RESUMEN

BACKGROUND: Chagas disease (CD), a neglected parasitic disease caused by Trypanosoma cruzi, poses a significant health threat in Latin America and has emerged globally because of human migration. Trypanosoma cruzi infects humans and over 100 other mammalian species, including dogs, which are important sentinels for assessing the risk of human infection. Nonetheless, the serodiagnosis of T. cruzi in dogs is still impaired by the absence of commercial tests. In this study, we investigated the diagnostic accuracy of four chimeric recombinant T. cruzi IBMP antigens (IBMP-8.1, IBMP-8.2, IBMP-8.3, and IBMP-8.4) for detecting anti-T. cruzi antibodies in dogs, using latent class analysis (LCA). METHODS: We examined 663 canine serum samples, employing indirect ELISA with the chimeric antigens. LCA was utilized to establish a latent variable as a gold standard for T. cruzi infection, revealing distinct response patterns for each antigen. RESULTS: The IBMP (Portuguese acronym for the Molecular Biology Institute of Paraná) antigens achieved area under the ROC curve (AUC) values ranging from 90.9% to 97.3%. The highest sensitivity was attributed to IBMP-8.2 (89.8%), while IBMP-8.1, IBMP-8.3, and IBMP-8.4 achieved 73.5%, 79.6%, and 85.7%, respectively. The highest specificity was observed for IBMP-8.4 (98.6%), followed by IBMP-8.2, IBMP-8.3, and IBMP-8.1 with specificities of 98.3%, 94.4%, and 92.7%, respectively. Predictive values varied according to prevalence, indicating higher effectiveness in endemic settings. CONCLUSIONS: Our findings underscore the remarkable diagnostic performance of IBMP-8.2 and IBMP-8.4 for the serodiagnosis of Trypanosoma cruzi in dogs, representing a promising tool for the diagnosis of CD in dogs. These chimeric recombinant antigens may not only enhance CD surveillance strategies but also hold broader implications for public health, contributing to the global fight against this neglected tropical disease.

Asunto(s)

RESUMEN

Pathogenic bacterial membrane proteins (MPs) are a class of vaccine and antibiotic development targets with widespread clinical application. However, the inherent hydrophobicity of MPs poses a challenge to fold correctly in living cells. Herein, we present a comprehensive method to improve the soluble form of MP antigen by rationally designing multi-epitope chimeric antigen (ChA) and screening two classes of protein-assisting folding element. The study uses a homologous protein antigen as a functional scaffold to generate a ChA possessing four epitopes from transferrin-binding protein A of Glaesserella parasuis. Our engineered strain, which co-expresses P17 tagged-ChA and endogenous chaperones groEL-ES, yields a 0.346 g/L highly soluble ChA with the property of HPS-positive serum reaction. Moreover, the protein titer of ChA reaches 4.27 g/L with >90% soluble proportion in 5-L bioreactor, which is the highest titer reported so far. The results highlight a timely approach to design and improve the soluble expression of MP antigen in industrially viable applications.

Asunto(s)

RESUMEN

The risk of reactivation in patients with chronic or past/resolved hepatitis B virus (HBV) infection receiving chemotherapy or immunosuppressive drugs is a well-known possibility. The indication of antiviral prophylaxis with nucleo(t)side analogue is given according to the risk of HBV reactivation of the prescribed therapy. Though the advent of new drugs is occurring in all the field of medicine, in the setting of hematologic malignancies the last few years have been characterized by several drug classes and innovative cellular treatment. As novel therapies, there are few data about the rate of HBV reactivation and the decision of starting or not an antiviral prophylaxis could be challenging. Moreover, patients are often treated with a combination of different drugs, so evaluating the actual role of these new therapies in increasing the risk of HBV reactivation is difficult. First results are now available, but further studies are still needed. Patients with chronic HBV infection [hepatitis B surface antigen (HBsAg) positive] are reasonably all treated. Past/resolved HBV patients (HBsAg negative) are the actual area of uncertainty where it could be difficult choosing between prophylaxis and pre-emptive strategy.

