Specific Secretory Immunoglobulin Research Articles

Bacillus subtilis expressing duck Tembusu virus E protein induces immune protection in ducklings

Duck Tembusu virus (DTMUV) is an infectious disease that emerged in China in 2010. It has caused serious economic losses to the poultry industry and may pose a threat to public health. We aimed to develop a new Bacillus subtilis (B. subtilis)-based oral vaccine to control DTMUV transmission among poultry; to this end, we constructed a B. subtilis strain that can secrete DTMUV E protein. Ducklings were orally immunized, and serum antibodies, mucosal antibodies, and splenic cytokines were detected. The results showed that, in addition to high levels of specific IgG, there were also high levels of specific secretory immunoglobulin A (sIgA) in ducklings orally treated with recombinant B. subtilis. In addition, the levels of IFN-γ, IL-2, IL-4, and IL-10 in spleens were significantly boosted by recombinant B. subtilis. Recombinant B. subtilis could effectively enhance ducklings resistance to DTMUV and significantly reduce viral load (p<0.01), along with pathological damage in the brain, heart, and spleen. This is the first study to apply a B. subtilis live-vector vaccine platform for DTMUV disease prevention and control, and our results suggest that B. subtilis expressing DTMUV E protein may be a candidate vaccine against DTMUV.

Oral administration of recombinant Bacillus subtilis expressing a multi-epitope protein induces strong immune responses against Salmonella Enteritidis

The S. Enteritidis causes serious economic losses to the poultry industry every year. Vaccines that induce a mucosal immune response may be successful against an S. Enteritidis infection because mucosa plays an important role in preventing S. Enteritidis from entering the body. In order to develop novel and potent oral vaccines based on Bacillus subtilis (B. subtilis) to control the spread of S. Enteritidis in the poultry industry, we constructed a B. subtilis that can secrete a multi-epitope protein (OmpC-FliC-SopF-SseB-IL-18). Oral immunization of chickens was performed, and serum antibodies, mucosal antibodies, specific cellular immunity and serum cytokines were detected. Immunizing chicks with S. Enteritidis was evaluated. The results showed high levels of specific IgG in addition to high levels of specific secretory immunoglobulin A (sIgA) in chickens who received oral administrations of recombinant B. subtilis. Additionally, recombinant B. subtilis may significantly increase the levels of IL-2 and T cell-mediated immunity. Recombinant B. subtilis effectively protected chickens against S. Enteritidis and reduced pathological damage to the spleen and jejunum. Our study’s outcomes indicate that the expression of the multi-epitope protein OmpC-FliC-SopF-SseB-IL-18 by B. subtilis could generate a mucosal vaccine candidate for animals to defend against S. Enteritidis in the future.

Oral vaccine of recombinant Lactococcus lactis expressing the VP1 protein of duck hepatitis A virus type 3 induces mucosal and systemic immune responses

Duck hepatitis A virus (DHAV) is the major pathogen of duck viral hepatitis, which has caused great economic losses to duck breeding industry. As an effective delivery tool for protein antigens, Lactococcus lactis (L. lactis) has been successfully used to stimulate mucosal and systemic immune response. In this study, a recombinant L. lactis named NZ3900-VP1 was constructed, which could express VP1 protein of DHAV type 3 (DHAV-3) by using a nisin-controlled expression (NICE) system. The animal experiment in both mice and ducklings were performed to detect the immune response and protection effect of oral vaccination by the recombinant L. lactis. The results showed that oral vaccination with L. lactis NZ3900-VP1 significantly induced specific anti-VP1 IgG antibodies and mucosal secretory immunoglobulin A (sIgA) of DHAV-3 in mice and ducklings, and cytokines including interleukin-2 (IL-2), interferon gamma (IFN-γ), interleukin-10 (IL-10) and interleukin-4 (IL-4). Notably, the ducklings vaccinated with L. lactis NZ3900-VP1 were effectively protected when facing natural infestation of DHAV-3, which indicated that the recombinant L. lactis could serve as an effective vaccine to prevent DHAV-3 infection in ducklings.

Immune responses induced by recombinant Lactobacillus plantarum expressing the spike protein derived from transmissible gastroenteritis virus in piglets.

