Development of a colloidal gold-based immunochromatographic assay for rapid detection of nasal mucosal secretory IgA against SARS-CoV-2.

Introduction: Infection with SARS-CoV-2 begins in the upper respiratory tract and can trigger the production of mucosal spike-specific secretory IgA (sIgA), which provides protection against reinfection. It has been recognized that individuals with high level of nasal spike-specific IgA have a lower risk of reinfection. However, mucosal spike-specific sIgA wanes over time, and different individuals may have various level of spike-specific sIgA and descending kinetics, leading to individual differences in susceptibility to reinfection. A method for detecting spike-specific sIgA in the nasal passage would be valuable for predicting the risk of reinfection so that people at risk can have better preparedness. Methods: In this study, we describe the development of a colloidal gold-based immunochromatographic (ICT) strip for detecting SARS-CoV-2 Omicron spike-specific sIgA in nasal mucosal lining fluids (NMLFs). Results: The ICT strip was designed to detect 0.125 µg or more spike-specific sIgA in 80 µL of NMLFs collected using a nasal swab. Purified nasal sIgA samples from individuals who recently recovered from an Omicron BA.5 infection were used to demonstrate that this ICT strip can specifically detect spike-specific sIgA. The signal levels positively correlated with neutralizing activities against XBB. Subsequent analysis revealed that people with low or undetectable levels of spike-specific sIgA in the nasal passage were more susceptible to SARS-CoV-2 reinfection. Conclusions: This nasal spike-specific sIgA ICT strip provides a non-invasive, rapid, and convenient method to assess the risk of reinfection for achieving precision preparedness.

Intestinal fungi and antifungal secretory immunoglobulin A in Crohn's disease.

The human gastrointestinal tract harbors trillions of commensal microorganisms. Emerging evidence points to a possible link between intestinal fungal dysbiosis and antifungal mucosal immunity in inflammatory bowel disease, especially in Crohn's disease (CD). As a protective factor for the gut mucosa, secretory immunoglobulin A (SIgA) prevents bacteria from invading the intestinal epithelium and maintains a healthy microbiota community. In recent years, the roles of antifungal SIgA antibodies in mucosal immunity, including the regulation of intestinal immunity binding to hyphae-associated virulence factors, are becoming increasingly recognized. Here we review the current knowledge on intestinal fungal dysbiosis and antifungal mucosal immunity in healthy individuals and in patients with CD, discuss the factors governing antifungal SIgA responses in the intestinal mucosa in the latter group, and highlight potential antifungal vaccines targeting SIgA to prevent CD.

Assessment of human milk samples obtained pre and post-influenza vaccination reveals a poor boosting of seasonally-relevant, hemagglutinin-specific antibodies.

Introduction: Influenza (flu) vaccination prevented over 100,000 hospitalizations and 7000 deaths from flu over the 2019-2020 season in the USA. Infants <6 months are the most likely to die from flu, though flu vaccines are only licensed for infants >6 months old. Therefore, it is recommended that flu vaccination occur during pregnancy, as this reduces severe complications; however, vaccination rates are suboptimal, and vaccination is also recommended postpartum. For breast/chest-fed infants, the vaccine is believed to elicit protective and robust seasonally-specific milk antibody (Ab). Few comprehensive studies exist examining Ab responses in milk after vaccination, with none measuring secretory Ab (sAb). Determining whether sAbs are elicited is critical, as this Ab class is highly stable in milk and mucosae. Methods: In the present study, our aim was to determine to what extent specific Ab titers in the milk of lactating people were boosted after seasonal influenza vaccination. Over the 2019-2020 and 2020-2021 seasons, milk was obtained pre- and post-vaccination and assessed for specific IgA, IgG, and sAb against relevant hemagglutinin (HA) antigens by a Luminex immunoassay. Results: IgA and sAb were not found to be significantly boosted, while only IgG titers against B/Phuket/3073/2013, included in vaccines since 2015, exhibited an increase. Across the 7 immunogens examined, as many as 54% of samples exhibited no sAb boost. No significant differences for IgA, sAb, or IgG boosting were measured between seasonally-matched versus mismatched milk groups, indicating boosting was not seasonally-specific. No correlations between IgA and sAb increases were found for 6/8 HA antigens. No boost in IgG- or IgA-mediated neutralization post vaccination was observed. Discussion: This study highlights the critical need to redesign influenza vaccines with the lactating population in mind, wherein the aim should be to elicit a potent seasonally-specific sAb response in milk. As such, this population must be included in clinical studies.

A candidate glycoconjugate vaccine induces protective antibodies in the serum and intestinal secretions, antibody recall response and memory T cells and protects against both typhoidal and non-typhoidal Salmonella serovars.

