PA-specific IgG Research Articles

Sublingual targeting of STING with 3′3′-cGAMP promotes systemic and mucosal immunity against anthrax toxins

Anthrax is caused by Bacillus anthracis, a zoonotic bacterial pathogen affecting humans and livestock worldwide. The current human anthrax vaccine, anthrax vaccine adsorbed (AVA), is an injected vaccine with a cumbersome administration schedule and fails to promote mucosal immunity. Bacterial enterotoxins, which stimulate production of the cyclic nucleotide cAMP are effective experimental mucosal vaccine adjuvants, but their inherent toxicity has precluded their use in humans. We investigated whether cyclic dinucleotides that target Stimulator of Interferon Gamma Genes (STING) in mammalian cells could represent an alternative to bacterial enterotoxins as adjuvant for sublingual immunization and promotion of mucosal immunity and secretory IgA responses in addition to systemic immunity. We found that sublingual immunization of mice with Bacillus anthracis protective antigen (PA) and the STING ligand 3′3′-cGAMP promotes PA-specific serum IgG Ab responses of the same magnitude as those induced after immunization with PA and the experimental adjuvant cholera toxin (CT). Interestingly, this STING ligand also promoted serum anti-PA IgA and IgA-producing cells in the bone marrow. Furthermore, the saliva of mice immunized with the STING ligand exhibited similar levels of PA-specific IgA Abs as groups immunized with CT as adjuvant. The adjuvant activity of 3′3′-cGAMP was associated with mixed Th1, Th2, and Th17 responses. This STING ligand also induced rapid IFN-β and IL-10 responses in sublingual tissues and cervical lymph nodes, and TGF-β responses in the cervical lymph nodes, which could contribute to promoting IgA responses after sublingual immunization.

Humoral and Cell-Mediated Immune Responses to Alternate Booster Schedules of Anthrax Vaccine Adsorbed in Humans.

Protective antigen (PA)-specific antibody and cell-mediated immune (CMI) responses to annual and alternate booster schedules of anthrax vaccine adsorbed (AVA; BioThrax) were characterized in humans over 43 months. Study participants received 1 of 6 vaccination schedules: a 3-dose intramuscular (IM) priming series (0, 1, and 6 months) with a single booster at 42 months (4-IM); 3-dose IM priming with boosters at 18 and 42 months (5-IM); 3-dose IM priming with boosters at 12, 18, 30, and 42 months (7-IM); the 1970 licensed priming series of 6 doses (0, 0.5, 1, 6, 12, and 18 months) and two annual boosters (30 and 42 months) administered either subcutaneously (SQ) (8-SQ) or IM (8-IM); or saline placebo control at all eight time points. Antibody response profiles included serum anti-PA IgG levels, subclass distributions, avidity, and lethal toxin neutralization activity (TNA). CMI profiles included frequencies of gamma interferon (IFN-γ)- and interleukin 4 (IL-4)-secreting cells and memory B cells (MBCs), lymphocyte stimulation indices (SI), and induction of IFN-γ, IL-2, IL-4, IL-6, IL-1β, and tumor necrosis factor alpha (TNF-α) mRNA. All active schedules elicited high-avidity PA-specific IgG, TNA, MBCs, and T cell responses with a mixed Th1-Th2 profile and Th2 dominance. Anti-PA IgG and TNA were highly correlated (e.g., month 7,r(2)= 0.86,P< 0.0001, log10 transformed) and declined in the absence of boosters. Boosters administered IM generated the highest antibody responses. Increasing time intervals between boosters generated antibody responses that were faster than and superior to those obtained with the final month 42 vaccination. CMI responses to the 3-dose IM priming remained elevated up to 43 months. (This study has been registered at ClinicalTrials.gov under registration no. NCT00119067.).

Protective antigen-specific memory B cells persist years after anthrax vaccination and correlate with humoral immunity.

