Antibody In Lymphocyte Supernatant Research Articles

Evaluation of the Safety, Tolerability and Immunogenicity of ShigETEC, an Oral Live Attenuated Shigella-ETEC Vaccine in Placebo-Controlled Randomized Phase 1 Trial.

Background: Shigella spp. and enterotoxigenic Escherichia coli (ETEC) cause high morbidity and mortality worldwide, yet no licensed vaccines are available to prevent corresponding infections. A live attenuated non-invasive Shigella vaccine strain lacking LPS O-antigen and expressing the ETEC toxoids, named ShigETEC was characterized previously in non-clinical studies. Methods: ShigETEC was evaluated in a two-staged, randomized, double-blind and placebo-controlled Phase I clinical trial. A single dose of increasing amounts of the vaccine was given to determine the maximum tolerated dose and increasing number of immunizations were administered with an interval based on the duration of shedding observed. Results: Oral immunization with ShigETEC was well tolerated and safe up to 4-time dosing with 5 × 1010 colony forming units. ShigETEC induced robust systemic immune responses against the Shigella vaccine strain, with IgA serum antibody dominance, as well as mucosal antibody responses evidenced by specific IgA in stool samples and in ALS (Antibodies in Lymphocyte Supernatant). Anti- ETEC toxin responses were detected primarily in the 4-times immunized cohort and for the heat-labile toxin correlated with neutralizing capacity. Conclusion: ShigETEC is a promising vaccine candidate that is scheduled for further testing in controlled human challenge studies for efficacy as well as in children in endemic setting for safety and immunogenicity.

Antibody in Lymphocyte Supernatant (ALS) responses after oral vaccination with live Shigella sonnei vaccine candidates WRSs2 and WRSs3 and correlation with serum antibodies, ASCs, fecal IgA and shedding.

The levels of antigen-specific Antibodies in Lymphocyte Supernatant (ALS) using an ELISA are being used to evaluate mucosal immune responses as an alternate to measuring the number of Antibody Secreting Cells (ASCs) using an ELISpot assay. A recently completed trial of two novel S. sonnei live oral vaccine candidates WRSs2 and WRSs3 established that both candidates were safe, well tolerated and immunogenic in a vaccine dose-dependent manner. Previously, mucosal immune responses were measured by assaying IgA- and IgG-ASC in peripheral blood mononuclear cells (PBMCs). In this report, the magnitude of the S. sonnei antigen-specific IgA- and IgG-ALS responses was measured and correlated with previously described ASCs, serum antibodies, fecal IgA and vaccine shedding. Overall, the magnitude of S. sonnei anti-Invaplex50 ALS was higher than that of LPS or IpaB, and both vaccines demonstrated a more robust IgA-ALS response than IgG; however, compared to WRSs3, the magnitude and percentage of responders were higher among WRSs2 recipients for IgA- or IgG-ALS. All WRSs2 vaccinees at the two highest doses responded for LPS and Invaplex50-specific IgA-ALS and 63–100% for WRSs3 vaccinees responded. Regardless of the vaccine candidate, vaccine dose or detecting antigen, the kinetics of ALS responses were similar peaking on days 7 to 9 and returning to baseline by day 14. The ALS responses were vaccine-specific since no responses were detected among placebo recipients at any time. A strong correlation and agreement between responders/non-responders were noted between ALS and other mucosal (ASC and fecal IgA) and systemic (serum antibody) immune responses. These data indicate that the ALS assay can be a useful tool to evaluate mucosal responses to oral vaccination, an observation noted with trials of other bacterial diarrheal pathogens. Furthermore, this data will guide the list of immunological assays to be conducted for efficacy trials in different populations. It is hoped that an antigen-specific-ALS titer may be a key mucosal correlate of protection, a feature not currently available for any Shigella vaccines candidates. https://clinicaltrials.gov/show/NCT01336699.

Assessment of an Antibody-in-Lymphocyte Supernatant Assay for the Etiological Diagnosis of Pneumococcal Pneumonia in Children.

