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Immunoglobulin A1 (IgA1) protease is a critical virulence factor of Haemophilus influenzae that facilitates bacterial mucosal infection. This study investigates the effect of iga gene polymorphism on the enzymatic activity of H. influenzae IgA1 protease. The IgA1 protease activity was examined in the H. influenzae Rd KW20 strain and 51 isolates. Genetic variations in iga and deduced amino acid substitutions affecting IgA1 protease activity were assessed. Machine learning tools and functional complementation assays were used to analyze the effects of identified substitutions on the stability and activity of IgA1 protease, respectively. All 51 isolates exhibited similar iga expression levels. No igaB expression was detected. According to comparisons with the reference Rd KW20 strain, four substitutions in the protease domain, 26 in the nonprotease passenger domain, and two in the β-barrel domain were associated with the change in IgA1 protease activity. No substitutions in the catalytic site of IgA1 protease were observed. Logistic regression, receiver operating characteristic curves, Venn diagrams, and protein stability analyses revealed that the substitutions Asn352Lys, Pro353Ala, Lys356Asn, Gln916Lys, and Gly917Ser, which were located in the nonactive site of the passenger domain, were associated with decreases in IgA1 protease activity and stability, whereas Asn914Lys was associated with an increase in these events. Functional complementation assays revealed that the Asn914Lys substitution increased IgA1 protease activity in the Rd KW20 strain. This study identified substitutions in the nonactive site of the passenger domain that affect both the activity and stability of H. influenzae IgA1 protease., Competing Interests: The authors declare no conflict of interest.

Activation of Th17 cell responses, including the production of IL-17A and IL-21, contributes to host defense and inflammatory responses by coordinating adaptive and innate immune responses. IL-17A and IL-17F signal through a multimeric receptor, which includes the IL-17 receptor A (IL-17RA) subunit and the IL-17RC subunit. IL-17RA is expressed by many cell types, and data from previous studies suggest that loss of IL-17 receptor is required to limit immunopathology in the Helicobacter pylori model of infection. Here, an Il17ra -/- mouse was generated on the FVB/n background, and the role of IL-17 signaling in the maintenance of barrier responses to H. pylori was investigated. Generating the Il17ra -/- on the FVB/n background allowed for the examination of responses in the paragastric lymph node and will allow for future investigation into carcinogenesis. While uninfected Il17ra -/- mice do not develop spontaneous gastritis following H. pylori infection, Il17ra -/- mice develop severe gastric inflammation accompanied by lymphoid follicle production and exacerbated production of Th17 cytokines. Increased inflammation in the tissue, increased IgA levels in the lumen, and reduced production of Muc5ac in the corpus correlate with increased H. pylori- induced paragastric lymph node activation. These data suggest that the cross talk between immune cells and epithelial cells regulates mucin production, IgA production, and translocation, impacting the integrity of the gastric mucosa and therefore activating of the adaptive immune response., Competing Interests: The authors declare no conflict of interest.

Secretory IgA (SIgA) is the most abundant antibody type in intestinal secretions where it contributes to safeguarding the epithelium from invasive pathogens like the Gram-negative bacterium, Salmonella enterica serovar Typhimurium (STm). For example, we recently reported that passive oral administration of the recombinant monoclonal SIgA antibody, Sal4, to mice promotes STm agglutination in the intestinal lumen and restricts bacterial invasion of Peyer's patch tissues. In this report, we sought to recapitulate Sal4-mediated protection against STm in human Enteroids and human intestinal organoids (HIOs) as models to decipher the molecular mechanisms by which antibodies function in mucosal immunity in the human gastrointestinal tract. We confirm that Enteroids and HIO-derived monolayers are permissive to STm infection, dependent on HilD, the master transcriptional regulator of the SPI-I type three secretion system (T3SS). Stimulation of M-like cells in both Enteroids and HIOs by the addition of RANKL further enhanced STm invasion. The apical addition of Sal4 mouse IgA, as well as recombinant human Sal4 dimeric IgA (dIgA) and SIgA resulted a dose-dependent reduction in bacterial invasion. Moreover, basolateral application of Sal4 dIgA to Enteroid and HIO monolayers gave rise to SIgA in the apical compartment via a pathway dependent on expression of the polymeric immunoglobulin receptor (pIgR). The resulting Sal4 SIgA was sufficient to reduce STm invasion of Enteroid and HIO epithelial cell monolayers by ~20-fold. Recombinant Sal4 IgG was also transported in the Enteroid and HIOs, but to a lesser degree and via a pathway dependent on the neonatal Fc receptor (FCGRT). The models described lay the foundation for future studies into detailed mechanisms of IgA and IgG protection against STm and other pathogens.

The intracellularly active bacterial toxin TcdB is a major Clostridioides difficile virulence factor that contributes to inflammation and tissue damage during disease. Immunization with an inactive TcdB fragment prevents C. difficile infection (CDI)-associated pathology. The protective immune response against inactive TcdB involves development of antigen-specific memory B cells and long-lived plasma cells that encode TcdB-neutralizing antibodies. Unlike the response to inactive TcdB, very little is known about the host humoral immune response to C. difficile and TcdB during primary and recurrent infection. Here, we used a murine model of C. difficile disease recurrence to demonstrate that an initial infection induced a serum IgM and mucosal IgA response against the toxin, but a low serum IgG response, which is associated with a lack of protection against disease during reinfection. Infection induced a partial expansion of the T follicular helper cell compartment, essential for B cell memory responses, and, consistent with that, failed to significantly expand the memory B cell compartment. Further, infection failed to stimulate the memory B cell compartment in preimmunized mice, although they were protected against associated disease. These results delineate the key humoral immune events that follow primary and recurrent C. difficile infection and provide a compelling inverse correlation between B cell memory and disease recurrence., (Copyright © 2020 American Society for Microbiology.)

Food allergy is a life-threatening response to specific foods, and microbiota imbalance (dysbiosis) in gut is considered a cause of this disease. Meanwhile, the host immune response also plays an important role in the disease. Notably, interleukin 33 (IL-33) released from damaged or necrotic intestinal epithelial cells facilitates IL-2-producing CD4 helper T (Th2) responses. However, causal relationships between the gut and oral dysbiosis and food allergy remain unknown. In this study, we analyzed effects of gut and oral dysbiosis on development of food allergy. A murine model of food allergy was established via ovalbumin (OVA) injection in BALB/c mice. Viable fecal bacteria were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). il33 expression in colon-26 mouse colon cells stimulated by isolated fecal bacteria was quantified by real-time PCR. Intestinal T cells from the mice were analyzed by flow cytometry. Salivary IgA levels were quantified by enzyme-linked immunosorbent assay (ELISA), and IgA-bound oral bacteria were detected by flow cytometry. Among fecal bacteria, the abundance of Citrobacter sp. increased in the feces of allergic mice and induced il33 expression in colon-26 cells. Orally administered Citrobacter koseri JCM1658 exacerbated systemic allergic symptoms and reduced intestinal Th17 cells. Salivary IgA and IgA-bound oral bacteria increased in the allergic mice. Based on the results described above, food allergy induced both gut and oral dysbiosis. Citrobacter sp. aggravated allergy symptoms by inducing IL-33 release from intestinal epithelial cells., (Copyright © 2019 Matsui et al.)

