Filamentous phage random peptide libraries were used to identify the epitopes of Burkholderia pseudomallei protease by panning against IgG polyclonal sera that exhibited protease neutralizing properties. The isolated fusion peptides presented a consensus peptide sequence, TKSMALSG, which closely resembles part of the active site sequence, 435GTSMATPHVAG445, of B. pseudomallei serine metalloprotease. By comparing the consensus sequence, TKSMALSG, with the predicted three-dimensional molecular model of B. pseudomallei serine metalloprotease, it appears that the potential antibody binding epitope was buried within the molecule. This active site was conformational whereby one continuous sub-region (SMA) was located between two discontinuous sub-regions, supplied by the flanking residues in the same polypeptide. All phages selected from the biopanning with IgG polyclonal sera showed good binding towards the polyclonal antibodies when compared to the negative control. In addition, these peptide-bearing phages showed competitive inhibition of B. pseudomallei serine metalloprotease binding to the polyclonal IgG.
Anti-filarial IgG4 antibody has been shown to be a good marker for detection of lymphatic filaria infection. Previous studies demonstrated that anti-filarial IgG4 assay using BmR1 recombinant antigen was highly specific and sensitive for detection of brugian filariasis. For bancroftian filariasis, an equivalent assay employing recombinant antigen expressed from the ORF of SXP1 gene has been reported. In order to detect infections by all species of lymphatic filarial, BmR1 and BmSXP recombinant antigens were employed in the development of a pan LF-ELISA.
In the global effort to eliminate lymphatic filariasis (LF), rapid field-applicable tests are useful tools that will allow on-site testing to be performed in remote places and the results to be obtained rapidly. Exclusive reliance on the few existing tests may jeopardize the progress of the LF elimination program, thus the introduction of other rapid tests would be useful to address this issue. Two new rapid immunochromatographic IgG4 cassette tests have been produced, namely WB rapid and panLF rapid, for detection of bancroftian filariasis and all three species of lymphatic filaria respectively. WB rapid was developed using BmSXP recombinant antigen, while PanLF rapid was developed using BmR1 and BmSXP recombinant antigens. A total of 165 WB rapid and 276 panLF rapid tests respectively were evaluated at USM and the rest were couriered to another university in Malaysia (98 WB rapid, 129 panLF rapid) and to universities in Indonesia (56 WB rapid, 62 panLF rapid), Japan (152 of each test) and India (18 of each test) where each of the tests underwent independent evaluations in a blinded manner. The average sensitivities of WB rapid and panLF rapid were found to be 97.6% (94%-100%) and 96.5% (94%-100%) respectively; while their average specificities were both 99.6% (99%-100%). Thus this study demonstrated that both the IgG4 rapid tests were highly sensitive and specific, and would be useful additional tests to facilitate the global drive to eliminate this disease.
BACKGROUND: The recombinant antigen BmR1 has been extensively employed in both ELISA and immunochromatographic rapid dipstick (Brugia Rapid) formats for the specific and sensitive detection of IgG4 antibodies against the lymphatic filarial parasites Brugia malayi and Brugia timori. In sera of individuals infected with Wuchereria bancrofti the IgG4 reactivity to BmR1 is variable, and cross-reactivity of sera from individuals infected with Onchocerca volvulus or Loa loa was observed only in single cases. In order to characterize the homologs of the BmR1 antigen in W. bancrofti (Wb-BmR1), O. volvulus (Ov-BmR1) and L. loa (Ll-BmR1) the cDNA sequences were identified, the protein expressed and the antibody reactivity of patients' sera was studied. METHODS: PCR methodology was used to identify the cDNA sequences from cDNA libraries and/or genomic DNA of W. bancrofti, O. volvulus and L. loa. The clones obtained were sequenced and compared to the cDNA sequence of BmR1. Ov-BmR1 and Ll-BmR1 were expressed in E. coli and tested using an IgG4-ELISA with 262 serum samples from individuals with or without B. malayi, W. bancrofti, O. volvulus and L. loa infections or various other parasitic infections. BmR1, Ov-BmR1 and Ll-BmR1 were also tested for reactivity with the other three IgG subclasses in patients' sera. RESULTS: Wb-BmR1 