Asunto(s)

RESUMEN

Human toxocariasis is one of the neglected helminthiases and it is caused by the zoonotic roundworm species Toxocara canis and Toxocara cati. Diagnosis of human toxocariasis is based on the combination of clinical, parasitological, and epidemiological criteria, as well as serology tests that detect anti-Toxocara antibodies. Notwithstanding, due to the absence of pathognomonic symptoms and signs of the disease, serology is the key evidence to support a conclusive diagnosis. TES-ELISA is the most widely used serological test for diagnosis. However, cross-reaction of TES antigens with antibodies produced to other helminth antigens is a major drawback for its application in countries with high parasitic prevalence. T. canis recombinant antigens have been described as an alternative to native TES for diagnosis. Nevertheless, the selection of antigenic proteins is a complex process that requires validation. In this paper, we developed an eGFP carrier-based system to express and purify blocks of recombinant polypeptides of T. canis antigenic proteins. Intense cross-reaction polypeptides were detected by Immunoblot and avoided to finally produce a chimeric prototype protein. Additionally, a control chimeric protein that harbors the complete tested proteins was produced. Purified chimeric antigens were tested in ELISA and Immunoblot assays with 310 sera samples of negative and positive control individuals. Our results showed that chimeric rCHITC0 and rCHITC1 antigens (with sensitivities of 62% 58%, 38% and 16% in IB-rCHITC0, ELISA-rCHITC0, ELISA-rCHITC1 and IB-rCHITC1 respectively for OLMS) can perform better in terms of specificity (being 91%, 89%, 87% and 76% for ELISA-rCHITC1, IB-rCHITC1, ELISA-rCHITC0 and IB-rCHITC0 respectively for OLMS) than T. canis TES-ELISA (with 61% specificity), giving a higher signal with serum samples of infected individuals as well the possibility to discriminate false positive cases with other parasitic infections. Our data suggest that T. canis chimeric proteins, represent candidate antigens for phase II studies.

RESUMEN

BACKGROUND: The continuous monitoring of malaria transmission intensity is still required to maintain elimination status after reaching the malaria elimination stage. In this study, serological surveillance with multiepitope artificial antigen was used to assess the transmission of Plasmodium falciparum in Yunnan, China, where malaria elimination has just been achieved, to provide data to support malaria control in the postelimination period. METHODS: Samples were collected in three border counties and one inland county in Yunnan Province in 2016 using a stratified whole-group sampling method. Fingerstick blood was collected from all participants, and antibodies to Malaria Random Constructed Antigen-1 (M. RCAg-1) were detected by indirect ELISA. The transmission intensity of P. falciparum malaria was estimated using a catalytic conversion model based on the maximum likelihood of generating a community seroconversion rate (SCR). RESULTS: A total of 5566 samples were collected. There was no statistically significant difference in antibody level between the inland county and the nonendemic area, but the antibody level in border counties was significantly higher than those in the inland county and the nonendemic control area. No seropositive cases were found in Yanjin County, and the seropositivity rate increased with age in the three border counties. The highest intensity of P. falciparum malaria transmission was in Zhenkang County (SCR = 0.0030, CI: 0.0029, 0.0031), followed by Gengma County (SCR = 0.0013, CI: 0.0012, 0.0015) and Yingjiang County (SCR = 0.00088, CI: 0.00083, 0.00090). CONCLUSION: The transmission intensity of P. falciparum malaria in Yunnan Province has obviously decreased in recent years, but for the border areas where malaria has just been eliminated, the transmission intensity will not immediately drop to zero, and it still needs to be monitored for a period of time to maintain malaria elimination status.

Asunto(s)

RESUMEN

PURPOSE: Brucellosis as a worldwide zoonotic illness affect domestic animals and humans doesn't have any vaccine for the prevention of infection in humans yet. The aim of this study was to evaluate the specific immune response following the administration of glycine nanoparticles as adjuvant and delivery system of a chimeric antigen contained trigger factor, Omp31, and Bp26 in murine model. MATERIALS AND METHODS: The chimeric antigen of Brucella was cloned and expressed in Escherichia coli (E. coli) BL21 (DE3). Purification and characterization of recombinant protein was conducted through Ni-NTA (nickel-nitrilotriacetic acid) agarose, SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), and Western blot. Nanoparticle characteristics including morphology, particle size distribution, zeta potential, protein retention rate, and release rate were measured in vitro. Subsequently, nanoparticle contained antigen was administered to mice and blood sample was taken to measured the antibody level. RESULTS: The protein retention in the nanoparticles was successfully done and the nanoparticle characteristics were appropriate. The average size of glycine particles containing antigen was about 174 nm, and the absorption of protein was approximately 61.27% of the initial value, with a release rate of approximately 70% after 8 hours. Enzyme-linked immunosorbent assay result proved that the immunized sera of mice which were administered with nano-formula contains high levels of antibodies (immunoglobulin G) against recombinant chimeric antigen and also a high level of mucosal antibody (immunoglobulin A) in the oral group, which showed a desirable immunity against Brucella. CONCLUSION: The results showed that chimeric antigen-loaded glycine nanoparticles can act as a vaccine candidate for inducing the cellular and humoral immune response against brucellosis.