Infection with Giardia duodenalis is one of the most common causes of diarrheal disease in the world. While numerous studies have identified important contributions of adaptive immune responses to parasite control, much less work has examined innate immunity and its connections to the adaptive response during this infection. We explored the role of complement in immunity to Giardia using mice deficient in mannose-binding lectin (Mbl2) or complement factor 3a receptor (C3aR). Both strains exhibited delayed clearance of parasites and a reduced ability to recruit mast cells in the intestinal submucosa. C3aR-deficient mice had normal production of antiparasite IgA, butex vivo T cell recall responses were impaired. These data suggest that complement is a key factor in the innate recognition of Giardia and that recruitment of mast cells and activation of T cell immunity through C3a are important for parasite control., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The unbound drug hypothesis states that only unbound drug concentrations are active and available for clearance, and highly variable results regarding unbound vancomycin fractions have been reported in the literature. We have determined the unbound vancomycin fractions in four different patient groups by a liquid chromatography tandem mass spectrometry (LC-MS/MS) method and identified factors that modulate vancomycin binding. We have further developed and validated a prediction model to estimate unbound vancomycin concentrations. Vancomycin (unbound and total) concentrations were measured in 90 patients in four different hospital wards (hematology [n = 33 samples], intensive care unit [ICU] [n = 51], orthopedics [n = 44], and pediatrics [age range, 6 months to 14 years; n = 18]) by a validated LC-MS/MS method. Multiple linear mixed model analysis was performed to identify patient variables that were predictive of unbound vancomycin fractions and concentrations. The variables included in the model were patient age, ward, number of coadministered drugs with high protein binding, kidney function (estimated glomerular filtration rate [determined by Chronic Kidney Disease Epidemiology Collaboration formula]), alpha-1-acid glycoprotein, albumin, total bilirubin, IgA, IgM, urea, and total vancomycin concentrations. In the pediatric cohort, the median unbound vancomycin fraction was 81.3% (range, 61.9 to 95.9%), which was significantly higher (P < 0.01) than the unbound fraction found in the three adult patient cohorts (hematology, 60.6% [48.7 to 90.6%]; ICU, 61.7% [47.0 to 87.6%]; orthopedics, 56.4% [45.9 to 78.0%]). The strongest significant predictor of the unbound vancomycin concentration was the total drug concentration, completed by albumin in the pediatric cohort and albumin and IgA in the adult cohorts. Validation of our model was performed with data from 13 adult patients. A mean difference of 0.3 mg/liter (95% confidence interval [CI], -1.3 to 0.7 mg/liter; R(2) = 0.99 [95% CI, 0.95 to 0.99]) between measured and calculated unbound vancomycin concentrations demonstrated that the predictive performance of our model was favorable. Unbound vancomycin fractions vary significantly between pediatric and adult patients. We developed a formula to estimate the unbound fraction derived from total vancomycin, albumin, and IgA concentrations in adult patients., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Infants born to HIV-1-infected mothers in resource-limited areas where replacement feeding is unsafe and impractical are repeatedly exposed to HIV-1 throughout breastfeeding. Despite this, the majority of infants do not contract HIV-1 postnatally, even in the absence of maternal antiretroviral therapy. This suggests that immune factors in breast milk of HIV-1-infected mothers help to limit vertical transmission. We compared the HIV-1 envelope-specific breast milk and plasma antibody responses of clade C HIV-1-infected postnatally transmitting and nontransmitting mothers in the control arm of the Malawi-based Breastfeeding Antiretrovirals and Nutrition Study using multivariable logistic regression modeling. We found no association between milk or plasma neutralization activity, antibody-dependent cell-mediated cytotoxicity, or HIV-1 envelope-specific IgG responses and postnatal transmission risk. While the envelope-specific breast milk and plasma IgA responses also did not reach significance in predicting postnatal transmission risk in the primary model after correction for multiple comparisons, subsequent exploratory analysis using two distinct assay methodologies demonstrated that the magnitudes of breast milk total and secretory IgA responses against a consensus HIV-1 envelope gp140 (B.con env03) were associated with reduced postnatal transmission risk. These results suggest a protective role for mucosal HIV-1 envelope-specific IgA responses in the context of postnatal virus transmission. This finding supports further investigations into the mechanisms by which mucosal IgA reduces risk of HIV-1 transmission via breast milk and into immune interventions aimed at enhancing this response., Importance: Infants born to HIV-1-infected mothers are repeatedly exposed to the virus in breast milk. Remarkably, the transmission rate is low, suggesting that immune factors in the breast milk of HIV-1-infected mothers help to limit transmission. We compared the antibody responses in plasma and breast milk of HIV-1-transmitting and -nontransmitting mothers to identify responses that correlated with reduced risk of postnatal HIV-1 transmission. We found that neither plasma nor breast milk IgG antibody responses were associated with risk of HIV-1 transmission. In contrast, the magnitudes of the breast milk IgA and secretory IgA responses against HIV-1 envelope proteins were associated with reduced risk of postnatal HIV-1 transmission. The results of this study support further investigations of the mechanisms by which mucosal IgA may reduce the risk of HIV-1 transmission via breastfeeding and the development of strategies to enhance milk envelope-specific IgA responses to reduce mother-to-child HIV transmission and promote an HIV-free generation., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The natural immune response to Helicobacter pylori neither clears infection nor prevents reinfection. However, the ability of secretory antibodies to influence the course of H. pylori infection has not been determined. We compared the natural progression of H. pylori infection in wild-type C57BL/6 mice with that in mice lacking the polymeric immunoglobulin receptor (pIgR) that is essential for the secretion of polymeric antibody across mucosal surfaces. H. pylori SS1-infected wild-type and pIgR knockout (KO) mice were sampled longitudinally for gastrointestinal bacterial load, antibody response, and histological changes. The gastric bacterial loads of wild-type and pIgR KO mice remained constant and comparable at up to 3 months postinfection (mpi) despite SS1-reactive secretory IgA in the intestinal contents of wild-type mice at that time. Conversely, abundant duodenal colonization of pIgR KO animals contrasted with the near-total eradication of H. pylori from the intestine of wild-type animals by 3 mpi. H. pylori was cultured only from the duodenum of those animals in which colonization in the distal gastric antrum was of sufficient density for immunohistological detection. By 6 mpi, the gastric load of H. pylori in wild-type mice was significantly lower than in pIgR KO animals. While there was no corresponding difference between the two mouse strains in gastric pathology results at 6 mpi, reductions in gastric bacterial load correlated with increased gastric inflammation together with an intestinal secretory antibody response in wild-type mice. Together, these results suggest that naturally produced secretory antibodies can modulate the progress of H. pylori infection, particularly in the duodenum.

To establish a safer and more effective vaccine against pneumococcal respiratory infections, current knowledge regarding the antigens common among pneumococcal strains and improvements to the system for delivering these antigens across the mucosal barrier must be integrated. We developed a pneumococcal vaccine that combines the advantages of pneumococcal surface protein A (PspA) with a nontoxic intranasal vaccine delivery system based on a nanometer-sized hydrogel (nanogel) consisting of a cationic cholesteryl group-bearing pullulan (cCHP). The efficacy of the nanogel-based PspA nasal vaccine (cCHP-PspA) was tested in murine pneumococcal airway infection models. Intranasal vaccination with cCHP-PspA provided protective immunity against lethal challenge with Streptococcus pneumoniae Xen10, reduced colonization and invasion by bacteria in the upper and lower respiratory tracts, and induced systemic and nasal mucosal Th17 responses, high levels of PspA-specific serum immunoglobulin G (IgG), and nasal and bronchial IgA antibody responses. Moreover, there was no sign of PspA delivery by nanogel to either the olfactory bulbs or the central nervous system after intranasal administration. These results demonstrate the effectiveness and safety of the nanogel-based PspA nasal vaccine system as a universal mucosal vaccine against pneumococcal respiratory infection.

Proteins in human saliva are thought to modulate bacterial colonization of the oral cavity. Yet, information is sparse on how salivary proteins interact with systemic pathogens that transiently or permanently colonize the oral environment. Staphylococcus aureus is a pathogen that frequently colonizes the oral cavity and can cause respiratory disease in hospitalized patients at risk. Here, we investigated salivary protein binding to this organism upon exposure to saliva as a first step toward understanding the mechanism by which the organism can colonize the oral cavity of vulnerable patients. By using fluorescently labeled saliva and proteomic techniques, we demonstrated selective binding of major salivary components by S. aureus to include DMBT1(gp-340), mucin-7, secretory component, immunoglobulin A, immunoglobulin G, S100-A9, and lysozyme C. Biofilm-grown S. aureus strains bound fewer salivary components than in the planctonic state, particularly less salivary immunoglobulins. A corresponding adhesive component on the S. aureus surface responsible for binding salivary immunoglobulins was identified as staphylococcal protein A (SpA). However, SpA did not mediate binding of nonimmunoglobulin components, including mucin-7, indicating the involvement of additional bacterial surface adhesive components. These findings demonstrate that a limited number of salivary proteins, many of which are associated with various aspects of host defense, selectively bind to S. aureus and lead us to propose a possible role of saliva in colonization of the human mouth by this pathogen.