was found to be identical to BmR1. Ov-BmR1 and Ll-BmR1 were found to be identical to each other and share 99.7% homology with BmR1. The pattern of IgG4 recognition of all serum samples to BmR1, Ov-BmR1 and Ll-BmR1 were identical. This included weak IgG4 reactivities demonstrated by L. loa- and O. volvulus-infected patients tested with Ov-BmR1 and Ll-BmR1 (or BmR1). With respect to reactivity to other IgG subclasses, sera from O. volvulus- and L. loa-infected patients showed positive reactions (when tested with BmR1, Ov-BmR1 or Ll-BmR1 antigens) only with IgG1. No reactivity was observed with IgG2 or with IgG3. Similarly, ELISAs to detect reactivity to other anti-filarial IgG subclasses antibodies showed that sera from individuals infected with B. malayi or W. bancrofti (active infections as well as patients with chronic disease) were positive with BmR1 only for IgG1 and were negative when tested with IgG2 and with IgG3 subclasses. CONCLUSIONS: This study demonstrates that homologs of the BmR1 antigen are present in W. bancrofti, O. volvulus and L. loa and that these antigens are highly conserved. Recognition of this antigen by patients' sera is similar with regard to IgG1, IgG2 and IgG3, but different for IgG4 antibodies. We conclude that the BmR1 antigen is suitable for detection of IgG4 antibodies in brugian filariasis. However, its homologs are not suitable for IgG4-based diagnosis of other filarial infections.
BACKGROUND: Brugia malayi is endemic in several Asian countries with the highest prevalence in Indonesia. Determination of prevalence of lymphatic filariasis by serology has been performed by various investigators using different kinds of antigen (either soluble worm antigen preparations or recombinant antigens). This investigation compared the data obtained from IgG4 assays using two different kinds of antigen in a study on prevalence of antibodies to B. malayi. METHODS: Serum samples from a transmigrant population and life long residents previously tested with IgG4 assay using soluble worm antigen (SWA-ELISA), were retested with an IgG4 assay that employs BmR1 recombinant antigen (BmR1 dipstick [Brugia Rapid trade mark ]). The results obtained with the two antigens were compared, using Pearson chi-square and McNemar test. RESULTS: There were similarities and differences in the results obtained using the two kinds of antigen (SWA and BmR1). Similarities included the observation that assays using both antigens demonstrated an increasing prevalence of IgG4 antibodies in the transmigrant population with increasing exposure to the infection, and by six years living in the area, antibody prevalence was similar to that of life-long residents. With regards to differences, of significance is the demonstration of similar antibody prevalence in adults and children by BmR1 dipstick whereas by SWA-ELISA the antibody prevalence in adults was higher than in children. CONCLUSIONS: Results and conclusions made from investigations of prevalence of anti-filarial IgG4 antibody in a population would be affected by the assay employed in the study.
Burkholderia pseudomallei is resistant to a diverse group of antimicrobials including third generation cephalosporins whilst quinolones and aminoglycosides have no reliable effect. As therapeutic options are limited, development of more effective forms of immunotherapy is vital to avoid a fatal outcome. In an earlier study, we reported on the B. pseudomallei serine MprA protease, which is relatively stable over a wide pH and temperature range and digests physiological proteins. The present study was carried out to evaluate the immunogenicity and protective efficacy of the MprA as a potential vaccine candidate. In BALB/c mice immunized with recombinant MprA protease (smBpF4), a significantly high IgG titer was detectable. Isotyping studies revealed that the smBpF4-specific antibodies produced were predominantly IgG(1), proposing that immunization with smBpF4 triggered a Th2 immune response. Mice were immunized with smBpF4 and subsequently challenged with B. pseudomallei via the intraperitoneal route. Whilst control mice succumbed to the infection by day 9, smBpF4-immunized mice were protected against the lethal challenge and survived beyond 25 days post-infection. In conclusion, MprA is immunogenic in melioidosis patients whilst also eliciting a strong immune response upon bacterial challenge in mice and presents itself as a potential vaccine candidate for the treatment of melioidosis.