Anthrax Vaccine Adsorbed (AVA) generates short-lived protective antigen (PA) specific IgG that correlates with in vitro toxin neutralization and protection from Bacillus anthracis challenge. Animal studies suggest that when PA-specific IgG has waned, survival after spore challenge correlates with an activation of PA-specific memory B cells. Here, we characterize the quantity and the longevity of AVA-induced memory B cell responses in humans. Peripheral blood mononuclear cells (PBMCs) from individuals vaccinated ≥3 times with AVA (n = 50) were collected early (3–6 months, n = 27) or late after their last vaccination (2–5 years, n = 23), pan-stimulated, and assayed by ELISPOT for total and PA-specific memory B cells differentiated into antibody secreting cells (ASCs). PA-specific ASC percentages ranged from 0.02% to 6.25% (median: 1.57%) and did not differ between early and late post-vaccination individuals. PA-specific ASC percentages correlated with plasma PA-specific IgG (r = 0.42, p = 0.03) and toxin neutralization (r = 0.52, p = 0.003) early post vaccination. PA-specific ASC percentages correlated with supernatant anti-PA both early (r = 0.60, p = 0.001) and late post vaccination (r = 0.71, p < 0.0001). These data suggest PA-specific memory B cell responses are long-lived and can be estimated after recent vaccination by the magnitude and neutralization capacity of the humoral response.

Comparison of two treatment regimens for eradication of Pseudomonas aeruginosa infection in children with cystic fibrosis

In patients with cystic fibrosis (CF), treatment of new Pseudomonas aeruginosa (Pa) infection postpones the occurrence of chronic infection, but the best eradication regimen is unknown . Aim of the studyCompare 2 Pa eradication regimens in children with new Pa infection. MethodsChildren with CF (0–18years) and a new isolation of Pa from sputum, cough swab or BAL were randomized to treatment with tobramycin inhalation solution for 28days (TIS) or inhaled sodiumcolistimethate (2×2millU/day) plus oral ciprofloxacin (30mg/kg/day) for 3months (CC). Airway cultures were taken for 6 consecutive months, then every 3months. The primary outcome was Pa eradication at the end of treatment. Secondary outcome parameters were: time to Pa relapse from end of treatment, total and Pa specific IgG, FEV1, BMI and Pa status at 2year follow-up. Results58 patients with new Pa isolation were randomized. Their median age was 9years (IQR 4.7–13.1) and their median FEV1 98% predicted (IQR 87–107). Eighteen treatments concerned the first Pa isolation ‘ever’ (TIS: 8; CC: 10). For the remaining, median time since previous Pa was 19months (IQR 9–41). Eradication at end of treatment was similar for both treatments: 26/29 CC and 23/29 in TOBI treated patients (p=0.47). Median time to recurrence of Pa was 9months (95% CI 0.0–19.0) for CC and 5months (95% CI 1.7–8.3) for TIS (p=0.608). After 1year, the 2 groups did not differ in change in total and Pa specific IgG, FEV1 and BMI. After 2years, 10% of patients had chronic Pa infection. ConclusionIn children with CF and new Pa infection, inhalation of TIS (28days) or CC (3months) resulted in similar eradication success at the end of treatment (80 and 90% respectively) and similar clinical evolution during the first 2years of follow-up.

Mucosal priming of newborn mice with S. Typhi Ty21a expressing anthrax protective antigen (PA) followed by parenteral PA-boost induces B and T cell-mediated immunity that protects against infection bypassing maternal antibodies