New diagnostic tests for the etiology of childhood pneumonia are needed. We evaluated the antibody-in-lymphocyte supernatant (ALS) assay to detect immunoglobulin (Ig) G secretion from ex vivo peripheral blood mononuclear cell (PBMC) culture, as a potential diagnostic test for pneumococcal pneumonia. We enrolled 348 children with pneumonia admitted to Patan Hospital, Kathmandu, Nepal between December 2015 and September 2016. PBMCs sampled from participants were incubated for 48 h before harvesting of cell culture supernatant (ALS). We used a fluorescence-based multiplexed immunoassay to measure the concentration of IgG in ALS against five conserved pneumococcal protein antigens. Of children with pneumonia, 68 had a confirmed etiological diagnosis: 12 children had pneumococcal pneumonia (defined as blood or pleural fluid culture-confirmed; or plasma CRP concentration ≥60 mg/l and nasopharyngeal carriage of serotype 1 pneumococci), and 56 children had non-pneumococcal pneumonia. Children with non-pneumococcal pneumonia had either a bacterial pathogen isolated from blood (six children); or C-reactive protein <60 mg/l, absence of radiographic consolidation and detection of a pathogenic virus by multiplex PCR (respiratory syncytial virus, influenza viruses, or parainfluenza viruses; 23 children). Concentrations of ALS IgG to all five pneumococcal proteins were significantly higher in children with pneumococcal pneumonia than in children with non-pneumococcal pneumonia. The concentration of IgG in ALS to the best-performing antigen discriminated between children with pneumococcal and non-pneumococcal pneumonia with a sensitivity of 1.0 (95% CI 0.73–1.0), specificity of 0.66 (95% CI 0.52–0.78) and area under the receiver-operating characteristic curve (AUROCC) 0.85 (95% CI 0.75–0.94). Children with pneumococcal pneumonia were older than children with non-pneumococcal pneumonia (median 5.6 and 2.0 years, respectively, p < 0.001). When the analysis was limited to children ≥2 years of age, assay of IgG ALS to pneumococcal proteins was unable to discriminate between children with pneumococcal pneumonia and non-pneumococcal pneumonia (AUROCC 0.67, 95% CI 0.47–0.88). This method detected spontaneous secretion of IgG to pneumococcal protein antigens from cultured PBMCs. However, when stratified by age group, assay of IgG in ALS to pneumococcal proteins showed limited utility as a test to discriminate between pneumococcal and non-pneumococcal pneumonia in children.

Comparative Performance of Modified Kenneth Jones Criteria Scoring, World Health Organization Criteria, and Antibodies in Lymphocyte Supernatant for Diagnosing Tuberculosis in Severely Malnourished Children Presenting With Pneumonia.

Background: The diagnosis of childhood tuberculosis (TB) can be difficult in severely malnourished children. This is mainly due to the fact of our perceived notion that clinical signs of TB are often subtle in severely malnourished children and we may rely on laboratory investigation for the diagnosis. However, comparative data on the performance of clinical and laboratory diagnostics of TB in such population are also very limited.Objectives: To compare the performance of composite clinical criteria and a technique that measures antibodies in lymphocyte supernatant (ALS) for the diagnosis of TB in severely malnourished children with pneumonia.Methods: Severely malnourished children under five with radiological pneumonia admitted to the Dhaka Hospital of International Centre for Diarrhoeal Disease Research, Bangladesh were enrolled consecutively following informed consent. We collected venous blood for ALS, gastric lavage fluid and induced sputum for microscopy, mycobacterial culture, and real-time PCR by Xpert MTB/RIF. We compared the sensitivity, specificity, positive, and negative predictive values, and accuracy of modified Kenneth Jones criteria (MKJC) score, World Health Organization (WHO) criteria, and ALS in diagnosing TB in severely malnourished children with pneumonia for “Confirmed TB” and “All TB” (“Confirmed TB” plus “Probable TB”) vs. “Not TB.”Results: Compared to culture confirmed TB, the sensitivity, and specificity (95% CI) for MKJC were 60 (27–86) and 84 (79–87)% and for WHO criteria were 40 (14–73) and 84 (80–87)%, respectively. Compared to culture and/or Xpert MTB/RIF positive TB, the sensitivity and specificity (95% CI) for the criteria were 37 (20–58) and 84 (79–87)%; and 22 (9–43) and 83 (79–87)%, respectively. For both these comparisons, the sensitivity and specificity of ALS were 50 (14–86) and 60 (53–67)%, respectively.Conclusion: Our data suggest that WHO criteria and MKJC scoring mainly based on clinical criteria are more useful than ALS in diagnosing TB in young severely malnourished children with pneumonia. The results underscore the importance of using clinical criteria for the diagnosis of TB in severely malnourished children that may help to minimize the chance of over treatment with anti-TB in such population, especially in resource limited TB endemic settings.