Human Salmonella infections pose significant public health challenges globally, primarily due to low diagnostic yield of systemic infections, emerging and expanding antibiotic resistance of both the typhoidal and non-typhoidal Salmonella strains and the development of asymptomatic carrier state that functions as a reservoir of infection in the community. The limited long-term efficacy of the currently licensed typhoid vaccines, especially in smaller children and non-availability of vaccines against other Salmonella serovars necessitate active research towards developing a multivalent vaccine with wider coverage of protection against pathogenic Salmonella serovars. We had earlier reported immunogenicity and protective efficacy of a subunit vaccine containing a recombinant outer membrane protein (T2544) of Salmonella Typhi in a mouse model. This was achieved through the robust induction of serum IgG, mucosal secretory IgA and Salmonella-specific cytotoxic T cells as well as memory B and T cell response. Here, we report the development of a glycoconjugate vaccine, containing high molecular weight complexes of Salmonella Typhimurium O-specific polysaccharide (OSP) and recombinant T2544 that conferred simultaneous protection against S. Typhi, S. Paratyphi, S. Typhimurium and cross-protection against S. enteritidis in mice. Our findings corroborate with the published studies that suggested the potential of Salmonella OSP as a vaccine antigen. The role of serum antibodies in vaccine-mediated protection is suggested by rapid seroconversion with high titers of serum IgG and IgA, persistently elevated titers after primary immunization along with a strong antibody recall response with higher avidity serum IgG against both OSP and T2544 and significantly raised SBA titers of both primary and secondary antibodies against different Salmonella serovars. Elevated intestinal secretory IgA and bacterial motility inhibition by the secretory antibodies supported their role as well in vaccine-induced protection. Finally, robust induction of T effector memory response indicates long term efficacy of the candidate vaccine. The above findings coupled with protection of vaccinated animals against multiple clinical isolates confirm the suitability of OSP-rT2544 as a broad-spectrum candidate subunit vaccine against human infection due to typhoidal and non-typhoidal Salmonella serovars.

Colostrum IgA1 antibodies recognize antigens from Helicobacter pylori and prevent cytoskeletal changesin human epithelial cells.

Helicobacter pylori is a Gram-negative bacterium found on the luminal surface of the gastric mucosa in at least 50% of the world's human population. The protective effect of breastfeeding against H. pylori infection has been extensively reported; however, the mechanisms behind this protection remain poorly understood. Human IgA from colostrum has reactivity against H. pylori antigens. Despite that IgA1 and IgA2 display structural and functional differences, their reactivity against H. pylori had not been previously determined. We attested titers and reactivity of human colostrum-IgA subclasses by ELISA, immunoblot, and flow cytometry. Colostrum samples from healthy mothers had higher titers of IgA; and IgA1 mostly recognized H. pylori antigens. Moreover, we found a correlation between IgA1 reactivity and their neutralizing effect determined by inhibition of cytoskeletal changes in AGS cells infected with H. pylori. In conclusion, colostrum-IgA reduces H. pylori infection of epithelial gastric cells, suggesting an important role in preventing the bacteria establishment during the first months of life. As a whole, these results suggest that IgA1 from human colostrum provides protection that may help in the development of the mucosal immune system of newborn children.

Intranasal Vaccination With Multiple Virulence Factors Promotes Mucosal Clearance of Streptococcus suis Across Serotypes and Protects Against Meningitis in Mice.

BACKGROUND: Streptococcus suis is an emerging zoonotic agent. Its natural habitat is the tonsils, which are the main portals of S. suis entry into the bloodstream of pigs. The remarkable variability of the bacteria and complex pathogenic mechanisms make the development of a vaccine a difficult task. METHOD: Five conserved virulence factors involved in critical events of S. suis pathogenesis were combined and used as an intranasal vaccine (V5). The effect of V5 was investigated with intranasal and systemic challenge models. RESULTS: V5 induced antibody and T-cell responses at the mucosal site and systemically. The immunity promoted clearance of S. suis from the nasopharynx independent of S. suis serotypes and reduced lethality after systemic challenge with S. suis serotype 2. Moreover, mice that survived sepsis from intravenous infection developed meningitis, whereas none of these mice showed neuropathological symptoms after V5 receipt. CONCLUSION: Intranasal immunization with multiple conserved virulence factors decreases S. suis colonization at the nasopharynx across serotypes and inhibits the dissemination of the bacteria in the host. The protective mucosal immunity effects would potentially reduce the S. suis reservoir and prevent S. suis disease in pigs.

Evaluation of Mucosal Humoral and Cellular Immune Responses to HIV in External Secretions and Mucosal Tissues.