We developed means to deliver multiple heterologous antigens on dual plasmids with non-antibiotic-resistance markers in a single recombinant attenuated vaccine strain of Salmonella enterica serotype Typhimurium. The first component of this delivery system is a strain of S. Typhimurium carrying genomic deletions in alr, dadB, and asd, resulting in obligate requirements for diaminopimelic acid (DAP) and d-alanine for growth. The second component is the Asd(+)-DadB(+) plasmid pair carrying wild-type copies of asdA and dadB, respectively, to complement the mutations. To evaluate the protection efficacy of the dual-plasmid vaccine, S. Typhimurium strain χ9760 (a strain with multiple attenuating mutations: Δasd Δalr ΔdadB ΔrecF) was transformed with Asd(+) and DadB(+) plasmids specifying pneumococcal antigens PspA and PspC, respectively. Both plasmids were stable in χ9760 for 50 generations when grown in nonselective medium. This was significantly (P < 0.05) greater than the stability seen in its recF(+) counterpart χ9590 and could be attributed to reduced interplasmid recombination in χ9760. Oral immunization of BALB/c mice with 1 × 10(9) CFU of χ9760 (carrying Asd(+)-PspA and DadB(+)-PspC plasmids) elicited a dominant Th1-type serum IgG response against both antigens and protected mice against intraperitoneal challenge with 200 50% lethal doses (LD(50)s) of virulent Streptococcus pneumoniae strain WU2 or intravenous challenge with 100 LD(50)s of virulent S. pneumoniae strain L81905 or intranasal challenge with a lethal dose of S. pneumoniae A66.1 in a pneumonia model. Protection offered by χ9760 was superior to that offered by the mixture of two strains, χ9828 (Asd(+)-PspA) and χ11026 (DadB(+)-PspC). This novel dual-plasmid system marks a remarkable improvement in the development of live bacterial vaccines.

Influenza A viruses cause significant morbidity in swine, resulting in a substantial economic burden. Swine influenza virus (SIV) infection also poses important human public health concerns. Vaccination is the primary method for the prevention of influenza virus infection. Previously, we generated two elastase-dependent mutant SIVs derived from A/Sw/Saskatchewan/18789/02(H1N1): A/Sw/Sk-R345V (R345V) and A/Sw/Sk-R345A (R345A). These two viruses are highly attenuated in pigs, making them good candidates for a live-virus vaccine. In this study, the immunogenicity and the ability of these candidates to protect against SIV infection were evaluated in pigs. We report that intratracheally administrated R345V and R345A induced antigen-specific humoral and cell-mediated immunity characterized by increased production of immunoglobulin G (IgG) and IgA antibodies in the serum and in bronchoalveolar lavage fluid, high hemagglutination inhibition titers in serum, an enhanced level of lymphocyte proliferation, and higher numbers of gamma interferon-secreting cells at the site of infection. Based on the immunogenicity results, the R345V virus was further tested in a protection trial in which pigs were vaccinated twice with R345V and then challenged with homologous A/Sw/Saskatchewan/18789/02, H1N1 antigenic variant A/Sw/Indiana/1726/88 or heterologous subtypic H3N2 A/Sw/Texas/4199-2/9/98. Our data showed that two vaccinations with R345V provided pigs with complete protection from homologous H1N1 SIV infection and partial protection from heterologous subtypic H3N2 SIV infection. This protection was characterized by significantly reduced macroscopic and microscopic lung lesions, lower virus titers from the respiratory tract, and lower levels of proinflammatory cytokines. Thus, elastase-dependent SIV mutants can be used as live-virus vaccines against swine influenza in pigs.

Escherichia coli O157:H7 is an important pathogen of humans. Cattle are most frequently identified as the primary source of infection, and therefore, reduction in E. coli O157:H7 prevalence in cattle by vaccination represents an attractive strategy for reducing the incidence of human disease. H7 flagella have been implicated in intestinal-epithelial colonization of E. coli O157:H7 and may represent a useful target for vaccination. In this study, calves were immunized either systemically with H7 flagellin by intramuscular injection or mucosally via the rectum with either H7 or H7 incorporated into poly(DL-lactide-co-glycolide) microparticles (PLG:H7). Systemic immunization resulted in high levels of flagellin-specific immunoglobulin G (IgG) and IgA in both serum and nasal secretions and detectable levels of both antibody isotypes in rectal secretions. Rectal administration of flagellin resulted in levels of rectal IgA similar to those by the intramuscular route but failed to induce any other antibody response, whereas rectal immunization with PLG:H7 failed to induce any H7-specific antibodies. Following subsequent oral challenge with E. coli O157:H7, reduced colonization rates and delayed peak bacterial shedding were observed in the intramuscularly immunized group compared to nonvaccinated calves, but no reduction in total bacterial shedding occurred. Rectal immunization with either H7 or PLG:H7 had no effect on subsequent bacterial colonization or shedding. Furthermore, purified H7-specific IgA and IgG from intramuscularly immunized calves were shown to reduce intestinal-epithelial binding in vitro. These results indicate that H7 flagellin may be a useful component in a systemic vaccine to reduce E. coli O157:H7 colonization in cattle.

Immunogenic vaccines against group B Streptococcus (GBS) have been created by coupling the GBS capsular polysaccharides (CPS) to carrier proteins. The GBS beta C protein (BCP) serves as an effective carrier while inducing protective immunity against BCP-expressing strains. BCP also binds human immunoglobulin A (IgA), a characteristic that may be undesirable for use in humans. Here, we examined the immunogenicity and protective efficacy of a recombinant GBS BCP (rBCP), an rBCP modified to eliminate its IgA-binding site (rBCP(DeltaIgA)), and their corresponding GBS serotype III CPS conjugates (III-rBCP and III-rBCP(DeltaIgA)). Deletion of the IgA-binding site or conjugation to CPS did not alter antigenic BCP epitopes. Recombinant proteins and conjugates elicited specific, high-titered IgG in mice. Antisera to rBCP, rBCP(DeltaIgA), III-rBCP, and III-rBCP(DeltaIgA) opsonized GBS strains A909 (Ia/BCP(+)) and H36B (Ib/BCP(+)) for killing by HL-60 cells; antiserum to III-rBCP and III-rBCP(DeltaIgA) also opsonized strain M781 (III/BCP(-)). Vaccination of female mice with either rBCP or rBCP(DeltaIgA) protected approximately 40% of their pups challenged with GBS strain A909. Pups born to III-rBCP- or III-rBCP(DeltaIgA)-vaccinated dams survived at rates of 56% and 66%, respectively. Over 90% of pups born to dams that received the type III CPS conjugates survived challenge with GBS strain M781. In summary, rBCP and rBCP(DeltaIgA) proteins and the conjugates containing them were immunogenic in mice, inducing both CPS- and protein-specific functional IgG. These results suggest that the rBCP(DeltaIgA) could be used as a carrier to augment the immunogenicity of the CPS while expanding coverage to GBS strains bearing BCP.

Respiratory pathogens, such as Neisseria meningitidis, secrete site-specific proteases able to cleave human immunoglobulin A1 (IgA1), the first line of defense at mucosal membranes. Bacterial isolates show wide variability in IgA1 protease activity, and those isolated from patients with clinical infection possess the highest levels of activity. A feature of this enzyme is the self-cleavage required for secretion of the mature extracellular form. Known cleavage targets contain a proline-rich consensus recognition sequence, Pro-Pro-Ser-Pro, residing in the variable linker region that connects the protease and translocator domains. Here, we report the sequence of the NMB IgA1 protease and the unexpected self-cleavage and subsequent extracellular release of mature IgA1 protease from mutants lacking the previously defined consensus cleavage site. We investigated the possible link between enzyme secretion and variability in the linker sequence segment using site-directed mutagenesis and linker domain swapping to construct mutated and chimeric forms of the IgA1 protease from N. meningitidis strain NMB. The observed change in secreted activity levels compared to the wild-type clone indicated that the precise amino acid sequence of the intervening region, between mature IgA1 protease and the beta-core translocator domain, influences the efficacy of autoproteolytic processing. The broader specificity uncovered for the NMB IgA1 protease suggests that it could cleave a far wider range of human proteins than previously appreciated.

Francisella tularensis is a gram-negative intracellular bacterium that is considered to be a potential category A biological weapon due to its extreme virulence. Although vaccination with the attenuated live vaccine strain (LVS) of F. tularensis can protect against lethal challenge, use of inactivated or subunit forms as vaccine candidates for induction of protective antibody responses has not been fully evaluated. In the present study, we examined whether immune protection in the lung could be stimulated by intranasal administration of inactivated LVS together with interleukin-12 (IL-12) as an adjuvant. LVS was inactivated by heat, paraformaldehyde treatment, or exposure to UV, and inactivation of the preparations was confirmed by assessing bacterial growth and the survival of mice after direct inoculation. We found that mucosal vaccination with inactivated LVS provided 90 to 100% protection in mice after lethal intranasal challenge with 10(4) CFU of LVS, and this protection was dependent on inclusion of exogenous IL-12 during vaccine administration. Survival of vaccinated mice after live bacterial challenge was correlated with reduced bacterial burden, decreased pulmonary inflammation, increased serum antibody titers, and lower levels of gamma interferon (IFN-gamma), tumor necrosis factor alpha, and IL-6 in the lungs, livers, and spleens. Whereas NK cells were primarily responsible for the production of IFN-gamma in unvaccinated, challenged animals, vaccinated mice had increased levels of lung IFN-gamma+ CD4+ T cells after challenge. Significantly, mice genetically deficient in immunoglobulin A (IgA) expression were unable to survive lethal challenge after vaccination. These results are the first results to demonstrate that IgA-mediated protection against lethal respiratory tularemia occurs after mucosal vaccination with inactivated F. tularensis LVS.