Development of a novel multiplex electrochemiluminescent-based immunoassay to aid enterotoxigenic Escherichia coli vaccine development and evaluations

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of bacterial diarrhea both among children in low and middle income countries and in travelers to these regions. Although there are several approaches to develop an effective vaccine for ETEC, no licensed vaccines are currently available. The most advanced ETEC vaccine candidates include multiple colonization factors along with the heat labile toxin B subunit. In the absence of known correlates of protection, and to understand the mechanism of protection, monitoring immune responses to a majority of the vaccine associated antigens using various types of samples is needed. Unfortunately, a traditional ELISA is time consuming, labor intensive and requires substantial amounts of antigens and sample volumes. To address these constraints, we developed and validated a novel high throughput electrochemiluminescent (ECL) - based multiplex immunoassay using Meso Scale Discovery (MSD) platform for analyzing immune responses to ETEC antigens. The ETEC multiplex ECL assay is an 8-plex assay which includes the ETEC colonization factor antigens (CFA/I, CS1, CS2, CS3, CS5 and CS6) along with the two subunits of heat labile toxin (LTA and LTB). Our data suggested that a single dilution of sample provides a quantifiable result for a wide range of sample titers. To compare ETEC multiplex ECL with ELISA, we carried out assays using the same antigens with the two immunoassay platforms using a common sample set of serum and ALS (antibodies in lymphocyte supernatant) specimens. The MSD platform achieved excellent correlations with ELISA for the antigens tested, consistently detecting comparable antibody levels in the samples. The ETEC multiplex ECL can serve as a fundamental platform in evaluating performances of candidate ETEC vaccines in future field trials.

A phase I trial of WRSS1, a Shigella sonnei live oral vaccine in Bangladeshi adults and children

ABSTRACT Shigella sonnei live vaccine candidate, WRSS1, which was previously evaluated in US, Israeli and Thai volunteers, was administered orally to Bangladeshi adults and children to assess its safety, clinical tolerability and immunogenicity. In a randomized, placebo-controlled, dose-escalation, age-descending study, 39 adults (18–39 years) and 64 children (5–9 years) were enrolled. Each adult cohort (n = 13) received one dose of 3x104, or three doses of 3 × 105 or 3 × 106 colony forming unit (CFU) of WRSS1 (n = 10) or placebo (n = 3). Each child cohort (n = 16) received one dose of 3x103, or three doses of 3x104, 3x105, or 3 × 106 CFU WRSS1 (n = 12) or placebo (n = 4). WRSS1 elicited mostly mild and transient reactogenicity events in adults and children. In the 3 × 106 dose group, 50% of the adults shed the vaccine; no shedding was seen in children. At the highest dose, 100% of adults and 40% of children responded with a ≥ 4-fold increase of S. sonnei LPS-specific IgA antibody in lymphocyte supernatant (ALS). At the same dose, 63% of adults and 70% of children seroconverted with IgA to LPS, while in placebo, 33% of adults and 18% of children seroconverted. Both the vaccinees and placebos responded with fecal IgA to LPS, indicating persistent exposure to Shigella infections. In conclusion, WRSS1 was found safe up to 106 CFU dose and immunogenic in adults and children in Bangladesh. These data indicate that live, oral Shigella vaccine candidates, including WRSS1 can potentially be evaluated in toddlers and infants (<2 years of age), who comprise the target population in an endemic environment.