Transmissible gastroenteritis coronavirus (TGEV) is one of the most severe threats to the swine industry. In this study, we constructed a suite of recombinant Lactobacillus plantarum with surface displaying the spike (S) protein coming from TGEV and fused with DC cells targeting peptides (DCpep) to develop an effective, safe, and convenient vaccine against transmissible gastroenteritis. Our research results found that the recombinant Lactobacillus plantarum (NC8-pSIP409-pgsA-S-DCpep) group expressing S fused with DCpep could not only significantly increase the percentages of MHC-II+CD80+ B cells and CD3+CD4+ T cells but also the number of IgA+ B cells and CD3+CD4+ T cells of ileum lamina propria, which elevated the specific secretory immunoglobulin A (SIgA) titers in feces and IgG titers in serum. Taken together, these results suggest that NC8-pSIP409-pgsA-S-DCpep expressing the S of TGEV fused with DCpep could effectively induce immune responses and provide a feasible original strategy and approach for the design of TGEV vaccines.

Development of a new DNA vaccine based on mycobacterial ESAT-6 antigen delivered by recombinant invasive Lactococcus lactis FnBPA+.

The use of the food-grade bacterium Lactococcus lactis as a vehicle for the oral delivery of DNA vaccine plasmids constitutes a promising strategy for vaccination. The delivery of DNA plasmids into eukaryotic cells is of critical importance for subsequent DNA expression and effectiveness of the vaccine. In this context, the use of the recombinant invasive L. lactis FnBPA+ (fibronectin-binding protein A) strain for the oral delivery of the eukaryotic expression vector vaccination using lactic acid bacteria (pValac), coding for the 6-kDa early secreted antigenic target (ESAT-6) gene of Mycobacterium tuberculosis, could represent a new DNA vaccine strategy against tuberculosis. To this end, the ESAT-6 sequence was cloned into the pValac vector; the L. lactis fibronectin-binding protein A (FnBPA)+ (pValac:ESAT-6) strain was obtained, and its immunological profile was checked in BALB/c mice. This strain was able to significantly increase interferon gamma (IFN-γ) production in spleen cells, showing a systemic T helper 1 (Th1) cell response. The mice also showed a significant increase in specific secretory immunoglobulin A (sIgA) production in colon tissue and fecal extracts. Thus, this is the first time that L. lactis has been used to deliver a plasmid DNA harboring a gene that encodes an antigen against tuberculosis through mucous membranes.

Evaluation of the live vaccine efficacy of virulence plasmid-cured, and phoP- or aroA-deficient Salmonella enterica serovar Typhimurium in mice.

We evaluated the protective efficacy of 94-kb virulence plasmid-cured, and phoP- or aroA-deficient strains of Salmonella enterica serovar Typhimurium (ΔphoP or ΔaroA S. Typhimurium) as oral vaccine candidates in BALB/c mice. Two weeks after the completion of 3 oral immunizations with 1 × 108 colony-forming units (CFU) of virulence plasmid-cured, and ΔphoP or ΔaroA S. Typhimurium at 10-day intervals, S. Typhimurium lipopolysaccharide (LPS)-specific mucosal secretory immunoglobulin A (s-IgA) antibody titers were detected in the cecal homogenate, bile and lung lavage fluid, but not in the intestinal lavage fluid. In addition, the increases in S. Typhimurium LPS-specific immunoglobulin G (IgG) and IgA antibody titers in the serum were also observed 2 weeks after completing 3 oral immunizations with virulence plasmid-cured, and ΔphoP or ΔaroA S. Typhimurium. The series of 3 oral immunizations protected the mice against an oral challenge with 5 × 108 CFU of the virulent strain of S. Typhimurium, suggesting that both the virulence plasmid-cured, and ΔphoP and ΔaroA S. Typhimurium strains are promising candidates for safe and effective live S. Typhimurium vaccines.

Rotavirus specific plasma secretory immunoglobulin in children with acute gastroenteritis and children vaccinated with an attenuated human rotavirus vaccine

Rotavirus (RV)–specific secretory immunoglobulin (RV-SIg) has been previously detected in serum of naturally RV infected children and shown to reflect the intestinal Ig immune response. Total plasma SIgA and plasma RV-SIg were evaluated by ELISA in children with gastroenteritis due or not due to RV infection and in 50 children vaccinated with the attenuated RIX4414 human RV vaccine and 62 placebo recipients. RV-SIg was only detected in children with evidence of previous RV infection or with acute RV gastroenteritis. Vaccinees had higher RV-SIg titers than placebo recipients and RV-SIg titers increased after the second vaccine dose. RV-SIg measured after the second dose correlated with protection when vaccinees and placebo recipients were analyzed jointly. RV-SIg may serve as a valuable correlate of protection for RV vaccines.