RESUMEN

An estimated 400 million people are infected by parasites of the genus Ascaris and the existing control measures are inefficient. Vaccine development using B cell antigens is a promising strategy for increased protection against this parasite. The present study aimed at developing a chimeric protein capable of conferring protection against infection by Ascaris sp. For this purpose, we performed B-cell epitope predictions on previously described vaccine candidate proteins from Ascaris suum and the corresponding peptides were used to construct a chimeric protein. Female BALB / c mice were immunized subcutaneously in three doses at 10 day intervals with a vaccine formulation comprised of the chimeric protein together with monophosphoryl lipid A (MPLA). Control groups included protein alone, MPLA, or PBS. After challenge infection, animals vaccinated with chimeric protein plus MPLA showed a reduction of 73.54% of larval load in the lung compared to control group animals. Animals immunized with chimeric protein plus MPLA also display higher IgG response and a reduction in lung inflammation. Our study highlights how chimeric proteins containing more than one B cell epitope can enhance immune protection against helminthic infection and offer new approaches to the development of Ascaris vaccines.

Asunto(s)

RESUMEN

Chimeric T. cruzi antigens have been proposed as a diagnostic tool for chronic Chagas disease (CD) in both settings where Chagas disease is endemic and those where it is not endemic. Antibody response varies in accordance to each T. cruzi strain, presenting challenges to the use of antigens lacking demonstrated cross-reactivity with Leishmania spp. Our group expressed four chimeric proteins (IBMP-8.1, IBMP-8.2, IBMP-8.3, and IBMP-8.4) and previously assessed their diagnostic performance to determine cross-reactivity with Leishmania spp. Here, we validated our findings using serum samples from different Brazilian geographic areas reporting endemic Chagas disease, endemic visceral or American cutaneous leishmaniasis (ACL), or both. Overall, 829 serum samples were evaluated using commercial and IBMP enzyme-linked immunosorbent assays. Due to the absence of a reference assay to diagnosis CD, latent class analysis (LCA) was performed through the use of a statistical model. The incidence of cross-reactivity for ACL-positive samples varied from 0.35% (IBMP-8.3) to 0.70% (IBMP-8.1 and IBMP-8.2). Regarding visceral leishmaniasis (VL)-positive samples, the IBMP-8.2 and IBMP-8.3 antigens cross-reacted with six (3.49%) and with only one sample (0.58%), respectively. No cross-reactivity with either ACL or VL was observed for the IBMP-8.4 antigen. Similarly, no cross-reactions were found when VL-positive samples were assayed with IBMP-8.1. The agreement among the results obtained using IBMP antigens ranged from 97.3% for IBMP-8.2 and 99% for IBMP-8.1 and IBMP-8.3 to 100% for IBMP-8.4, demonstrating almost perfect agreement with LCA. Accordingly, in light of the negligible cross-reactivity with both ACL and VL, we suggest the use of IBMP antigens in regions where T. cruzi and Leishmania spp. are coendemic.

Asunto(s)

RESUMEN

BACKGROUND: Chronic Chagas Disease (CD) diagnosis is based on serological methods employing crude, semipurified or recombinant antigens, which may result in low sensitivity or cross-reactivity. To reduce these restrictions, we developed a strategy involving use of molecules containing repetitive fragments of Trypanosoma cruzi conserved proteins. Diagnostic performance of IBMP-8.1 and IBMP-8.4 chimeric antigens (Molecular Biology Institute of Paraná - IBMP in Portuguese acronym) was assessed to diagnose T. cruzi-infected and non-infected immigrants living in Barcelona (Spain), a non-endemic setting for Chagas disease. METHODS: Reactivity of IBMP-8.1 and IBMP-8.4 was assessed using an in-house automated ELISA with 347 positive and 331 negative individuals to Chagas disease. Antigenic cross-reactivity was measured with sera samples from pregnant women with Toxoplasma gondii (n = 98) and Zika virus (n = 75) antibodies. RESULTS: The area under the curve values was 1 and 0.99 for the IBMP-8.1 and IBMP-8.4 proteins, respectively, demonstrating excellent diagnostic accuracy. The reactivity index was higher for IBMP-8.1 than IBMP-8.4 in positive samples and no significant difference in reactivity index was observed in negative samples. Sensitivity ranged from 99.4% for IBMP-8.1 to 99.1% for IBMP-8.4 and was not statistically different. Specificity for IBMP-8.1 reached 100 and 99.7% for IBMP-8.4, both nearly 100% accurate. No antigenic cross-reactivity was observed and reactivity index was similar to that for negative Chagas disease individuals. CONCLUSIONS: Our results showed an outstanding performance of IBMP-8.1 and IBMP-8.4 chimeric antigens by ELISA and suggest both chimeric antigens could also be used for Chagas disease diagnosis in immigrants living in non-endemic settings.