Evaluation of cytokines in peripheral blood mononuclear cell supernatants for the diagnosis of tuberculosis.

IntroductionThere is active interest in leveraging host immune responses as biomarkers of tuberculosis (TB) disease activity. We had previously evaluated an immunodiagnostic test called the antibody in lymphocyte supernatant (ALS) assay. Here, we aimed to evaluate a panel of inflammatory mediators and associate the responses with the ALS results to identify a biosignature to distinguish TB cases from controls.MethodologyIn this case–control study, adults with TB were compared to controls who were hospitalized for non-infectious conditions. Blood was collected at baseline and after 4 weeks of TB treatment (from TB cases only). Peripheral blood mononuclear cells were isolated and cultured without antigenic stimulation for 72 hours. Inflammatory mediators were measured using the Multiplex cytokine kit and compared between TB cases and controls; among TB cases, responses were compared over time. ALS and inflammatory mediator results were evaluated using generalized discriminant analysis to identify the optimal biosignature to predict TB.ResultsWhen comparing inflammatory mediators between groups, IL-1ra, IL-1β, and granulocyte macrophage-colony stimulating factor (GM-CSF) were lower in TB cases (P<0.002). Fibroblast growth factor-basic significantly increased from baseline to week-4 (P=0.002). Generalized discriminant analysis yielded a model with IL-2, tumor necrosis factor-alpha, vascular endothelial growth factor, and ALS, providing a sensitivity of 82.2% and specificity of 76.2%.ConclusionOur results suggest that IL-1ra, IL-1β, and GM-CSF might be used as diagnostic biomarkers to distinguish between TB cases and non-TB cases. We could not identify a group of mediators that outperformed the diagnostic accuracy of the ALS alone.

Assessment and Translation of the Antibody-in-Lymphocyte Supernatant (ALS) Assay to Improve the Diagnosis of Enteric Fever in Two Controlled Human Infection Models and an Endemic Area of Nepal.

New diagnostic tests for enteric fever are urgently needed to assist with timely antimicrobial treatment of patients and to measure the efficacy of prevention measures such as vaccination. In a novel translational approach, here we use two recently developed controlled human infection models (CHIM) of enteric fever to evaluate an antibody-in-lymphocyte supernatant (ALS) assay, which can detect recent IgA antibody production by circulating B cells in ex vivo mononuclear cell culture. We calculated the discriminative ability of the ALS assay to distinguish diagnosed cases in the two CHIM studies in Oxford, prior to evaluating blood culture-confirmed diagnoses of patients presenting with fever to hospital in an endemic areas of Kathmandu, Nepal. Antibody responses to membrane preparations and lipopolysaccharide provided good sensitivity (>90%) for diagnosing systemic infection after oral challenge with Salmonella Typhi or S. Paratyphi A. Assay specificity was moderate (~60%) due to imperfect sensitivity of blood culture as the reference standard and likely unrecognized subclinical infection. These findings were augmented through the translation of the assay into the endemic setting in Nepal. Anti-MP IgA responses again exhibited good sensitivity (86%) but poor specificity (51%) for detecting blood culture-confirmed enteric fever cases (ROC AUC 0.79, 95%CI 0.70–0.88). Patients with anti-MP IgA ALS titers in the upper quartile exhibited a clinical syndrome synonymous with enteric fever. While better reference standards are need to assess enteric fever diagnostics, routine use of this ALS assay could be used to rule out infection and has the potential to double the laboratory detection rate of enteric fever in this setting over blood culture alone.

Kinetics of antibody-secreting cell and fecal IgA responses after oral cholera vaccination in different age groups in a cholera endemic country