Antibodies of the immunoglobulin A (IgA) class react with capsular polysaccharides of Streptococcus pneumoniae and support complement-dependent opsonophagocytosis (OPC) of the organism by phagocytes. We characterized the biologic impact of the molecular forms of human monoclonal capsule-specific IgA (monomeric IgA [mIgA], polymeric IgA [pIgA], and secretory IgA [SIgA]) on OPC and susceptibility to cleavage by IgA1 protease. The efficiency of SIgA in support of OPC of S. pneumoniae was comparable to that of pIgA, and both forms exceeded that of mIgA by a fivefold margin. This structure-function relationship was associated with three factors. First, the avidities, or functional affinities, of both pIgA and SIgA for pneumococcal capsules exceeded those of mIgA. Second, both pIgA and SIgA required less complement to achieve similar levels of bacterial OPC than did mIgA, indicating that secretory component does not hinder the effect of complement. Third, both pIgA and SIgA mediated agglutination of the organism, whereas mIgA did not. All three forms of capsule-specific IgA showed comparable susceptibilities to cleavage and functional inhibition by bacterial IgA1 protease, demonstrating that secretory component does not prevent the proteolytic degradation of IgA1 by IgA1 protease. IgA1 cleavage results in formation of identical Fab fragments for each of the molecular forms, thereby abolishing the contribution of multivalence of pIgA and SIgA. In summary, the polymeric forms of IgA (both pIgA and SIgA) provide a substantial advantage in binding, agglutination, and OPC of the organism.

A group 2 human coronavirus designated HECV-4408 was isolated from a child with acute diarrhea and is antigenically and genetically more closely related to bovine coronavirus (BCoV) than to human coronavirus OC43 (X. M. Zhang, W. Herbst, K. G. Kousoulas, and J. Storz, J. Med. Virol. 44:152-161, 1994). To determine whether HECV-4408 infects gnotobiotic calves and induces cross-protective immunity against the virulent enteric BCoV DB2 strain, gnotobiotic calves (n = 4) were orally inoculated with HECV-4408 and then challenged with BCoV DB2 at postinoculation day (PID) 21. All calves inoculated with HECV-4408 developed diarrhea at PID 3 to 4 lasting 5 to 9 days. Fecal and nasal virus shedding were first detected by reverse transcription-PCR at PID 3 to 4 and at PID 2 to 4, respectively. After challenge with bovine coronavirus, no diarrhea or virus shedding was detected in calves inoculated with HECV-4408, but a mock-inoculated calf developed diarrhea and fecal and nasal shedding. Fecal immunoglobulin A (IgA) and serum IgG antibodies were first detected at PID 7 and PID 14, respectively. At postchallenge day 7, serum IgG and fecal IgA antibody titers remained the same or increased only twofold compared to prechallenge titers. An additional two gnotobiotic calves were inoculated with HECV-4408 and euthanized at PID 5. Moderate villous atrophy was observed in the small intestines, and viral antigen was detected in villous enterocytes of the small and large intestines by immunohistochemistry. These results support and extend the previous report that HECV-4408 is likely a variant of bovine coronavirus. They confirm its infectivity for calves and complete cross-protection against a bovine coronavirus (DB2 strain) showing 98.2% amino acid identity to HECV-4408 in the S protein.

We have compared the use of five nonvaccine antigens to the use of conventional vaccine antigens, pertussis toxin (PT), and filamentous hemagglutinin (FHA) for the serological diagnosis of pertussis by enzyme-linked immunosorbent assay (ELISA). The nonvaccine antigens included the catalytic region of adenylate cyclase toxin (CatACT), the C-terminal region of FHA (C-FHA), lipooligosaccharide (LOS), the peptidoglycan-associated lipoprotein (PAL), and the BrkA protein. The serological responses of individuals with culture-confirmed pertussis were compared to those of adults with no recent history of a coughing disease. An immunoglobulin G (IgG) ELISA for PT was the most sensitive (92.2%) test for the serodiagnosis of pertussis. Of the nonvaccine antigens, ELISA for IgG responses to CatACT (sensitivity, 62.8%), C-FHA (sensitivity, 39.2%), and LOS IgA (sensitivity, 29.4%) were less sensitive but could also distinguish culture-positive individuals from control individuals. The use of a combination of multiple ELISA targets improved the sensitivity of the assay for serological diagnosis. Elevated IgG and IgA antibody titers persisted for more than a year in the individuals with culture-confirmed pertussis.

Neisseria meningitidis secretes a protease that specifically cleaves the hinge region of immunoglobulin A1 (IgA1), releasing the effector (Fc) domain of IgA1 from the antigen binding (Fab) determinants. Theoretically, the remaining Fab fragments can block pathogen receptors or toxins and still provide protection. Here, we describe binding of V-gene-matched human IgA1 and IgA2 to PorA of strain H44/76. On live meningococci, efficient cleavage of IgA1, but not cleavage of IgA2, was observed, and up to approximately 80% of the IgA1 Fc tails were lost from the meningococcal surface within 30 min. No cleavage of IgA1 was found on an isogenic H44/76 strain lacking IgA1 protease. Furthermore, our data indicate that PorA-bound IgA1 is masked by the serogroup B polysaccharide capsule, rendering the IgA1 less accessible to degradation by secreted IgA1 protease present in the bacterial surroundings. Experiments with protein synthesis inhibitors showed that de novo production of IgA1 protease was responsible for cleavage of PorA-bound IgA1 on encapsulated bacteria. Finally, our data suggest that cleavage of IgA1 by IgA1 protease releases a significant proportion of Fab fragments from the bacterium, probably as a result of their reduced avidity compared to that of whole antibodies.

Components of the human immunoglobulin A1 (IgA1) hinge governing sensitivity to cleavage by bacterial IgA1 proteases were investigated. Recombinant antibodies with distinct hinge mutations were constructed from a hybrid comprised of human IgA2 bearing half of the human IgA1 hinge region. This hybrid antibody and all the mutant antibodies derived from it were resistant to cleavage by the IgA1 proteases from Streptococcus oralis and Streptococcus mitis biovar 1 strains but were cleaved to various degrees by those of Streptococcus pneumoniae, some Streptococcus sanguis strains, and the type 1 and 2 IgA1 proteases of Haemophilus influenzae, Neisseria meningitidis, and Neisseria gonorrhoeae. Remarkably, those proteases that cleave a Pro-Ser peptide bond in the wild-type IgA1 hinge were able to cleave mutant antibodies lacking a Pro-Ser peptide bond in the hinge, and those that cleave a Pro-Thr peptide bond in the wild-type IgA1 hinge were able to cleave mutant antibodies devoid of a Pro-Thr peptide bond in the hinge. Thus, the enzymes can cleave alternatives to their preferred postproline peptide bond when such a bond is unavailable. Peptide sequence analysis of a representative antibody digestion product confirmed this conclusion. The presence of a cleavable peptide bond near the CH2 end of the hinge appeared to result in greater cleavage than if the scissile bond was at the CH1 end of the hinge. Proline-to-serine substitution at residue 230 in a hinge containing potentially cleavable Pro-Ser and Pro-Thr peptide bonds increased the resistance of the antibody to cleavage by many IgA1 proteases.

To efficiently colonize and persist in the lower respiratory tract, bacteria must survive multiple host immune mechanisms. Bordetella bronchiseptica is a gram-negative respiratory pathogen that naturally infects mice and persists in the lower respiratory tract for up to 49 days postinoculation. In this work, we examined the effect of mutation of the pagP gene on the persistence of B. bronchiseptica in the lower respiratory tract of mice. The pagP gene encodes a palmitoyl transferase that is responsible for the addition of a palmitoyl group to the lipid A region of B. bronchiseptica lipopolysaccharide. Data presented here confirm that a B. bronchiseptica deltapagP mutant demonstrates defective persistence in the lower respiratory tract of wild-type mice. We hypothesized that the defective persistence of the B. bronchiseptica deltapagP mutant was due to an increased susceptibility of this mutant to a host immune response. In vivo data indicate that both B cells and the complement component C3 are required for the reduced bacterial numbers of the deltapagP mutant on day 14 postinoculation. In addition, an in vitro complement killing assay demonstrated that B. bronchiseptica exhibits pagP-dependent resistance to antibody-mediated complement killing at low concentrations of immune serum. Taken together, these results suggest that pagP is required for B. bronchiseptica to resist antibody-mediated complement lysis during respiratory infection.