Intranasal immunization of Balb/c mice against prion protein attenuates orally acquired transmissible spongiform encephalopathy

To test whether prion protein (PrP) specific secretory immunoglobulin A (sIgA) can be induced and protect against oral transmission of spongiform encephalopathy (SE) we immunized Balb/c mice either intragastrically or intranasally (i.n.) with a recombinant PrP-fragment (PrP90-231) and cholera toxin (CT) adjuvant. Since PrP90-231 was rapidly digested in intestinal lavage, aprotinin was added to some vaccine formulations. While an anti-CT response was elicited via both routes, solely i.n. immunization without aprotinin induced PrP-specific sIgA. They recognize predominantly PrP-oligomers as the antigen was aggregated in the vaccine formulations. Challenge experiments showed that the immune response induced by our protocol could not prevent disease, but increases the median survival of the animals. We conclude that PrP-specific sIgA reduce the infectivity of the inoculum and that complete protection against transmission of SE should be achievable by optimized immunization regimens.

Role of secretory antibodies in the defence against infections

AbstractAdaptive immunity mediated by secretory antibodies is important in the defence against mucosal infections. Specific secretory immunoglobulin A (SIgA) can inhibit initial pathogen colonization by performing immune exclusion both on the mucosal surface and within virus-infected secretory epithelial cells without causing tissue damage. Resistance against toxin-producing bacteria such as Vibrio cholerae appears to be particularly dependent on SIgA antibodies. Like natural infections, live topical vaccines or adequate combinations of inactivated vaccines and mucosal adjuvants give rise not only to SIgA antibodies, but also to longstanding serum IgG and IgA responses. The intranasal route of vaccine application could be particularly attractive to achieve this result, but only if successful stimulation is obtained without the use of toxic adjuvants. The degree of protection after vaccination may range from complete inhibition of reinfection to reduction of symptoms. In this scenario it is generally difficult to determine unequivocally the relative importance of SIgA versus serum antibodies. However, infection models in knockout mice strongly support the notion that SIgA exerts a decisive role in protection and cross-protection against a variety of infectious agents.

Effect of antimicrobial factors in human milk on rhinoviruses and milk-borne cytomegalovirus in vitro.

Various antimicrobial factors present in human milk were tested for in-vitro antiviral activity against three rhinoviruses (two clinical isolates and rhinovirus 2) and an isolate of cytomegalovirus (CMV) from human milk. These factors included the gangliosides GM1, 2 and 3, sialyl-lactose, chondroitin sulphates A, B and C, prostaglandins E2 and F2alpha, monolaurin, vitamin A and the protein lactoferrin. All were tested for their ability to inhibit growth of the viruses in cell culture. Human milk was also tested for antiviral activity against these viruses. Only vitamin A, monolaurin and lactoferrin inhibited the growth of CMV, whereas both prostaglandins enhanced the growth of this virus at least four-fold. CMV infects infants from milk but, nevertheless, the milk-borne CMV isolate showed no special resistance to any of the antiviral factors tested. None of the compounds inhibited or enhanced the growth of the rhinoviruses. However, human milk decreased the growth of some of the rhinoviruses and specific secretory immunoglobulin A (sIgA) neutralised the virus.

Correlations between antibody immune responses at different mucosal effector sites are controlled by antigen type and dosage.

Monitoring specific secretory immunoglobulin A (IgA) responses in the intestines after mucosal immunization or infection is impeded by the fact that sampling of small intestinal secretions requires invasive methods not feasible for routine diagnostics. Since IgA plasma cells generated after intragastric immunization are known to populate remote mucosal sites as well, secretory IgA responses at other mucosal surfaces may correlate to those in the intestines and could serve as proxy measures for IgA secretion in the gut. To evaluate the practicability of this approach, mice were immunized intragastrically with 0.2, 2, and 20 mg of ovalbumin plus 10 microg of cholera toxin, and the antigen-specific local secretory IgA responses in duodenal, ileal, jejunal, rectal, and vaginal secretions, saliva, urine, and feces, as well as serum IgG and IgA responses were analyzed by enzyme-linked immunosorbent assay. Correlation analysis revealed significant relationships between serum IgG and IgA, urinary IgA, salivary IgA, and secretory IgA in duodenal, jejunal, ileal, and rectal secretions for the 0.2-mg but not for the 20-mg ovalbumin dose. Fecal samples were poor predictors for intestinal antiovalbumin IgA responses, and no correlations could be established for cholera toxin, neither between local anti-cholera toxin levels nor to the antiovalbumin responses. Thus, specific IgA in serum, saliva, or urine can serve as a predictor of the release of specific IgA at intestinal surfaces after intragastric immunization, but the lack of correlations for high ovalbumin doses and for cholera toxin indicates a strong dependency on antigen type and dosage for these relationships.