Immune responses to oral enteric vaccines in children and infants may be influenced by factors such as age, previous priming with related microorganisms and breast feeding. In this study, we aimed to determine optimal time points to assess immune responses to oral enteric vaccines in different clinical specimens. This was done by investigating antibody secreting cell (ASC) and fecal antibody responses on different days after vaccination using the licensed oral cholera vaccine Dukoral, containing cholera toxin B-subunit (rCTB) and inactivated Vibrio cholerae bacteria, as a model vaccine.Two vaccine doses were given 2weeks apart to infants (6–11months), young children (12–18months), toddlers (19months–5years) and adults in a cholera endemic country (Bangladesh). IgA ASC responses, as determined by the antibodies in lymphocyte supernatant (ALS) assay, plasma IgA and IgG responses and secretory IgA (SIgA) responses in extracts of fecal samples were evaluated 4/5 and 7days after each vaccination.After the first vaccine dose, anti-CTB ALS IgA responses in adults and toddlers were high and comparable on day 5 and 7, while responses were low and infrequent in young children. After the second dose, highest ALS responses were detected on day 5 among the time points studied in all age groups and the responses declined until day 7. In contrast, plasma IgA and IgG anti-CTB responses were high both on day 5 and 7 after the second dose. Fecal SIgA responses in young children and infants were highest on day 7 after the second dose.Our results suggest that ASC/ALS responses to two doses of the oral cholera vaccine Dukoral and related oral vaccines should be analyzed earlier than previously recommended (day 7) at all ages. Fecal antibody responses should preferably be analyzed later than ASC/ALS responses to detect the highest antibody responses.

Laboratory Diagnosis of Tuberculosis- an Update

Annually about two million deaths occur globally due to tuberculosis (TB). Bangladesh ranks the sixth position among 22 highest burden TB countries in the world and also one of the 27 high multidrug resistant tuberculosis (MDR-TB) burden countries where about 70,000 people die every year due to TB. Among six key components of Stop TB Strategy (STS) Plan, the first one includes increase case notification of all forms of TB and improve diagnosis of new smear negative, extrapulmonary cases and TB in children by 2016. As TB can affect any organ in human body, the TB cases are managed by any discipline in medical community. Unfortunately diagnostic accuracy is not satisfactory and is not only due to uniform unavailability of the latest diagnostic facilities but also due to inadequate knowledge of the professionals about currently available modern laboratory techniques to diagnose TB. Light-emitting diode (LED) microscopy with fluorescence (auramine-rhodamine staining) should be preferred than conventional microscopy with Zeihl-neelsen (acid fast) staining to identify TB bacilli. Mantoux test (MT) indicates only infection by TB bacilli, does not necessarily the active disease. It may be positive in latent TB and in BCG (Bacillus Calmette-Guerin) vaccinated cases. Antibodies from Lymphocyte Secretion or Antibodies in Lymphocyte Supernatant (ALS) assay can detect active TB cases within three days of sample collection. The test is very useful to diagnose TB in children where sputum collection is difficult. Interferon gamma release assay (IGRA) tests are not advocated in low and middle-income countries, typically those with a high TB and/or HIV burden. Anti TB IgG/IgM/IgA tests should be avoided because these are being misinterpreted by someone as active TB cases. Adenosine Deaminase Assay (ADA) is a reliable test to diagnose tuberculous pleural effusion together with other evidences. ADA in pleural fluid &lt;40 IU/L is considered negative for TB. The more the ADA level, the more possibility to be tuberculous effusion. Level &gt;100 IU/L is highly specific for TB origin. Gene Xpert MTB/RIF, an Xpert test for mycobacterium tuberculosis (MTB) and Rifampicin (RIF) resistance, is used for rapid identification for TB bacilli, specially when MDR-TB is suspected, in human immunodeficiency virus ( HIV) infected cases and highly suspected sputum negative cases ( as a follow on test ) where microscopy frequently failed due to low bacterial load. The test exhibits high sensitivity and specificity for detecting pulmonary TB.Faridpur Med. Coll. J. Jul 2015;10(2): 71-75

Characterization of Mucosal Immune Responses to Enterotoxigenic Escherichia coli Vaccine Antigens in a Human Challenge Model: Response Profiles after Primary Infection and Homologous Rechallenge with Strain H10407.