The cleavage of human serum monomeric immunoglobulin A1 (IgA1) and human secretory IgA1 (S-IgA1) by IgA1 proteinase of Neisseria meningitidis and cleavage by the proteinase from Proteus mirabilis have been compared. For serum IgA1, both proteinases cleaved only the alpha chain. N. meningitidis proteinase cleaved only in the hinge. P. mirabilis proteinase sequentially removed the tailpiece, the CH3 domain, and the CH2 domain. The cleavage of S-IgA1 by N. meningitidis proteinase occurred only in the hinge and was as rapid as that of serum IgA1. P. mirabilis proteinase predominantly cleaved the secretory component (SC) of S-IgA1. The SC of S-IgA1, whether cleaved or not, appeared to protect the alpha1 chain. Purified Fc fragment derived from the cleavage of serum IgA1 by N. meningitidis proteinase stimulated a respiratory burst in neutrophils through Fcalpha receptors, whereas the (Fcalpha1)(2)-SC fragment from digested S-IgA1 did not. The loss of the tailpiece from serum IgA1 treated with P. mirabilis proteinase had little effect, but the loss of the CH3 domain was concurrent with a rapid loss in the ability to bind to Fcalpha receptors. S-IgA1 treated with P. mirabilis proteinase under the same conditions retained the ability to bind to Fcalpha receptors. The results are consistent with the Fcalpha receptor binding site being at the CH2-CH3 interface. These data shed further light on the structure of S-IgA1 and indicate that the binding site for the Fcalpha receptor in S-IgA is protected by SC, thus prolonging its ability to activate phagocytic cells at the mucosal surface.

Secretory immunoglobulin A (IgA) protects the mucosal surfaces against inhaled and ingested pathogens. Many pathogenic bacteria produce IgA1 proteases that cleave in the hinge of IgA1, thus separating the Fab region from the Fc region and making IgA ineffective. Here, we show that Haemophilus influenzae type 1 and Neisseria gonorrhoeae type 2 IgA1 proteases cleave the IgA1 hinge in the context of the constant region of IgA1 or IgA2m(1) but not in the context of IgG2. Both C(alpha)2 and C(alpha)3 but not C(alpha)1 are required for the cleavage of the IgA1 hinge by H. influenzae and N. gonorrhoeae proteases. While there was no difference in the cleavage kinetics between wild-type IgA1 and IgA1 containing only the first GalNAc residue of the O-linked glycans, the absence of N-linked glycans in the Fc increased the ability of the N. gonorrhoeae protease to cleave the IgA1 hinge. Taken together, these results suggest that, in addition to the IgA1 hinge, structures in the Fc region of IgA are required for the recognition and cleavage of IgA1 by the H. influenzae and N. gonorrhoeae proteases.

The amino acid sequence requirements in the hinge of human immunoglobulin A1 (IgA1) for cleavage by IgA1 proteases of different species of Streptococcus were investigated. Recombinant IgA1 antibodies were generated with point mutations at proline 227 and threonine 228, the residues lying on either side of the peptide bond at which all streptococcal IgA1 proteases cleave wild-type human IgA1. The amino acid substitutions produced no major effect upon the structure of the mutant IgA1 antibodies or their functional ability to bind to Fcalpha receptors. However, the substitutions had a substantial effect upon sensitivity to cleavage with some streptococcal IgA1 proteases, with, in some cases, a single point mutation rendering the antibody resistant to a particular IgA1 protease. This effect was least marked with the IgA1 protease from Streptococcus pneumoniae, which showed no absolute requirement for either proline or threonine at residues 227 to 228. By contrast, the IgA1 proteases of Streptococcus oralis, Streptococcus sanguis, and Streptococcus mitis had an absolute requirement for proline at 227 but not for threonine at 228, which could be replaced by valine. There was evidence in S. mitis that proteases from different strains may have different amino acid requirements for cleavage. Remarkably, some streptococcal proteases appeared able to cleave the hinge at a distant alternative site if substitution prevented efficient cleavage of the original site. Hence, this study has identified key residues required for the recognition of the IgA1 hinge as a substrate by streptococcal IgA1 proteases, and it marks a preliminary step towards development of specific enzyme inhibitors.

Protective host defense mechanisms against vaginal Candida albicans infections are poorly understood. Although cell-mediated immunity (CMI) is the predominant host defense mechanism against most mucosal Candida infections, the role of CMI against vaginal candidiasis is uncertain, both in humans and in an experimental mouse model. The role of humoral immunity is equally unclear. While clinical observations suggest a minimal role for antibodies against vaginal candidiasis, an experimental rat model has provided evidence for a protective role for Candida-specific immunoglobulin A (IgA) antibodies. Additionally, Candida vaccination-induced IgM and IgG3 antibodies are protective in a mouse model of vaginitis. In the present study, the role of infection-induced humoral immunity in protection against experimental vaginal candidiasis was evaluated through the quantification of Candida-specific IgA, IgG, and IgM antibodies in serum and vaginal lavage fluids of mice with primary and secondary (partially protected) infection. In naïve mice, total, but not Candida-specific, antibodies were detected in serum and lavage fluids, consistent with lack of yeast colonization in mice. In infected mice, Candida-specific IgA and IgG antibodies were induced in serum with anamnestic responses to secondary infection. In lavage fluid, while Candida-specific antibodies were detectable, concentrations were extremely low with no anamnestic responses in mice with secondary infection. The incorporation of alternative protocols-including infections in a different strain of mice, prolongation of primary infection prior to secondary challenge, use of different enzyme-linked immunosorbent assay capture antigens, and concentration of lavage fluid-did not enhance local Candida-specific antibody production or detection. Additionally, antibodies were not removed from lavage fluids by being bound to Candida during infection. Together, these data suggest that antibodies are not readily present in vaginal secretions of infected mice and thus have a limited natural protective role against infection.

A T helper type 1 (Th1) response is essential for resolving genital infections with the mouse pneumonitis biovar of Chlamydia trachomatis (MoPn). However, T-cell-dependent anti-chlamydial antibody is produced and may also contribute to protective immunity. We produced a MoPn-specific CD4 Th2 clone (Th2-MoPn) to study the role of a Th2 response during infection. We found that Th2-MoPn was unable to eradicate chlamydiae from the genital tract (GT) when it was transferred into MoPn-infected nude mice. Mice that received Th2-MoPn produced greater titers of MoPn-specific serum immunoglobulin G (IgG) antibody than mice that received a MoPn-specific Th1 clone (Th1-MoPn) (log(10) titers, 1.89 +/- 0.84 and 0.58 +/- 0.76 [mean +/- standard deviation], respectively [P < 0.01]). Also, the IgG isotypes were different for the two groups; whereas IgG1 was associated with Th2-MoPn, IgG2a was associated with Th1-MoPn. Also, infected nude mice that received Th2-MoPn produced higher levels of IgA in vaginal secretions. Although clone Th2-MoPn was detected in the GT, it was less efficient at migrating (112 +/- 35.6 labeled Th2 clone cells/10(5) GT cells) than Th1-MoPn (505 +/- 51.6 Th1 clone cells/10(5) GT cells) (P < 0.001, as determined by a t test). This may have been due to reduced expression of alpha4beta7 and P-selectin ligand 1 on Th2-MoPn. However, Th2-MoPn cells were retained in the GT during chronic infection and comprised 10 to 15% of the total GT cells 80 days after transfer. The data show that the MoPn-specific Th2 cells are important for serum and vaginal antibody production and may accumulate in the GT during chronic infection.