A cross-sectional study was conducted to determine the seroepidemiology of Toxoplasma infection and its risk association among people having close contact with animals. A total of 312 blood samples were collected from veterinary personnel (veterinarian, technicians, and students) and pet owners from veterinary clinics and hospitals in the area of Klang Valley, Malaysia. About 4 cc of blood samples drawn from agreed participants were processed for measurement of anti-Toxoplasma IgG and IgM antibodies as well as avidity test of Toxoplasma IgG by ELISA I, II, and III kits. Meanwhile, the demographic profiles and possible risk factors of these participants were also recorded in the standardized data collection sheets. Overall seroprevalence of toxoplasmosis was observed in 62 (19.9%) participants being 7 (18.4%) in veterinarians, 15 (33.3%) in veterinary technicians, 29 (14.9%) in veterinary students, and 11 (31.4%) in pet owners. Of 19.9% Toxoplasma seropositive samples, 18.3% was positive for IgG antibody, 1.0% for IgM antibody, and 0.6% for both IgG and IgM antibodies. Of three different IgG avidity ELISA kits, ELISA III showed high avidity in all five seropositive samples (IgM and IgG/IgM antibodies) indicating chronic Toxoplasma infection which is consistent with no evidence of clinical toxoplasmosis diagnosed during the time of this study. Univariate analysis showed that age group, gender, study population, gardening, task performance, and working duration were significantly associated with Toxoplasma seropositivity. Further analysis by multivariate analysis using logistic regression showed that age group of ≥30 years old (OR = 0.34, 95% CI = 0.18-0.63, p = 0.001) and working or study duration of >10 years having close contact with animals (OR = 5.07, 95% CI = 1.80-14.24, p = 0.002) were identified as significant risks for Toxoplasma infection. Based on the results obtained, a comprehensive Toxoplasma screening and health surveillance program on toxoplasmosis should be implemented among people having close contact with animals in general and confirmed Toxoplasma seronegative individuals in particular to prevent seroconversion.
Background: Anaphylaxis is an acute and life-threatening allergic response. Classically and most commonly, it can be mediated by the crosslinking of allergens to immunoglobulin E (IgE)- high affinity IgE receptor (FcεRI) complex found mostly on mast cells. However, there is another pathway of anaphylaxis that is less well-studied. This pathway known as the alternative pathway is mediated by IgG and its Fc gamma receptor (Fcγ). Though it was not documented in human anaphylaxis, a few studies have found that IgG-mediated anaphylaxis can happen as demonstrated in rodent models of anaphylaxis. In these studies, a variety of soluble mediators were being evaluated and they differ from each study which causes confusion in the suitability, and reliability of choice of soluble mediators to be analyzed for diagnosis or therapeutic purposes. Hence, the objective of this meta-analysis is to identify the potential soluble mediators that are involved in an IgG-mediated anaphylaxis reaction. Methods: Studies related to IgG-mediated anaphylaxis were sourced from five search engines namely PubMed, Scopus, Ovid, Cochrane Library, and Center for Agricultural Bioscience International (CABI) regardless of publication year. Relevant studies were then reviewed based on specific inclusion factors. The means and standard deviations of each soluble mediator studied were then extracted using ImageJ or Get Data Graph Digitiser software and the data were subjected to meta-analysis. Results: From our findings, we found that histamine, serotonin, platelet activating factor (PAF), β-hexosaminidase, leukotriene C4 (LTC4), mucosal mast cell protease-1 (MMCP-1), interleukins (IL)-4,-6, and-13; tumor necrosis factor alpha (TNF-α), and macrophage inflammatory protein-1α (MIP-1α) were often being analyzed. Out of these soluble mediators, histamine, PAF, β-hexosaminidase, IL-6, and-13, MIP-1α and TNF-α were more significant with positive effect size and p < 0.001. As study effect was relatively small, we performed publication bias and found that there was publication bias and this could be due to the small sample size studied. Conclusion: As such, we proposed that through meta-analysis, the potential soluble mediators involved in rodent IgG-mediated anaphylaxis to be histamine, PAF, β-hexosaminidase, IL-6 and-13 and MIP-1α, and TNF-α but will require further studies with larger sample size.