Enterotoxigenic Escherichia coli (ETEC) bacteria are the most common bacterial cause of diarrhea in children in resource-poor settings as well as in travelers. Although there are several approaches to develop an effective vaccine for ETEC, no licensed vaccines are currently available. A significant challenge to successful vaccine development is our poor understanding of the immune responses that correlate best with protection against ETEC illness. In this study, ETEC-specific mucosal immune responses were characterized and compared in subjects challenged with ETEC strain H10407 and in subjects rechallenged with the homologous organism. IgA responses to lipopolysaccharide (LPS), heat-labile toxin B subunit (LTB), and colonization factor antigen I (CFA/I) in antibody in lymphocyte supernatant (ALS), feces, lavage fluid, and saliva samples were evaluated. In all assay comparisons, ALS was the most sensitive indicator of a local immune response, but serum IgA was also a useful indirect marker of immune response to oral antigens. Volunteers challenged and then rechallenged with strain H10407 were protected from illness following rechallenge. Comparing mucosal antibody responses after primary and homologous rechallenge, protection against disease was reflected in reduced antibody responses to key ETEC antigens and in reduced fecal shedding of the H10407 challenge strain. Subjects challenged with strain H10407 mounted stronger antibody responses to LPS and LTB than subjects in the rechallenge group, while responses to CFA/I in the rechallenge group were higher than in the challenge group. We anticipate that this study will help provide an immunological benchmark for the evaluation of ETEC vaccines and immunization regimens in the future.

Evaluation of ALS Assay of TB-SA for Diagnosis of Pulmonary Tuberculosis

The study evaluated tuberculosis specific antigen (TB-SA) antibody in lymphocyte supernatant (ALS) assay for diagnosis of pulmonary tuberculosis (PTB). The mean concentration of TB-SA ALS in PTB patients was significantly higher than that of two control groups (non-PTB patients and healthy subjects, P < 0.001). The area under the curve of the ALS assay were 0.960 (0.904–0.988, PTB vs healthy subjects), and were 0.822 (0.739–0.887, PTB vs Non-PTB patients), respectively. Our data indicated the ALS assay for TB-SA can be used as a screening test and is a useful adjunct to current tests for diagnosis of PTB.

Immunoproteomic Analysis of Antibody in Lymphocyte Supernatant in Patients with Typhoid Fever in Bangladesh

We have previously shown that an assay based on detection of anti-Salmonella enterica serotype Typhi antibodies in supernatant of lymphocytes harvested from patients presenting with typhoid fever (antibody in lymphocyte supernatant [ALS] assay) can identify 100% of patients with blood culture-confirmed typhoid fever in Bangladesh. In order to define immunodominant proteins within the S. Typhi membrane preparation used as antigen in these prior studies and to identify potential biomarkers unique to S. Typhi bacteremic patients, we probed microarrays containing 2,724 S. Typhi proteins with ALS collected at the time of clinical presentation from 10 Bangladeshis with acute typhoid fever. We identified 62 immunoreactive antigens when evaluating both the IgG and IgA responses. Immune responses to 10 of these antigens discriminated between individuals with acute typhoid infection and healthy control individuals from areas where typhoid infection is endemic, as well as Bangladeshi patients presenting with fever who were subsequently confirmed to have a nontyphoid illness. Using an ALS enzyme-linked immunosorbent assay (ELISA) format and purified antigen, we then confirmed that immune responses against the antigen with the highest immunoreactivity (hemolysin E [HlyE]) correctly identified individuals with acute typhoid or paratyphoid fever in Dhaka, Bangladesh. These observations suggest that purified antigens could be used with ALS and corresponding acute-phase activated B lymphocytes in diagnostic platforms to identify acutely infected patients, even in areas where enteric fever is endemic.