The eib genes of Escherichia coli encode surface-exposed proteins which bind immunoglobulins (Ig) such as the Fc fragment of human IgG (IgG Fc) in a nonimmune manner. The Eib proteins belong to a family which includes YadA of Yersinia, UspA2 of Moraxella, and DsrA of Haemophilus ducreyi. This family of surface-exposed proteins shares several features, such as the ability to impart resistance to human serum complement and a tendency to exist as stable multimers. Four genes, eibA, eibC, eibD and eibE, were previously identified and cloned from ECOR-9, a strain from the E. coli reference collection. EibC, -D, and -E bind human serum IgA in addition to IgG, but no IgA binding has been observed for EibA. Here, we report the cloning of a new eib gene, eibF, from a second strain of E. coli, ECOR-2. The product, EibF, has a relatively strong preference for IgA. Like the other eib genes, eibF attenuates serum sensitivity, occurs as a stable multimer, and is associated with a prophage. By subcloning portions of the eibA and eibF genes, we have identified distinct sequence segments sufficient to cause Ig binding, multimerization, and discrimination between IgA and IgG. The ability to multimerize is associated with a sequence close to the C terminus that is homologous to other family members such as YadA. Binding of IgG Fc is associated with a sequence that is highly conserved among all Eib proteins but otherwise unique. Binding of IgA is associated with a sequence of EibF that is not similar to any EibA sequence.

Streptococcus pyogenes secretes a specific immunoglobulin G (IgG)-protease, SpeB, as well as the IgG glycan-hydrolyzing enzyme EndoS. Here we show that SpeB also degrades IgA, IgM, IgD, and IgE. We also show that EndoS only hydrolyzes the glycan moiety on native but not denatured IgG. Thus, SpeB has a broad immunoglobulin-degrading activity, while EndoS is highly specific for IgG.

Synthetic peptide vaccines which are derived from functional domains of Streptococcus mutans glucosyltransferases (GTF) have been shown to induce protective immunity in Sprague-Dawley rats after subcutaneous injection in the salivary gland region. Since mucosal induction of salivary immunity would be preferable in humans, we explored methods to induce mucosal antibody in the rat to the GTF peptide vaccines HDS and HDS-GLU after intranasal administration. Several methods of facilitation of the immune response were studied: the incorporation of peptides in bioadhesive poly(D,L-lactide-coglycolide) (PLGA) microparticles, the use of monoepitopic (HDS) or diepitopic (HDS-GLU) peptide constructs, or the use of mucosal adjuvants. Salivary immunoglobulin A (IgA) responses were not detected after intranasal administration of diepitopic HDS-GLU peptide constructs in alum or after incorporation into PLGA microparticles. However, significant primary and secondary salivary IgA and serum IgG antibody responses to HDS were induced in all rats when cholera holotoxin (CT) or a detoxified mutant Escherichia coli heat-labile enterotoxin (R192G LT) were intranasally administered with HDS peptide constructs in PLGA. Coadministration of LT with HDS resulted in predominantly IgG2a responses in the serum, while coadministration with CT resulted in significant IgG1 and IgG2a responses to HDS. Serum IgG antibody, which was induced to the HDS peptide construct by coadministration with these adjuvants, also bound intact mutans streptococcal GTF in an enzyme-linked immunosorbent assay and inhibited its enzymatic activity. Thus, immune responses which are potentially protective for dental caries can be induced to peptide-based GTF vaccines after mucosal administration if combined with the CT or LT R192G mucosal adjuvant.

To understand more about the factors influencing the cleavage of immunoglobulin A1 (IgA1) by microbial IgA1 proteases, a recombinant human IgA2/IgA1 hybrid molecule was generated. In the hybrid, termed IgA2/A1 half hinge, a seven-amino-acid sequence corresponding to one half of the duplicated sequence making up the IgA1 hinge was incorporated into the equivalent site in IgA2. Insertion of the IgA1 half hinge into IgA2 did not affect antigen binding capacity or the functional activity of the hybrid molecule, as judged by its ability to bind to IgA Fcalpha receptors and trigger respiratory bursts in neutrophils. Although the IgA2/A1 hybrid contained only half of the IgA1 hinge, it was found to be cleaved by a variety of different bacterial IgA1 proteases, including representatives of those that cleave IgA1 in the different duplicated halves of the hinge, namely, those of Prevotella melaninogenica, Streptococcus pneumoniae, S. sanguis, Neisseria meningitidis types 1 and 2, N. gonorrhoeae types 1 and 2, and Haemophilus influenzae type 2. Thus, for these enzymes the recognition site for IgA1 cleavage is contained within half of the IgA1 hinge region; additional distal elements, if required, are provided by either an IgA1 or an IgA2 framework. In contrast, the IgA2/A1 hybrid appeared to be resistant to cleavage with S. oralis and some H. influenzae type 1 IgA1 proteases, suggesting these enzymes require additional determinants for efficient substrate recognition.

Immunoglobulin A1 (IgA1) protease, an enzyme that selectively cleaves human IgA1, may be a virulence factor for pathogenic organisms such as Neisseria gonorrhoeae. Host protection from the effects of IgA1 protease includes antibody-mediated inhibition of IgA1 protease activity, and it is believed that the relative balance between IgA1 protease and inhibitory antibodies contributes to the pathogenesis of disease caused by IgA1 protease-producing organisms. We have examined the levels of these two opposing factors in genital tract secretions and sera from women with uncomplicated infection with N. gonorrhoeae. When IgA1 in cervical mucus was examined by Western blotting, no evidence of cleavage fragments characteristic of IgA1 protease activity was seen in gonococcus-infected or control patients. Cleavage fragments typical of IgA1 protease were detected, however, after the addition of exogenous IgA1 protease to cervical mucus. Degraded IgA1 was detected in some vaginal wash samples, but the fragment pattern was not typical of IgA1 protease activity. All N. gonorrhoeae isolates from the infected patients produced IgA1 protease in vitro. All but two serum samples and 16 of 65 cervical mucus samples displayed inhibitory activity against gonococcal IgA1 protease, but there was no significant difference in the level of inhibitory activity between gonococcus-infected and noninfected patients in either cervical mucus or serum. There was no difference in the levels of IgA1 protease-inhibitory activity in serum or cervical mucus collected from patients at recruitment and 2 weeks later. These results suggest that cleavage of IgA1 by gonococcal IgA1 protease within the lumen of the female lower genital tract is unlikely to be a significant factor in the pathogenesis of infections by N. gonorrhoeae.

Rotavirus (RV) strains infecting newborns often have unique neutralization antigens (P serotypes) on their outer capsids that are distinct from those found on RV strains that cause diarrhea in older children. We examined the hypothesis that unusual RV strains preferentially infect newborns because the newborns lack maternal neutralizing antibodies to these strains. To test this hypothesis, sera and saliva samples collected from neonates infected with 116E-like (P[11]G9) strains in the maternity ward of the All India Institute of Medical Sciences (AIIMS) hospital in New Delhi were tested for neutralizing antibodies against common RV strains and those infecting newborns and these titers were compared with those of newborns who did not become infected (controls). The infected neonates had significantly lower levels of cord blood neutralizing antibodies to 116E than the controls, suggesting that immunity to neonatal RV infection is acquired transplacentally through maternal antibodies. Further, this study confirmed the immunogenicity of the AIIMS neonatal strain 116E, a vaccine candidate, in its ability to evoke a potent RV-specific immunoglobulin A and neutralizing antibody response in serum and saliva among the infected babies. Our findings have important implications for the development of an effective RV vaccine. In India, where G9 strains are common in the community, the use of 116E as a vaccine, together with the rhesus tetravalent vaccine, may provide a broader protection against all the circulating RV serotypes, including serotype G9, which is not represented in the current rhesus RV tetravalent vaccine (G1-G4).

Oral Prevotella and Capnocytophaga species, regularly isolated from periodontal pockets and associated with extraoral infections, secret specific immunoglobulin A1 (IgA1) proteases cleaving human IgA1 in the hinge region into intact Fab and Fc fragments. To investigate whether these enzymes are subject to inhibition in vivo in humans, we tested 34 sera from periodontally diseased and healthy individuals in an enzyme-linked immunosorbent assay for the presence and titers of inhibition of seven Prevotella and Capnocytophaga proteases. All or nearly all of the sera inhibited the IgA1 protease activity of Prevotella buccae, Prevotella oris, and Prevotella loescheii. A minor proportion of the sera inhibited Prevotella buccalis, Prevotella denticola, and Prevotella melaninogenica IgA1 proteases, while no sera inhibited Capnocytophaga ochracea IgA1 protease. All inhibition titers were low, ranging from 5 to 55, with titer being defined as the reciprocal of the dilution of serum causing 50% inhibition of one defined unit of protease activity. No correlation between periodontal disease status and the presence, absence, or titer of inhibition was observed. The nature of the low titers of inhibition in all sera of the IgA1 proteases of P. buccae, P. oris, and P. loescheii was further examined. In size exclusion chromatography, inhibitory activity corresponded to the peak volume of IgA. Additional inhibition of the P. oris IgA1 protease was found in fractions containing both IgA and IgG. Purification of the IgG fractions of five sera by passage of the sera on a protein G column resulted in recovery of inhibitory IgG antibodies against all three IgA1 proteases, with the highest titer being for the P. oris enzyme. These finding indicate that inhibitory activity is associated with enzyme-neutralizing antibodies.