The currently licensed anthrax vaccine has several limitations and its efficacy has been proven only in adults. Effective immunization of newborns and infants requires adequate stimulation of their immune system, which is competent but not fully activated. We explored the use of the licensed live attenuated S. Typhi vaccine strain Ty21a expressing Bacillus anthracis protective antigen [Ty21a(PA)] followed PA-alum as a strategy for immunizing the pediatric population. Newborn mice primed with a single dose of Ty21a(PA) exhibited high frequencies of mucosal IgA-secreting B cells and IFN-γ-secreting T cells during the neonatal period, none of which was detected in newborns immunized with a single dose of PA-alum. Priming with Ty21a(PA) followed by PA-boost resulted in high levels of PA-specific IgG, toxin neutralizing and opsonophagocytic antibodies and increased frequency of bone marrow IgG plasma cells and memory B cells compared with repeated immunization with PA-alum alone. Robust B and T cell responses developed even in the presence of maternal antibodies. The prime-boost protected against systemic and respiratory infection. Mucosal priming with a safe and effective S. Typhi-based anthrax vaccine followed by PA-boost could serve as a practical and effective prophylactic approach to prevent anthrax early in life.

Sequence microheterogeneity of parvalbumin pI 5.0 of pike: A mass spectrometric study

Parvalbumin (PA) is a muscle and neuronal calcium-binding protein, the major fish and frog allergen. Its characteristic feature is the presence of multiple isoforms with significantly different amino acid sequences. Here we show that the major isoform of northern pike muscle PA (pI 5.0, α-PA) exhibits microheterogeneity of amino acid sequence. ESI Q-TOF mass-spectrometry (MS) analysis of α-PA sample showed the presence of two components with mass difference of 71 Da. Analysis of tryptic and endoproteinase Asp-N digests of α-PA by MALDI-TOF MS revealed peptides, corresponding to two different amino acid sequences. The sequence differences between variant proteins are limited to AB-domain and include substitutions K27A and L31K, and an extra Leu residue between K11 and K12. Since the affected residues comprise a cluster on the surface of PA, an involvement of the identified region into target recognition is suggested. The substitutions at positions 27 and 31 are located in the region of previously identified epitopes of parvalbumin relevant for PA-specific IgE and IgG binding, which suggests different immunoactivities of the variants. The found microheterogeneity of PA is suggested to be of importance for physiological adaptation of the propulsive musculature to developmental and/or environmental requirements and may contribute to PA allergenicity.

Intranasal Administration of Dry Powder Anthrax Vaccine Provides Protection Against Lethal Aerosol Spore Challenge

The use of an aerosolizable form of anthrax as a biological weapon is considered to be among the most serious bioterror threats. Intranasal (IN) delivery of a dry powder anthrax vaccine could provide an effective and non-invasive administration alternative to traditional intramuscular (IM) or subcutaneous (SC) injection. We evaluated a dry powder vaccine based on the recombinant Protective Antigen (rPA) of Bacillus anthracis for vaccination against anthrax via IN immunization in a rabbit model. rPA powders were formulated and administered IN using a prototype powder delivery device. We compared serum IgG and toxin neutralizing antibody (TNA) titers of rabbits immunized IN with 10 μg rPA of a powder formulation with those immunized with the same dose of liquid rPA vaccine, delivered either IN or by IM injection. In addition, each group was tested for survival after aerosol spore challenge. Our results showed that IN vaccination with rPA powders elicited serum PA-specific IgG and TNA titers that were equivalent to those raised by liquid rPA administered IN. Serum PA-specific IgG and TNA titers after IN delivery were lower than for IM injection, however, after aerosol spore challenge, rabbits immunized IN with powders displayed 100% protection versus 63% for the group immunized IN with the liquid vaccine and 86% for the group immunized by IM injection. The results suggest that an IN powder vaccine based on rPA is at least as protective as a liquid delivered by IM injection.

Immune responses to Bacillus anthracis protective antigen in patients with bioterrorism-related cutaneous or inhalation anthrax.