Safety and Immunogenicity of a Single Oral Dose of Recombinant Double Mutant Heat-Labile Toxin Derived from Enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) is a primary cause of traveler's diarrhea for which there is no licensed vaccine. This phase 1 trial determined the safety and immunogenicity of a recombinantly produced double mutant heat-labile enterotoxin (dmLT) of ETEC. It was administered as a single oral dose of dmLT in escalating doses of 5 μg, 25 μg, 50 μg, and 100 μg, followed by a 72-h inpatient observation, outpatient visits at 8, 14, and 28 days, and telephone calls at 2 and 6 months postvaccination. Safety was assessed by frequency of adverse events, and immune responses determined after immunization included dmLT-specific serum IgA and IgG, fecal IgA, antibody-secreting cells (ASC), and antibodies in lymphocyte supernatant (ALS) responses. All doses were well tolerated by the 36 healthy adults enrolled. Immune responses were limited in the 5- and 25-μg dose recipients. The 50-μg dose recipients trended toward stronger responses than the 100-μg dose recipients by serum IgA (67% versus 33%, P = 0.22), serum IgG (58% versus 33%, P = 0.41), and fecal IgA (58% versus 33%, P = 0.41). By day 14 postvaccination, there were significantly more positive responders (≥4-fold increase from baseline) among the 50- versus 100-μg dose recipients for serum IgA (P = 0.036) but not serum IgG (P = 0.21). In conclusion, a single oral dose of dmLT was well tolerated and immunogenic, with immune responses plateauing at the 50-μg dose. (This clinical trial is registered at www.clinicaltrials.gov, registration number NCT01147445.).

Different kinetics of circulating antibody-secreting cell responses after primary and booster oral immunizations: A tool for assessing immunological memory

We show that the kinetics of circulating IgA as well as IgG antibody-secreting cell (ASC) responses differs considerably after primary and booster vaccination with the oral cholera vaccine Dukoral®, as determined by the antibody in lymphocyte supernatant (ALS) as well as ELISPOT methods. Thus, whereas the antitoxin ASC responses did not peak until 7–9 days after primary vaccination, peak responses to a second dose given after two weeks, or a single booster dose given 6 months to 14 years later, were recorded already after 4–5 days and then rapidly declined. Our results indicate that many previous studies reporting ASC results 7–10 days after repeated immunization may have substantially underestimated the magnitudes of the responses. The results also suggest that detection of peak ASC responses at an early time point after booster immunization can be used as a simple tool to assess immunological memory.

BCG-specific IgG-secreting peripheral plasmablasts as a potential biomarker of active tuberculosis in HIV negative and HIV positive patients

BackgroundDiagnosis of active tuberculosis (TB) among sputum-negative cases, patients with HIV infection and extra-pulmonary TB is difficult. In this study, assessment of BCG-specific IgG-secreting peripheral plasmablasts, was used to identify...

Comparative evaluation of the antibody in lymphocyte supernatant (ALS) and enzyme-linked immunospot (ELISPOT) assays for measuring mucosal immune responses to Shigella antigens

Accurately assessing mucosal immune responses to candidate vaccines remains a technical challenge. ELISPOT is widely used as a surrogate of mucosal immune response by directly enumerating circulating antibody secreting cells (ASCs), while antibody in lymphocyte supernatant (ALS) titers the total amount of antibody secreted by ASC ex vivo using ELISA. ALS is more practical than ELISPOT because the ASC supernatant is frozen for ELISA that can be conducted at any time, with any antigen, and in any laboratory. We compared IgA and IgG responses to serotype-specific Shigella LPS using ELISPOT and ALS in subjects following vaccination or infection with Shigella. ALS results correlated well with ELISPOT results, and the ALS method was both sensitive and specific for the detection of antibody responses against Shigella LPS. Based on these observations, the ALS assay is a practical and flexible alternative to ELISPOT for measuring mucosal IgA responses to Shigella LPS antigen.

Validation of the ALS Assay in Adult Patients with Culture Confirmed Pulmonary Tuberculosis