Toxoplasmosis is one of the most common opportunistic parasitic diseases in patients living with HIV/AIDS. This study aimed to determine the seroprevalence of Toxoplasma infection in HIV-infected patients and to identify associated risk factors in Toxoplasma seropositive patients. This study was conducted at a regional public hospital in Hat Yai, southern Thailand during October 2009 to June 2010. Blood samples were collected from 300 HIV-infected patients. Each subject also answered a socio-demographic and risk factors associated with Toxoplasma infection. The prevalence of anti-Toxoplasma IgG antibodies in HIV-infected patients was 109 (36.3%), of which 83 (76.2%) had past infection and 26 (23.9%) had recently acquired Toxoplasma infection as indicated by their IgG avidity. Multivariate analysis using logistic regression showed that gender difference (adjusted OR = 1.69, 95% CI = 1.05-2.72) was the only factor associated with Toxoplasma infection. From the results obtained, these HIV-infected patients could be at high risk of developing clinical evidence of severe toxoplasmosis. Therefore, it is necessary to introduce primary behavioral practices to prevent Toxoplasma infection among HIV-infected patients.
Toxoplasmosis is a foodborne disease caused by Toxoplasma gondii, an obligate intracellular parasite. Severe symptoms occur in the immunocompromised patients and pregnant women leading to fatality and abortions respectively. Vaccination development is essential to control the disease. The T. gondii dense granule antigen 2 and 5 (GRA2 and GRA5) have been targeted in this study because these proteins are essential to the development of parasitophorous vacuole (PV), a specialized compartment formed within the infected host cell. PV is resistance to host cell endosomes and lysosomes thereby protecting the invaded parasite. Recombinant dense granular proteins, GRA2 (rGRA2) and GRA5 (rGRA5) were cloned, expressed, and purified in Escherichia coli, BL21 (DE3) pLysS. The potential of these purified antigens as subunit vaccine candidates against toxoplasmosis were evaluated through subcutaneous injection of BALB/c mice followed by immunological characterization (humoral- and cellular-mediated) and lethal challenge against virulent T. gondii RH strain in BALB/c mice. Results obtained demonstrated that rGRA2 and rGRA5 elicited humoral and cellular-mediated immunity in the mice. High level of IgG antibody was produced with the isotype IgG2a/IgG1 ratio of ≈0.87 (p < 0.001). Significant increase (p < 0.05) in the level of four cytokines (IFN-γ, IL-2, IL-4, and IL-10) was obtained. The antibody and cytokine results suggest that a mix mode of Th1/Th2-immunity was elicited with predominant Th1-immune response inducing partial protection against T. gondii acute infection in BALB/c mice. Our findings indicated that both GRA2 and GRA5 are potential candidates for vaccine development against T. gondii acute infection.
Nephrotic syndrome affects both children and adults. Idiopathic nephrotic syndrome is reported to be one of the most frequent renal pathologies in childhood. Nephrotic children are at high risk for severe pneumococcal infections as one of the life-threatening complications of nephrotic syndrome due to involvement of the immunosuppressive regimen and the acquired immune deficiency induced by nephrotic syndrome including decreased plasma IgG and low complement system components. Aiming to prevent pneumococcal infection is of paramount importance especially in this era of ever-increasing pneumococcal resistance to penicillins and cephalosporins. The pneumococcal vaccines currently available are inactivated vaccines-the two main forms in use are polysaccharide vaccines and conjugated vaccines. However, the data supporting the use of these vaccines and to guide the timing and dosage recommendations is still limited for nephrotic children. Thus, this review discusses the evidences of immunogenicity and safety profile of both vaccinations on nephrotic patients as well as the effect of nephrotic syndrome treatment on vaccine seroresponses.