The mucosal immune systems of the genital and intestinal tracts as the most frequent sites of HIV-1 entry, display remarkable immunological differences from the systemic immune compartment which must be considered in the evaluation of humoral and cellular immune responses to HIV-1. Marked differences in the fluids from the genital and intestinal tracts and in plasma with respect to the Ig isotypes, their levels, molecular forms and distinct effector functions must be taken into consideration in the evaluation and interpretation of humoral immune responses. Because of the low levels and highly pronounced variation in Ig content, HIV-1-specific antibody concentrations should be always related to the levels of total Ig of a given isotype. This practice will avoid inevitable differences due to the small volumes of collected fluids and sample dilution during the collection and processing of samples from external secretions. Furthermore, appropriate controls and immunochemical assays should be used to complement and confirm results generated by ELISA, which is prone to false positivity. In the evaluation of antibody-mediated virus neutralization in external secretions, precautions and rigorous controls must be used to exclude the effect of innate humoral factors. The evaluation of cell-mediated immune responses in mucosal tissues is difficult due to the low yields of cells obtained from tissue biopsies or cytobrush scrapings. Furthermore, tissue biopsies of, for example rectal mucosa, provide information pertinent exclusively to this local site, which due to the differences in distribution of cells of different phenotypes, do not provide information generalized to the entire intestinal tract. Importantly, studies concerning the kinetics of cellular responses are difficult to perform due to the limited availability of samples or to the inability of obtaining frequent repeated tissue biopsies. For sampling the female genital tract parallel collection of menstrual and peripheral blood yields high numbers of cells that permit their detailed phenotypic and functional analyses. In contrast to tissue biopsies, this non-traumatic collection procedure, results in high cell yields and repeated monthly sampling permits extensive and parallel functional studies of kinetics and unique characteristics of HIV-1-specific cellular responses in the female genital tract and peripheral blood.

The Multifaceted Nature of Immunoglobulin A and Its Complex Role in HIV.

IgA is the most abundant immunoglobulin in mucosal secretions, and understanding the role of IgA in both protection from HIV acquisition and modulation of HIV disease progression is a field of considerable controversy and renewed research interest. Analysis of the RV144 clinical trial associated plasma HIV envelope-specific monomeric IgA from vaccines with reduced vaccine efficacy. The RV144 trial, however, only assessed for plasma IgA, which was not further subclassed, and the role of mucosal IgA was not addressed as mucosal samples were not collected. On the other hand, several studies have detected envelope-specific IgA in mucosal secretions of highly exposed persistently seronegative cohorts, while recent macaque simian-HIV passive immunization studies have suggested a potentially protective role for mucosal IgA. It is well established that total IgA in serum appears to correlate with HIV disease progression. In contrast, a selective deficit of anti-HIV IgA responses in HIV infection is apparent, with a number of recent studies beginning to elucidate the mechanisms behind these dysfunctional IgA responses. In this review, we highlight the dichotomy that exists in the literature as to whether anti-HIV IgA is protective or harmful to the host. Herein, we emphasize the importance of distinguishing between monomeric, multimeric, and isoforms of IgA and review what is known about the complex and diverse interactions of various molecular forms of IgA with HIV in both the systemic circulation and mucosal compartments.

Effects of Hot Spring Bathing on Salivary Secretion and Secretory IgA secretion in Healthy Volunteers / 日本温泉気候物理医学会雑誌

This study examined the effect of hot-spring bathing (40 to 41°C) on salivary secretion and salivary secretory IgA (sIgA) in healthy volunteers. Ten volunteers (10 men, average 33.6±9.3 years old) bathed in a hot-spring for 10 minutes.<br>Saliva samples were collected before bathing, during bathing (from 5 to 7 min), and after bathing using the Saxon test. The saliva flow rates and sIgA concentration were determined and then the sIgA secretion rates were calculated.<br>The saliva flow rates increased significantly during the bathing (p<0.02) and decreased after bathing. The sIgA secretion rates during bathing were significantly higher than those before and after bathing (p<0.02).<br>The increases in saliva flow rates and sIgA secretion rates during bathing were considered to indicate the improvement of local immunity in the oral cavity and thus considered to be useful for preventing upper respiratory tract infections.

Development of two novel nontoxic mutants of Escherichia coli heat-labile enterotoxin

Escherichia coli heat-labile enterotoxin (LT) is composed of catalytic A and non-catalytic homo-pentameric B subunits and causes diarrheal disease in human and animals. In order to produce a nontoxic LT for vaccine and adjuvant development, two novel derivatives of LT were constructed by a site-directed mutagenesis of A subunit; Ser63 to Tyr63 in LTS63Y and Glu110, Glu112 were deleted in LT delta 110/112. The purified mutant LTs (mLTs) showed a similar molecular structural complex as AB5 to that of wild LT. In contrast to wild-type LT, mLTs failed to induce either elongation activity, ADP-ribosyltransferase activity, cAMP synthesis in CHO cells or fluid accumulation in mouse small intestine in vivo. Mice immunized with mLTs either intragastrically or intranasally elicited high titers of LT-specific serum and mucosal antibodies comparable to those induced by wild-type LT. These results indicate that substitution of Ser63 to Tyr63 or deletion of Glu110 and Glu112 eliminate the toxicity of LT without a change of AB5 conformation, and both mutants are immunogenic to LT itself. Therefore, both mLTs may be used to develop novel anti-diarrheal vaccines against enterotoxigenic E. coli.