Intranasal immunization with C5a peptidase prevents nasopharyngeal colonization of mice by the group A Streptococcus

Early inflammatory events are initiated by phased production of C5a and interleukin-8 in tissue. Most serotypes of group A streptococci express a surface-bound peptidase (SCPA) which specifically cleaves mouse and human C5a chemotaxins. This study investigates the impact of SCPA on colonization of the nasopharyngeal mucosa of mice and evaluates its potential to induce protective immunity. Two strains, serotypes M6 and M49, which contain insertion and deletion mutations in the SCPA gene (scpA) and represent the two major subdivisions of group A streptococci, were characterized and compared in a mouse intranasal infection model. In this model, SCPA mutants were more rapidly cleared from the nasopharynges of inoculated mice compared with wild-type strains. A 2,908-bp fragment of scpA49 gene, obtained by PCR, was ligated to the expression vector pGEX-4T-1 and expressed in Escherichia coli. The affinity-purified deltaSCPA49 protein proved to be highly immunogenic in mice and rabbits. Although the purified deltaSCPA49 immunogen lacked enzymatic activity, it induced high titers of rabbit antibodies which were able to neutralize peptidase activity associated with M1, M6, M12, and M49 streptococci in vitro. This result confirmed that antipeptidase antibodies lack serotype specificity. Intranasal immunization of mice with the deleted form of the SCPA49 protein stimulated significant levels of specific salivary secretory immunoglobulin A (IgA) and serum IgG antibodies and reduced the potential of wild-type M1, M2, M6, M11, and M49 streptococci to colonize. These experiments suggest a new approach to vaccine development for prevention of streptococcal pharyngitis.

Persistence of serum and salivary antibody responses after oral immunization with a bacterial protein antigen genetically linked to the A2/B subunits of cholera toxin.

Primary oral immunization of mice with a bacterial protein antigen genetically coupled to the A2 and B subunits of cholera toxin induced specific secretory immunoglobulin A and serum immunoglobulin G antibodies that persisted at substantial levels for at least 11 months. A subsequent single booster immunization did not further enhance the antibody responses. Long-term antibody persistence may be especially important in infections caused by common pathogens for which continuous immunity would be advantageous.

Oral immunization using live attenuated Salmonella spp. as carriers of foreign antigens.

A variety of techniques, including the use of live oral vaccines, have been used to deliver antigens to the gut-associated lymphoid tissues in an attempt to initiate production of specific secretory immunoglobulin A for protection against pathogens that colonize or cross mucosal surfaces to initiate infection. A number of attenuated Salmonella mutants are able to interact with the lymphoid tissues in the Peyer's patches but are not able to cause systemic disease. Some of these mutants are effective as live vaccines (i.e., able to protect against infection with the virulent Salmonella parent) and are candidates for use as carriers for virulence determinants of other mucosal pathogens. This has been shown to be an effective means of stimulating significant levels of specific mucosal secretory immunoglobulin A directed against the carrier strains and against a variety of heterologous antigens and has been shown to stimulate production of serum antibodies and cell-mediated responses as well. This review examines the history of this mechanism of vaccine delivery and summarizes the most recent applications of this evolving technology. This is a technique for vaccine delivery with significant potential for influencing the management of infectious diseases on a large scale. It can be used not only for vaccines against enteric bacterial pathogens but also for vaccines against a variety of other bacteria, viruses, and parasites. The results obtained to date are encouraging, and there is great potential for development of safe, effective, affordable vaccines.

Human Giardiasis: Correlation of Specific Secretory IgA levels in Duodenal Fluid to the Severity of Disease and Infestation by Giardia Lamblia

Giardia lamblia specific secretory immunoglobulin A (sIgA) levels in the duodenal fluid of adult giardiasis cases are reported for the first time. The sIgA levels in the study group were found to be significantly higher (p less than 0.01) than in the 20 age- and sex-matched controls comprising cases classified as non-ulcerative dyspepsia who did nor reveal any G. lamblia in their stools and the duodenal fluid. An inverse relationship between the clinical severity of giardiasis and the level of sIgA in the duodenal fluid was noted. Cases with a higher trophozoite load in duodenal aspirate tended to be associated with envanescent G. lamblia-specific antibodies.

Effects of sample processing on the measurement of specific intestinal IgA immune responses

The effects of techniques commonly used in the collection and processing of human intestinal fluid on the specific secretory immunoglobulin A (sIgA) response following oral immunization with the live typhoid vaccine Salmonella typhi Ty21a were examined. It was observed that the failure to adjust specific intestinal anti-typhoid lipopolysaccharide IgA antibody titres for total secretory IgA resulted in a false-negative detection rate of 19.8% and a false-positive detection rate of 7.4%. Furthermore, these specific responses were significantly diminished if the intestinal fluid was subjected to heat inactivation to reduce intestinal protease activity (p=0.0083), but were not affected if stored at −70°C for up to 1 year, without heat inactivation. It was concluded that in the processing of the intestinal fluid samples for specific sIgA determination heat inactivation significantly reduced specific sIgA titres, and that the failure to adjust absolute titres for total sIgA content resulted in a significant false-negative detection rate.