Measles (rubeola) is a highly contagious vaccine-preventable disease caused by the measles virus-a virus of the Paramyxoviridae family. The illness typically begins with fever, runny nose, cough, and pathognomonic enanthem (Koplik spots) followed by a characteristic erythematous, maculopapular rash. The rash classically begins on the face and becomes more confluent as it spreads cephalocaudally. Laboratory confirmation of measles virus infection can be based on a positive serological test for measles-specific immunoglobulin M antibody, a four-fold or greater increase in measles-specific immunoglobulin G between acute and convalescent sera, isolation of measles virus in culture, or detection of measles virus ribonucleic acid by reverse transcriptase-polymerase chain reaction. Complications occur in 10% to 40% of patients, and treatment is mainly symptomatic. Bacterial superinfections, if present, should be properly treated with antibiotics. To eradicate measles, universal childhood immunisation and vaccination of all susceptible individuals with measles vaccine would be ideal. In developed countries, routine immunisation with measles-containing vaccine is recommended, with the first and second doses at ages 12 to 15 months and 4 to 6 years, respectively. The World Health Organization recommends that the first and second doses of measles-containing vaccine be given at ages 9 months and 15 to 18 months, respectively, in countries with high rates of measles transmission.
Ezrin, radixin and moesin, which are strongly expressed in the Schwann cell microvilli, are putative targets for autoantibodies in acute or chronic inflammatory demyelinating polyneuropathy (AIDP or CIDP). An association between anti-moesin IgG antibodies and cytomegalovirus-related AIDP has been postulated. None of 41 AIDP patients, including 8 cytomegalovirus-related AIDP patients, and 23 CIDP had IgG or IgM antibodies to ezrin, radixin and moesin; whereas, one patient with cytomegalovirus-related AIDP had anti-ezrin IgM antibodies. Ezrin, radixin and moesin are unlikely targets for autoantibodies in AIDP and CIDP, and the association of anti-moesin antibodies with cytomegalovirus-related AIDP was not confirmed.
This study introduces a new approach for enhancing immunity toward mucosal vaccines. HEV71 killed vaccine that is formulated with nanosize calcium phosphate adjuvant and encapsulated onto chitosan and alginate delivery carriers was examined for eliciting antibody responses in serum and saliva collected at weeks 0, 1, 3, 5, 7 and 9 for viral-specific IgA & IgG levels and viral neutralizing antibody titers. The antibody responses induced in rabbits by the different formulations delivered by a single (buccal) route were compared to those of dual immunization (intradermal / mucosal) and un-immunized control. Chitosan-loaded vaccine adjuvant induced elevated IgA antibody, while Alginate-adjuvant irreversible bonding sequestered the vaccine and markedly reduced immunogenicity. The induced mucosal and parenteral antibody profiles appeared in an inverse manner of enhanced mucosal IgA antibody accompanied by lower systemic IgG following a single oral immunization route. The combined intradermal and oral dual-immunized group developed an elevated salivary IgA, systemic IgG, and virus neutralizing response. A reduced salivary neutralizing antibody titer was observed and attributed to the continual secretion exchanges in saliva. Designing a successful mucosal delivery formulation needs to take into account the vaccine delivery site, dosage, adjuvant and carrier particle size, charge, and the reversibility of component interactions. The dual immunization seems superior and is a important approach for modulating the antibody response and boosting mucosal protection against HEV71 and similar pathogens based on their transmission mode, tissue tropism and shedding sites. Finally, the study has highlighted the significant role of dual immunization for simultaneous inducing and modulating the systemic and mucosal immune responses to EV71.
Highly pathogenic avian influenza (HPAI) H5N1 is an ongoing global health concern due to its severe sporadic outbreaks in Asia, Africa and Europe, which poses a potential pandemic threat. The development of safe and cost-effective vaccine candidates for HPAI is considered the best strategy for managing the disease and addressing the pandemic preparedness. The most potential vaccine candidate is the antigenic determinant of influenza A virus, hemagglutinin (HA). The present research was aimed at developing optimized expression in Nicotiana benthamiana and protein purification process for HA from the Malaysian isolate of H5N1 as a vaccine antigen for HPAI H5N1. Expression of HA from the Malaysian isolate of HPAI in N. benthamiana was confirmed, and more soluble protein was expressed as truncated HA, the HA1 domain over the entire ectodomain of HA. Two different purification processes were evaluated for efficiency in terms of purity and yield. Due to the reduced yield, protein degradation and length of the 3-column purification process, the 2-column method was chosen for target purification. Purified HA1 was found immunogenic in mice inducing H5 HA-specific IgG and a hemagglutination inhibition antibody. This paper offers an alternative production system of a vaccine candidate against a locally circulating HPAI, which has a regional significance.