Anti-protective antigen (PA) immunoglobulin (Ig) G, toxin neutralization, and PA-specific IgG memory B cell responses were studied in patients with bioterrorism-related cutaneous or inhalation anthrax and in a patient with laboratory-acquired cutaneous anthrax. Responses were determined for >1 year after the onset of symptoms. Eleven days after the onset of symptoms (15 days after likely exposure), anti-PA IgG was detected in 16 of 17 patients with confirmed or suspected clinical anthrax who were tested. Anti-PA IgG remained detectable 8-16 months after the onset of symptoms in all 6 survivors of inhalation anthrax and in 7 of 11 survivors of cutaneous anthrax who were tested. Anti-PA IgG levels and serum toxin neutralizing activity were strongly associated (R2=0.83). PA-specific IgG memory B cells were detectable in all 6 survivors of inhalation anthrax but in only 2 of 7 patients with cutaneous anthrax who were tested. Anti-PA IgG is an important diagnostic marker of anthrax, a predictor of serum anti-toxin activity, and a marker of immunological memory against anthrax.

Characterization of the human immune response to the UK anthrax vaccine

The anthrax bipartite lethal toxin (protective antigen (PA) and lethal factor (LF))-specific antibody responses of humans receiving the UK licensed anthrax vaccine were determined. The PA-specific IgG response peaked two weeks post immunization and fell back to pre-boost levels by week 12. The heterogeneity of the host population modulated the extent of the PA-specific antibody response. Significantly lower levels of LF-specific antibodies were also detected. Vaccinated individuals recognized the same PA epitope as the protective mouse lethal toxin neutralizing monoclonal 2D3 suggesting that this may also be a target for human protection.

Effective mucosal immunity to anthrax: neutralizing antibodies and Th cell responses following nasal immunization with protective antigen.

Mucosal, but not parenteral, immunization induces immune responses in both systemic and secretory immune compartments. Thus, despite the reports that Abs to the protective Ag of anthrax (PA) have both anti-toxin and anti-spore activities, a vaccine administered parenterally, such as the aluminum-adsorbed anthrax vaccine, will most likely not induce the needed mucosal immunity to efficiently protect the initial site of infection with inhaled anthrax spores. We therefore took a nasal anthrax vaccine approach to attempt to induce protective immunity both at mucosal surfaces and in the peripheral immune compartment. Mice nasally immunized with recombinant PA (rPA) and cholera toxin (CT) as mucosal adjuvant developed high plasma PA-specific IgG Ab responses. Plasma IgA Abs as well as secretory IgA anti-PA Abs in saliva, nasal washes, and fecal extracts were also induced when a higher dose of rPA was used. The anti-PA IgG subclass responses to nasal rPA plus CT consisted of IgG1 and IgG2b Abs. A more balanced profile of IgG subclasses with IgG1, IgG2a, and IgG2b Abs was seen when rPA was given with a CpG oligodeoxynucleotide as adjuvant, suggesting a role for the adjuvants in the nasal rPA-induced immunity. The PA-specific CD4(+) T cells from mice nasally immunized with rPA and CT as adjuvant secreted low levels of CD4(+) Th1-type cytokines in vitro, but exhibited elevated IL-4, IL-5, IL-6, and IL-10 responses. The functional significance of the anti-PA Ab responses was established in an in vitro macrophage toxicity assay in which both plasma and mucosal secretions neutralized the lethal effects of Bacillus anthracis toxin.

Serum IgE and lung function in workers exposed to phthalic anhydride

A total of 23 phthalic anhydride (PA)-exposed workers (air levels up to 17 mg/m3) showed significantly (P less than 0.01) more workrelated symptoms in their eyes (48% vs 6%) and nose (39% vs 0) than did 18 unexposed control subjects. Two of the exposed workers had PA-associated asthma. Surprisingly, the control group exhibited significantly (P less than 0.05) more symptoms of nonspecific bronchial hyperreactivity (44% vs 13%). The exposed workers showed significantly higher levels of total serum IgE (medians, 32 vs 15 kIU/l, P less than 0.05), although values for specific IgE against PA did not differ. This may indicate that such exposure can facilitate the entry of common allergens. There was a significant difference in PA-specific IgG [enzyme-linked immunosorbent assay (ELISA) ratios 0.21 vs 0.12; P less than 0.01]. There were no differences in lung function with regard to vital capacity (VC) and forced expiratory volume (FEV1), closing volume expressed as a percentage of VC (CV%), volume of trapped gas (VTG) before and after inhalation of metacholine, or carbon monoxide transfer factor (TLCO), whereas exposed workers displayed significantly higher late expiratory flow rates (MEF50 and MEF25). In a [99mTc]-diethylenetriamine penta-acetate (DTPA) clearance test, there was no difference between exposed subjects and controls. In summary, exposure to PA did not cause subclinical effects of the lungs. Subjects with lower-airway symptoms showed lower FEV1, MEF50, and MEF25 values and higher VTG (after metacholine) than did those without symptoms.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of inhibition of glutathione synthesis on the immunogenicity of captopril and captoprilprotein conjugates in the mouse