The beta-antigen of the c protein complex of group B streptococci contains two immunoglobulin A (IgA)-binding domains called A and B. A 73-amino-acid segment in domain A is responsible for most of the IgA-binding activity. To identify the IgA binding motif, the 73-amino-acid domain was divided into 60 14-amino-acid overlapping peptides spot synthesized onto a cellulose membrane. A 20-residue putative antigenic epitope was identified and expressed as a fusion protein. The fusion protein was purified by fast protein liquid chromatography and used to raise rabbit antiserum. By use of a membrane with spot-synthesized peptide amino acids of decreasing length (from 14 to 6 amino acids), the major antigenic epitope recognized by the anti-fusion protein antibodies was mapped to motif MLKKIE. Anti-fusion protein antibodies inhibited the binding of IgA to group B streptococci. This inhibition could be blocked by the peptide containing the motif MLKKIE. These results indicate that the motif MLKKIE is located in the IgA-binding site. The IgA-binding domain of beta-antigen from three group B streptococcal strains reacted with the anti-fusion protein antibodies, and their coding sequences gave positive signals in Southern hybridization. The sequences of beta-antigen from these strains were amplified by PCR, and sequence analysis showed them to be identical. The results indicate that the motif MLKKIE is required for IgA binding and is present in different group B streptococcal strains.

Immunoglobulin A (IgA) proteases are bacterial enzymes with substrate specificity for human serum and secretory IgAs. To further define the basis of this specificity, we examined the ability of IgA proteases of Clostridium ramosum, Streptococcus pneumoniae (EC 3.4.24.13), Neisseria meningitidis (EC 3.4.21.72), and Haemophilus influenzae (EC 3.4.21.72) to cleave serum IgAs of gorillas, chimpanzees, and orangutans. All enzymes cleaved the IgAs of the three apes despite differences in ape IgA1 hinge sequence relative to the human prototype. To directly compare the ape and human hinge cleavage sites, the sites were identified in eight ape IgA digests. This analysis confirmed that ape proteins were all cleaved in the IgA hinge region, in all but one case after proline residues. The exception, C. ramosum protease, cleaved gorilla and chimpanzee IgAs at peptide bonds having no proline, but the scissile bonds were in the same hinge location as the Pro-221-Val-222 cleaved in human IgA1. These data indicate that proline is not an invariant substrate requirement for all IgA proteases and that the location of the scissile bond, in addition to its composition, is a critical determinant of cleavage specificity.

Pneumolysin, an intracellular protein toxin of all clinically relevant pneumococcal serotypes, is released in vivo during the autolysis of pneumococci and is believed to pave the way for intact pneumococci to invade and cause disease. Therefore, antibodies to pneumolysin should prevent its destructive function. We measured antibodies to pneumococcal pneumolysin in acute- and convalescent-phase nasopharyngeal aspirate samples of 120 children (median age, 2.5 years) with acute otitis media by enzyme immunoassay. Nasopharyngeal immunoglobulin M (IgM) and IgG class antibodies to pneumolysin were rarely detectable, whereas IgA class antibody was detected often, occurred independently of serum IgA antibody in serum, and correlated with the presence of the secretory component in pneumococcal antibody, indicating local production of IgA antibodies. Nasopharyngeal IgA antibody to pneumolysin was detected in 93% of the children already in the acute phase of otitis. Twenty percent of the children developed at least a threefold rise in the pneumolysin-specific IgA antibody concentration by the convalescent phase of otitis, with the youngest at 6 months of age, regardless of the pneumococcal findings in the nasopharynx or middle ear fluid. We suggest that nasopharyngeal IgA antibody to pneumolysin can be produced early in life by pneumococcal colonization.

Trichomonas vaginalis is a protozoan parasite that causes a widely distributed sexually transmitted disease (STD). Since immunoglobulin G (IgG) antibodies to specific trichomonad immunogens are found in serum and vaginal washes (VWs) from patients with trichomoniasis, a potential mechanism of immune evasion by this parasite might be the ability of T. vaginalis proteinases to degrade human immunoglobulins (Igs). Incubation of human IgG with lysates of T. vaginalis organisms resulted in time- and concentration-dependent degradation of the heavy chain. Secretory IgA was degraded similarly. Inhibitors of cysteine proteinases, when added to trichomonal lysates, abolished IgG and IgA degradation, while EDTA, a metalloproteinase inhibitor, did not. Substrate-gel electrophoresis with human IgG, IgM, or IgA copolymerized with acrylamide revealed several distinct cysteine proteinases in both lysates and culture supernatants from logarithmically growing parasites that degraded all classes of human antibodies. Trichomonal lysates and supernatants of numerous isolates tested all had Ig-degrading activity. Finally, proteolytic activity against IgG was detected in most (26 of 33; 78%) VWs from patients with trichomoniasis. In contrast, 18 of 28 (65%) VWs from women without trichomoniasis or from patients infected with other STDs had no detectable proteinases when tested in an identical manner. The other 10 of these 28 VWs (35%) had smaller amounts of detectable Ig-degrading proteinases. These differences in Ig-degrading proteinase activity between patients with and without trichomoniasis, regardless of coinfecting STDs, were statistically significant (P = 0.001). These results illustrate that T. vaginalis is capable of degrading human Igs.

We studied the immune response and the colonization pattern in vitamin A-deficient rats that were colonized with the Escherichia coli O6 K13 pOmp 21 strain, which is genetically manipulated to produce ovalbumin and to be resistant to ampicillin. In the vitamin A-deficient rats, the number of bacteria per gram of feces was about five times higher than in the paired fed control rats 4 weeks after colonization. In the control rats, the colon and the lower part of the ileum were colonized, while in the vitamin A-deficient rats all parts of the small intestine, as well as the colon, were heavily inhabited by bacteria. Furthermore, in 75% of the vitamin A-deficient rats, the E. coli bacteria were found in the mesenteric lymph nodes, and in 50% of the rats E. coli were found in the kidneys. These animals also developed severe arthritis. The levels of serum immunoglobulin G (IgG), IgM, IgE, and biliary IgA antibodies against the bacterial antigens were significantly higher in the vitamin A-deficient rats than in the control rats. The number of IgA-producing cells in the lamina propria of the small intestine was significantly lower in the vitamin A-deficient rats than in the control rats; however, there was an increase in the number of CD8+ cells and transforming growth factor beta-producing cells in the lamina propria of the vitamin A-deficient rats. Disturbances in T-cell function were demonstrated, since spleen cells from the vitamin A-deficient rats produced more gamma interferon and interleukin-2 in vitro than control spleen cells. In summary, vitamin A deficiency led to a decrease in the ability to control the localization of intestinal bacteria and an increase in translocation, which was followed by development of arthritis regardless of substantial levels of antibacterial antibodies. The bacterial invasion made the animals hyperresponsive to the bacterial antigens, despite the fact that vitamin A deficiency is normally associated with suppressed antibody production, as previously shown by us and others.

Malnourished rats during suckling were orally immunized with cholera toxin (CT) after different periods of refeeding. Intestinal fluids, sera, and supernatant fluids from cultured mesenteric lymph node (MLN) cells were obtained after rats were given three doses of CT and analyzed by enzyme-linked immunosorbent assay (ELISA) to evaluate the specific antibody response. Serum-specific immunoglobulin G (IgG), IgA, and IgM were severely diminished in malnourished rats immunized with three doses of CT after 1 week of refeeding when compared with those of controls. Also, a decreased IgA ELISA titer of the intestinal fluids and abrogation of the capacity to neutralize the CT in the intestinal ligated loop test were found. When a booster was given at 113 days of age, the immune response continued to be affected in the serum and the intestinal fluid. The results from the analysis of the supernatant fluids from cultured MLN cells were coincident with those mentioned above. When one dose of CT was administered into Peyer's patches (PP) after 1 week of refeeding, an impaired immune response was found in the intestinal fluid of malnourished rats during suckling compared with that of controls. This result together with the analysis of supernatant from MLN and PP cell cultures suggests that antigen triggering in the PP was affected. When the refeeding period was extended to 30 days and then the first dose of CT was administered, the antibody immune responses in intestinal fluid serum and supernatant fluid approached control values. These observations reinforce the fact that the gut-associated lymphoid tissue immaturity of the rats when they received the first CT dose (at 28 days old) was the main reason for the decreased immune response observed in the experimental group.