Subclass distribution of salivary secretory immunoglobulin A antibodies to oral streptococci

The ability of specific secretory immunoglobulin A (S-IgA) antibodies to inhibit bacterial colonization of mucosal surfaces may be neutralized by the activity of bacterial IgA1 proteases. Because of the resistance of the IgA2 subclass to these enzymes, the biological effect of IgA1 proteases in vivo may depend on the subclass distribution of the bacterium-specific antibodies. We have estimated the subclass distribution of S-IgA antibodies in saliva samples from 13 individuals against IgA1 protease-producing (Streptococcus sanguis and Streptococcus oralis) and nonproducing (Streptococcus gordonii and Streptococcus mitis bv. 2) oral streptococci. IgA1 was found to be the predominant subclass of antibodies against these four bacteria in most of the saliva samples, corroborating previous data suggesting a role of IgA1 proteases in plaque formation. However, variation in the subclass distribution of S-IgA antibodies against the same strain was observed. In one individual, IgA2 was the predominant subclass of antibodies against all four streptococci and of total salivary S-IgA, pointing to the possible significance of genetic variations. The study also addresses methodological problems related to the quantitation of salivary antibodies by solid-phase immunoassays.

An ‘antigen capture’ ELISA for secretory immunoglobulin A antibodies to hepatitis B surface antigen in human saliva

An enzyme-linked immunosorbent assay (ELISA) was developed which permits the assay of specific secretory immunoglobulin A (sIgA) antibodies against hepatitis B surface antigen (HBsAg) in saliva. The assay is based on the binding of sIgA antibodies present in saliva to microtitre plates coated with excess of F(ab′) 2 anti-secretory component antibodies, followed by the addition of specific antigen, HBsAg and finally peroxidase-labelled anti-HBsAg. The assay is fast, simple, reproducible and antigen specific as shown by total absence of inhibition of specific antigen by unrelated antigens but significant inhibition of labelled anti-HBsAg by unlabelled anti-HBsAg. The values obtained for hospital personnel exposed to hepatitis infections (0.068 ± 0.083 U/ml) and for post-icteric hepatitis B patients (0.062 ± 0.033 U/ml) were significantly higher than values in control subjects (0.013 ± 0.006 U/ml).

Protective secretory immunoglobulin A antibodies in humans following oral immunization with Streptococcus mutans.

Ingestion of a vaccine containing killed Streptococcus mutans, originally isolated from each volunteer, daily for 10 consecutive days induced increased levels of specific secretory immunoglobulin A (sIgA) antibodies to S. mutans cells and two cell surface proteins, glucosyltransferase and surface antigen I/II, in parotid saliva and tears of four healthy males and in parotid saliva, tears, colostrum, and milk of a pregnant woman. In addition, these antibodies inhibited glucosyltransferase activity. Both IgA1 and IgA2 antibodies were induced. The levels of IgA antibodies in all secretions remained significantly above preimmunization levels for more than 50 days after oral administration of antigen. A second series of immunizations for 7 consecutive days resulted in even higher levels of sIgA antibodies, which peaked earlier and persisted longer than those observed after the primary immunizations. No increase in levels of antibodies in serum were detected in any subject. Antibodies reactive with human heart and kidney antigens could not be detected in saliva, tears, colostrum, milk, or serum samples collected at any time during the immunization regimen. The numbers of viable S. mutans organisms in dental plaque and whole saliva decreased after each series of immunizations, which correlated with increased levels of IgA antibodies in saliva, suggesting that IgA antibodies in saliva were responsible for the reduced adherence of this bacterium. These results indicate that ingested S. mutans antigen induces secretion of specific IgA1 and IgA2 antibodies in saliva, tears, colostrum, and milk, providing further evidence for the existence of a common mucosal immune system.

An enzyme-linked immunosorbent assay (ELISA) for anti-chlamydial secretory immunoglobulin A in guinea pig tears

A method is described which permits the assay of specific secretory immunoglobulin A (sIgA) antibodies produced by guinea pigs in response to ocular infection with the guinea pig inclusion conjunctivitis strain of Chlamydia psittaci (GPIC agent). The enzyme-linked immunosorbent assay (ELISA) developed was shown to be more sensitive and less subjective than the micro-immunofluorescence assay as a means of assaying specific antibody.