Neutralizing antibodies (NTAb) play a significant role in preventing and protecting against SARS-CoV-2 virus infection. Identifying NTAb is undoubtedly imperative in understanding the immunity toward COVID-19 better. However, it is interesting to note that the production of NTAb varies among individuals, especially among healthcare workers (HCWs), as they are exposed to the virus daily. Hence, we would like to investigate factors affecting the production of S-RBD IgG and NTAb among different categories of HCWs, particularly after receiving the third dose of the BNT162b2 mRNA COVID-19 Vaccine. A total of 361 HCWs from our hospital were prospectively enrolled and had their S-RBD IgG and NTAb titers measured. They were studied in relation to the degree of exposure to COVID-19, breakthrough infections, gender, age, race, household income, housing type, household number, and education levels. HCWs with the highest risk of exposure to COVID-19, breakthrough infections, and male gender displayed the highest median titers of both S-RBD IgG and NTAb, and the differences were statistically significant (p
Prostate-specific antigen (PSA) is widely used as a diagnostic marker for the detection of prostate cancer in men. We have generated stable hybridomas producing specific monoclonal antibodies (MAbs) of the IgG class against PSA from fusions of splenocytes from immunized mice with myeloma cells. The hybridomas were adapted into serum-free media and cultured in CELLine CL-1000 bioreactors to produce milligram quantities of MAbs. Cross-reactivity study demonstrated that all the MAbs reacted did not cross-react with several other types of tumor antigens. Two of the MAbs were successfully radiolabeled by the iodogen method. The (125)I-labeled MAbs demonstrated strong binding to PSA on the surface of the LNCaP cells (Kd of 1.16 x 10(-9) M and 1.4 x 10(-9) M). Thus the (125)I-labeled MAbs retained their immunoreactivity and possessed high affinity and is potentially useful for binding to tumor cells. In conclusion, the MAbs can be used to develop radioimmunodiagnostic, radioimaging, and immunohistochemistry techniques for the early detection and treatment of prostate cancer.
The anti-phospholipid antibody (aPL) in 26 heat-inactivated normal human sera (NHS) was tested for IgG subclass in ELISA. The specific antibody in NHS included all four IgG antibody subclasses, as well as IgA. The incidence of IgG subclasses ranged from 50% (13/26) for IgG1 to 92% (24/26) for IgG2. Specific IgA anti-phospholipid antibody (aPL) was detected by ELISA in 38% (28/73) of normal human saliva. The salivary IgA aPL bound preferentially to anionic phospholipids including cardiolipin, phosphatidylserine and phosphatidic acid but not to phosphatidylcholine or sphingomyelin. Unlike aPL in normal human sera, aPL in saliva was predominantly not associated with the previously described heat-labile inhibitor of aPL. This may indicate a role of salivary IgA aPL in local immunity by binding to cross-reactive bacterial cell surface components including phospholipids.
Heat-sensitive serum masking cofactor(s) of antiphospholipid antibody (aPL) in normal human sera (NHS) are specifically inactivated at 56 degrees C. The degree of binding in ELISA by unmasked aPL in NHS was equivalent to that in non-heated, aPL-reactive autoimmune SLE sera. Previously "negative" SLE sera also reacted equally strongly in the aPL ELISA when similarly heat-inactivated. Isotype studies by ELISA of the heat-potentiated aPL in 36 NHS revealed the presence of specific IgG (34/36), IgM (11/36) and IgA (24/36) aPL antibodies. 11/36 (31%) NHS had all three aPL isotypes while 13/36 (36%) had both IgG and IgA antibodies to phospholipid.