As part of a longitudinal study of the relationship between bacterial colonization and the secretory immune response, 367 isolates of pioneer viridans streptococci collected from 40 breast- and bottle-fed neonates within the first month postpartum were tested for the production of immunoglobulin A1 (IgA1) protease and glycosidases. Fifty percent of the streptococci isolated produced IgA1 protease, including all isolates of Streptococcus oralis and S. sanguis, 60.7% of S. mitis biovar 1 isolates, and some isolates that could not be identified. Three cleavage patterns of alpha 1 heavy chains were observed. Six isolates of S. mitis biovar 1 that did not produce IgA1 protease attacked the alpha 1 chain. Incubation of IgA1 protease-negative S. mitis biovar 1 isolates with IgA1, either prior to or together with S. sanguis, rendered the IgA1 paraprotein resistant to cleavage by the IgA1 protease of S. sanguis. The ability of some pioneer streptococci in the human oral cavity to produce IgA1 protease and of others to modify the susceptibility of IgA1 to cleavage by IgA1 protease perhaps enhances their ability to survive in this habitat.

Many strains of group A streptococci are capable of binding human immunoglobulin A (IgA) by a nonimmune mechanism. M or M-like proteins constitute a family of structurally diverse molecules which form surface fibrillae, and some of the M or M-like protein forms are responsible for the IgA-binding activity. In this report, the binding site for IgA is localized within two structurally distinct M or M-like proteins, ML2.2 and Arp4. Apart from those structural domains which are common to all M and M-like proteins, ML2.2 and Arp4 lack significant levels of amino acid sequence homology, with the exception of a short segment (ALXGENXDLR) located at residues 21 to 30 of the mature ML2.2 protein. Recombinant fusion polypeptides containing portions of the ML2.2 and Arp4 proteins were expressed in Escherichia coli and tested for binding of human myeloma IgA. A 58-residue polypeptide containing residues 14 to 71 of ML2.2 bound human IgA. The IgA-binding site of Arp4 could be localized to a 53-residue polypeptide containing residues 43 to 95, which encompasses the ALXGENXDLR consensus sequence of Arp4 positioned at residues 50 to 59. Site-specific mutagenesis at three codons within the ALXGENXDLR coding sequence of both the ML2.2 and Arp4 recombinant polypeptides leads to a loss in IgA-binding activity. Thus, the ALXGENXDLR consensus sequence is essential for the nonimmune binding of IgA by both ML2.2 and Arp4. However, the failure to bind IgA by polypeptides which partially overlap the 58- and 53-residue IgA-binding polypeptides of ML2.2 and Arp4, yet contain the ALXGENXDLR consensus sequence, strongly suggests that flanking regions are also critical for IgA binding. In summary, the results indicate that common functional domains bearing significant sequence homology are distributed within regions of M or M-like molecules that are otherwise highly divergent.

A therapeutic dose of azithromycin was administered to test subjects and then the following lymphocyte functions were examined in vitro: proliferative lymphocyte response to stimulation with pokeweed mitogen, levels of immunoglobulins G, A, and M in serum, and the amount of the soluble interleukin 2 receptors in supernatants of mononuclear cell cultures stimulated with phytohemagglutinin and phorbol myristate acetate. The study was performed as a controlled clinical trial comparing an azithromycin-treated group (n = 21) and a placebo-treated control group (n = 10). Healthy female volunteers were placed into one of the two groups, and the study was performed as a double-blind trial. Although the findings of the present study showed that azithromycin significantly increased the proliferative lymphocyte response to pokeweed mitogen, the results could have been due to experimental variation. However, impairment of the lymphocyte function was not observed, which could represent valuable information. Likewise, no effect of azithromycin on levels of the immunoglobulins in serum was observed. The most marked effect of azithromycin on the lymphocyte function was demonstrated by an elevation in the amount of soluble interleukin 2 receptor production in mononuclear cell cultures. The lack of impairment or, perhaps, even a beneficial influence on the immunodefense system may be an important property of azithromycin, especially in immunocompromised individuals.

The proteinases secreted by pathogenic strains of Proteus mirabilis, P. vulgaris biotype 2, P. vulgaris biotype 3, and P. penneri were purified with almost 100% recovery by affinity chromatography on phenyl-Sepharose followed by anion-exchange chromatography. The proteinase purified from the urinary tract pathogen P. mirabilis, which we had previously shown to degrade immunoglobulins A and G, appeared as a composite of a single band and a double band (53 and 50 kDa, respectively) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The other Proteus proteinases had similar patterns but slightly different mobilities. In each case all proteinase activity in culture supernatants was demonstrated by gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be associated with only the triple-band complex; all three bands were proteolytically active. The P. mirabilis proteinase was resistant to inhibitors of both serine and thiol proteinases but strongly inhibited by metal chelators, although it was not affected by phosphoramidon, an inhibitor of the thermolysin group of bacterial metalloproteinases. Active proteinase was detected in urine samples from P. mirabilis-infected patients; this is consistent with our detection of immunoglobulin A fragments of a size suggestive of P. mirabilis proteinase activity.

Bacterial immunoglobulin A1 (IgA1) proteases cleaving IgA1 and secretory IgA1 molecules in the hinge region are believed to be important virulence factors. Previous studies have indicated that IgA of humans, gorillas, and chimpanzees are the exclusive substrates of these enzymes. In a recent study, IgA from the rhesus monkey was found to be susceptible to the IgA1 protease activity of Streptococcus pneumoniae. In an attempt to reproduce this observation, we found that neither five isolates of S. pneumoniae nor other IgA1 protease-producing bacteria representing different cleavage specificities caused cleavage of rhesus monkey IgA. Hence, the rhesus monkey does not appear to be a suitable animal model for studies of IgA1 proteases as virulence factors.

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

Atypical vancomycin pharmacokinetics were observed in an immunoglobulin A myeloma patient. Drug concentrations in serum were extremely elevated, the elimination half-life was prolonged despite normal renal function, and the vancomycin therapy was ineffective. Extensive binding of vancomycin, presumably by high concentrations of an aberrant immunoglobulin A protein, may have accounted for these observations.

Immunoglobulin A (IgA) antibodies against pertussis toxin (PT) and filamentous hemagglutinin (FHA) in 181 saliva samples obtained during various stages of pertussis from 112 patients were determined. Saliva samples obtained within 5 days after the onset of symptoms did not have detectable IgA antibodies against either of the two antigens. Of the samples obtained between 6 and 50 days after the onset of symptoms, 72% had antibodies against FHA but only 40% had antibodies against PT. With few exceptions, saliva samples obtained more than 50 days after the onset of symptoms contained antibodies against both antigens. In the 59 patients from whom paired saliva samples were obtained at intervals of 2 to 5 weeks, a significant increase in the geometric mean FHA antibody titers but not PT antibody titers occurred. However, increases that were fourfold or greater were observed against FHA in only 19 patients and against PT in 14 patients. Thus, IgA antibodies against FHA and PT in saliva develop during pertussis, and the importance of secretory IgA antibodies for protection against infection and disease should be investigated. Determination of these antibodies in paired saliva samples is, however, of little value for the laboratory diagnosis of pertussis.

Proteus mirabilis strains associated with human urinary tract infections have previously been shown to secrete an extracellular metalloproteinase which cleaves serum immunoglobulin A (IgA). The enzyme has now been purified to apparent homogeneity from culture supernatants of P. mirabilis 64676. The protease activity is associated with a 50-kilodalton (kDa) protein. Unlike that of the classic IgA1 proteases, the substrate specificity of the P. mirabilis protease has been found to extend to both sublcasses of IgA, IgG, and the nonimmunoglobulin substrates, secretory component and casein. Cleavage of IgA1 and IgA2 by the P. mirabilis protease yielded fragments on sodium dodecyl sulfate-polyacrylamide gel electrophoresis whose sizes were consistent with a cleavage site outside the hinge region. Both secretory IgA1 and IgA2 were also cleaved by P. mirabilis protease, although the secretory IgA2 molecule was less readily cleaved than secretory IgA1. Free and IgA-bound secretory components were degraded to some extent by P. mirabilis protease. Cleavage of IgG, however, occurred at the hinge region as a two-stage process. The first stage was pepsinlike and generated an F(ab')2 fragment of 120 kDa and a small pFc fragment detected on nonreduced polyacrylamide gels. In the second stage, the F(ab')2 product was cleaved to yield papainlike Fab and Fc fragments, visualized as a diffuse band of 40 to 50 kDa.