BackgroundWe have earlier shown that Bacille Calmette-Guérin (BCG) vaccine-specific IgG Antibodies in Lymphocyte Supernatant (ALS) can be used for diagnosis of active tuberculosis (TB) in adults and children.Methodology/Principal FindingsThe ALS method was validated in a larger cohort (n = 212) of patients with suspicion of pulmonary TB using multiple antigens (BCG, LAM, TB15.3, TB51A, CFP10-ESAT6-A, CFP, CW) from Mycobacterium tuberculosis. The sensitivity and specificity of the ALS assay was calculated using non-TB patients as controls. The sensitivity and the specificity were highest with BCG vaccine (90% and 88% respectively) followed by LAM (89% and 87% respectively). Simultaneous assessment of multiple antigen-specific antibodies increased sensitivity (91%) and specificity (88%). Using higher lymphocyte count in smaller volume of culture media increased detection and reduced the assay duration to ∼30 hrs. Twenty one patients with clinical findings strongly suggestive of TB finally diagnosed as non-TB patients were positive by the ALS assay, of which 9 (43%) were positive for 7 antigens and 19 (90%) for at least 3 antigens.Conclusions/SignificanceOur findings show that simultaneous detection of antigens improves the diagnostic potential of the ALS assay; the modified method increases sensitivity and can provide results in <48 hours, and enable detection of some cases of pulmonary TB that are not detectable by standard methods.

Comparison of clinical features and immunological parameters of patients with dehydrating diarrhoea infected with Inaba or Ogawa serotypes of Vibrio cholerae O1

Vibrio cholerae O1, Ogawa and Inaba serotypes, both cause severe cholera. We compared clinical and immunological features in patients in Bangladesh infected with these 2 serotypes. Blood was collected from hospitalized Ogawa (N=146) or Inaba (N=191) patients at the acute stage (day 2) and 5 and 19 days later. Ogawa patients were younger than Inaba, presented with shorter duration of diarrhoea, and had more frequent abdominal pain, vomiting and need for intravenous fluids (p<0.05). Inaba patients more frequently had dark-field positive stools (p<0.01). Inaba strains were more susceptible to tetracycline and erythromycin than Ogawa strains (p<0.001). Ogawa infection produced higher plasma vibriocidal as well as IgG responses to cholera toxin B subunit, toxin-coregulated pilus subunit and lipopolysaccharide (LPS); higher IgA responses to LPS in 'antibody in lymphocyte supernatant' (ALS) specimens were also seen. These results suggest that a cholera vaccine based on the Ogawa serotype needs to be further investigated.

Detection of Antibodies Secreted from CirculatingMycobacterium tuberculosis-Specific Plasma Cells in the Diagnosis of Pediatric Tuberculosis

Diagnosis of tuberculosis (TB) in children is difficult because symptoms are often nonspecific or absent in infected children, diagnostic specimens are difficult to obtain from younger children, and >50% have negative TB cultures. Thus, there is an urgent need for improved diagnosis of pediatric TB. This study aimed to evaluate the diagnostic value of a new serological method, the ALS (antibodies in lymphocyte supernatant) assay, for the diagnosis of active TB in children with clinically identified TB. The ALS test is based on the concept that antigen-specific plasma cells are present in the circulation only at times of acute infection and not in latency. A cross-sectional study of pediatric patients (age range, 11 to 167 months) who were clinically identified as TB (n = 58) or non-TB (n = 16) patients was conducted, and they were monitored for 6 months. Healthy children (n = 58) were enrolled as controls. Spontaneous release of TB antigen-specific antibodies by in vitro-cultured, unstimulated peripheral blood mononuclear cells was assessed by an enzyme-linked immunosorbent assay using Mycobacterium bovis bacillus Calmette-Guérin (BCG) as the detecting antigen. Of the patients clinically diagnosed with TB, 15% had culture-confirmed TB, 64% were positive for TB by clinically established scoring charts (K. Edwards, P. N. G. Med. J. 30: 169-178, 1987; G. Stegen, K. Jones, and P. Kaplan, Pediatrics 43: 260-263, 1969; and stop TB Partnership, Childhood TB subgroup, World Health Organization, Int. J. Tuberc. Lung Dis. 10: 1091-1097, 2006), and 91% were TB positive by the ALS method. All TB patients had significantly higher BCG-specific ALS titers at enrollment (optical density [OD], 1.06 +/- 0.32) than healthy-control children (OD, 0.18 +/- 0.06) and non-TB children (OD, 0.21 +/- 0.10) (P = 0.001). The ALS titers declined in children with active disease from enrollment through 6 months following anti-TB therapy (P = 0.001). The ALS assay is a novel diagnostic method with potential applications in the diagnosis of pediatric TB and in subsequent monitoring of treatment effectiveness.