The effect of glutathione (GSH) depletion on the immunogenicity of captopril (CP) and d-penicillamine (PA) was investigated in C57 mouse. Depletion of GSH was by dl-buthionine sulfoximine (BSO), a potent and specific inhibitor of GSH synthesis, injected on the days of immunization of the drug/drug-protein conjugates. Chronic BSO pretreatment for 5 consecutive days, before and including the 4-day immunization period, caused tissue GSH depletion (liver 60%, kidney 71%, lung 14%, spleen 14% and whole blood 36%) in C57 mouse. After chronic administration of CP (270 μmol/kg, i.p. or i.m.) or CP-HSA (200 μg, i.p. or i.m.), IgG antibody response to CP-derived antigen was detected by an enzyme-linked immunosorbent assay (ELISA). The IgG antibody recognised CP-OVA but not OVA, and was inhibited by CP in other protein conjugated form, thus confirming its specificity to CP. Depletion of GSH by BSO caused an increase in the CP-specific IgG antibody titre when CP/CP-HSA was immunized through i.p. (with or without Freund's Complete Adjuvant) but not i.m. administration. In contrast, chronic administration of PA (270 μmol kg , i.p.) or PA-HSA (200μg, i.p.), with or without BSO pre-treatment, did not lead to detectable PA-specific IgG antibody. The results again illustrate the difference in the intrinsic immunogenicity between CP and PA, in that depletion of GSH increased the humoral (Blymphocyte) anti-CP responses but not anti-PA responses under the experimental conditions. These findings suggest that GSH status, apart from its effects on the disposition of CP/CP-protein conjugates, should be considered as an important determinant of both the immunological and toxicological response to CP.

Drug-protein conjugates—XV: A study of the disposition of D-penicillamine in the rat and its relationship to immunogenicity

The disposition of [ 14C] d-penicillamine (PA) was investigated in vitro and in vivo with male Wistar rats. Irreversible binding of [ 14C]PA to isolated rat plasma proteins in vitro reached a maximum of 20.6% of total radioactivity at 6 hr. Irreversibly bound [ 14C]PA could be dissociated with dithiothreitol, demonstrating that conjugation was via disulphide linkage. Three hours after i.v. administration of [ 14C]PA (27 μmol/kg) to rats 100% of plasma radioactivity was irreversibly bound, representing approximately 3.5% of the dose. Further studies on the disposition of PA-plasma protein conjugates snowed that dissociation occurred readily in vivo: the plasma half-life of the conjugate was approximately 3 hr. Free [ 14C]PA was the major urinary metabolite after administration of both free and conjugated drug. These studies show that the disposition of PA is similar to that reported for the structurally related sulphydryl drug captopril (CP). Free PA (340 μmol/kg and 3.4 mmol/kg) administered i.p. and i.m. daily for 4 days at one monthly intervals, and also PA-KLH conjugate (100 μg/rat) administered by single i.p. injection at monthly intervals with and without Freund's complete adjuvant, failed to induce PA-specific IgG or IgM antibody responses detectable by ELISA. In contrast, CP (270μmol/kg), administered by the same protocol as free PA, induced a CP-specific IgG antibody response after the third series of monthly injections. These data suggest that the difference in immunogenicity between PA and CP arises from a difference in the intrinsic immunogenicity of the haptens, rather than from their disposition.