from subjects with severe malaria and healthy children contained antibodies that recognized predominantly synthetic Man 3-GPI and Man 4-GPIs. In contrast, antibodies in sera of children with mild malaria also showed substantial reactivity with truncated glycans comprising glucosamine–inositol moieties without mannose or with only one or two mannose residues.

Mast cell activators: a new class of highly effective vaccine adjuvants

Mast cells (MCs) have recently received recognition as prominent effectors in the regulation of immune cell migration to draining lymph nodes and lymphocyte activation. However, their role in the development of humoral immune responses is not clear. Here, we demonstrate that subcutaneous or nasal administration of small-molecule MC activators with vaccine antigens evokes large increases in antigen-specific serum immunoglobulin G (IgG) responses. These responses were MC dependent and correlated with increased dendritic cell and lymphocyte recruitment to draining lymph nodes. Nasal instillation of these formulations also evoked antigen-specific secretory IgA and provided protection against anthrax lethal toxin challenge in vitro and against vaccinia virus infection in vivo. Collectively, these results define the MC as an integral sensory arm of the adaptive immune system. Moreover, they highlight MC activators as a new class of vaccine adjuvants, capable of inducing protective antigen-specific immune responses through needle-free routes of administration.

Modulation of Airway Inflammation by Haemophilus influenzae Isolates Associated with Chronic Obstructive Pulmonary Disease Exacerbation

Chronic obstructive pulmonary disease (COPD) is associated with periodic exacerbations that manifest as worsening of lung function and increased dyspnea, cough, sputum production, and sputum purulence. COPD exacerbations are associated with increased markers of inflammation in the airway, including neutrophils, interleukin(IL)-6, and IL-8 (1, 2). These observations led to the hypothesis that factors that promote airway inflammation lead to worsening airway function in patients with COPD. Although infection clearly has the ability to induce airway inflammation, the role that bacteria play in COPD exacerbations remains controversial (3, 4). Bacteria commonly colonize the airway of patients with COPD at baseline, making the presence of bacteria insufficient to explain worsening airway function (5–7). However, COPD exacerbation could be precipitated by an increase in bacterial number; change in the airway compartment in which bacteria are located; or acquisition of new, more virulent, and/or more proinflammatory bacterial species or strain (3, 8, 9). Nontypeable Haemophilus influenzae is the bacterial species most commonly isolated from airway samples during COPD exacerbations (9, 10). The ability of this bacteria to cause intense airway inflammation and the association of exacerbations with development of humoral immune responses to H. influenzae acquired for the first time supports a potential role for infection by this organism in causing COPD exacerbations (11). However, patient and bacterial factors that determine whether airway infection results in worsening of airway function are poorly defined. We have previously reported an initial comparative study of H. influenzae isolates from patients with COPD who were followed as part of a prospective, longitudinal study at the Buffalo Veterans Affairs Medical Center (12). For this study, 17 bacterial isolates were obtained from 15 patients with COPD who underwent serial assessment of clinical status, sputum microbiology, and serum antibacterial antibody production. Exacerbation strains were defined as new isolates cultured from sputum during exacerbation of clinical symptoms with subsequent development of a homologous bactericidal serum antibody response. Colonization strains were not associated with an increase in symptoms or new antibody production. We found that strains of H. influenzae associated with exacerbations caused more airway neutrophil recruitment compared with colonization strains in an in vivo mouse model of airway infection. Experiments using an in vitro model of human primary airway epithelial cells revealed that exacerbation strains adhered significantly more to epithelial cells. In addition, exacerbation isolates induced greater release of IL-8 compared with colonization strains, a response likely mediated by increased activation of both the nuclear factor-κB and p38 mitogen-activated protein kinase signaling pathways. The results indicate that H. influenzae isolated from patients with exacerbations of COPD induce more airway inflammation, and likely have differences in virulence compared with colonizing strains. We have gone on to investigate selected characteristics of the interaction between H. influenzae and airway epithelial cells that could account for bacterial strain differences in induction of IL-8. Previous experiments in our laboratory indicate that multiple soluble factors are released during epithelial cell interaction with H. influenzae that augment both IL-8 release and expression of other inflammatory mediators. Accordingly, we questioned whether these factors might disproportionately augment epithelial cell IL-8 levels in experiments using exacerbation strains or suppress IL-8 levels after epithelial cell interaction with colonization strains. However, we found that differences between H. influenzae isolates associated with exacerbation versus colonization in patients with COPD were not attributable to the presence of soluble factors generated after epithelial cell–bacterial interaction. Interestingly, induction of IL-8 expression by airway epithelial cell interaction with both exacerbation and colonization strains of H. influenzae was inhibited by exposure of epithelial cells to the macrolide antibiotics erythromycin and azithromycin. This effect was not due to antibacterial properties of these antibiotics, as inhibition of IL-8 expression was seen when bacteria were killed before experiments by pretreatment with gentamicin. Taken together, our findings support the concept that H. influenzae infecting the airway during COPD exacerbations mediate increased airway inflammation and contribute to decreased airway function. Furthermore, inflammatory mediator generation by epithelial cells exposed to bacteria in the airway may be modulated pharmacologically, and this strategy could improve airway function in patients with COPD.