Asunto(s)

RESUMEN

Diagnosing chronic Chagas disease (CD) requires antibody-antigen detection methods, which are traditionally based on enzymatic assay techniques whose performance depend on the type and quality of antigen used. Previously, 4 recombinant chimeric proteins from the Instituto de Biologia Molecular do Paraná (IBMP-8.1 to 8.4) comprising immuno-dominant regions of diverse Trypanosoma cruzi antigens showed excellent diagnostic performance in enzyme-linked immunosorbent assays. Considering that next-generation platforms offer improved CD diagnostic accuracy with different T. cruzi-specific recombinant antigens, we assessed the performance of these chimeras in liquid microarrays (LMAs). The chimeric proteins were expressed in Escherichia coli and purified by chromatography. Sera from 653 chagasic and 680 healthy individuals were used to assess the performance of these chimeras in detecting specific anti-T. cruzi antibodies. Accuracies ranged from 98.1 to 99.3%, and diagnostic odds ratio values were 3,548 for IBMP-8.3, 4,826 for IBMP-8.1, 7,882 for IBMP-8.2, and 25,000 for IBMP-8.4. A separate sera bank (851 samples) was employed to assess cross-reactivity with other tropical diseases. Leishmania, a pathogen with high similarity to T. cruzi, showed cross-reactivity rates ranging from 0 to 2.17%. Inconclusive results were negligible (0 to 0.71%). Bland-Altman and Deming regression analysis based on 200 randomly selected CD-positive and negative samples demonstrated interchangeability with respect to CD diagnostic performance in both singleplex and multiplex assays. Our results suggested that these chimeras can potentially replace antigens currently used in commercially available assay kits. Moreover, the use of multiplex platforms, such as LMA assays employing 2 or more IBMP antigens, would abrogate the need for 2 different testing techniques when diagnosing CD.

Asunto(s)

RESUMEN

DNA vaccination is one method to protect farmed fish from viral and bacterial diseases. Chimeric antigens encoded by DNA vaccines have been shown to increase the resistance to viral diseases. Here, we sequenced the gene encoding lysosome-associated membrane protein-1 from Japanese flounder, Paralichthys olivaceus, (JfLAMP-1) and assessed its use in a chimeric DNA vaccine fused with the major capsule protein (MCP) from red seabream iridovirus (RSIV). JfLAMP-1 cDNA has a length of 1248 bp encoding 415 aa, which contains transmembrane and cytoplasmic domains. JfLAMP-1 is constitutively expressed in several tissues and its expression in spleen was upregulated following injection of formalin-killed cells (FKC) of Edwardsiella tarda. Immunofluorescence analysis showed that JfLAMP-1 is distributed in the small and large granules in the cytoplasm and groups close to the nucleus. The DNA encoding the luminal domain of JfLAMP-1 was replaced with the gene for the RSIV MCP, and the construct was cloned in an expression vector (pCIneo). Fish vaccinated with pCLAMP-MCP had significantly higher antibody levels than fish vaccinated with pCIneo vector harboring the MCP gene (p < 0.05) at day 30 post-vaccination.

Asunto(s)

RESUMEN

BACKGROUND: Following the decline of malaria transmission in many countries and regions, serological parameters have become particularly useful for estimating malaria transmission in low-intensity areas. This study evaluated a novel serological marker, Malaria Random Constructed Antigen-1 (M.RCAg-1), which contains 11 epitopes from eight Plasmodium falciparum antigens, as a tool for assessing malaria transmission intensity along the border area of China-Myanmar. METHOD: Serum from Plasmodium falciparum and P. vivax patients was used to detect the properties of M.RCAg-1 and antibody responses. Cross-sectional surveys were conducted at the China-Myanmar border and in Hainan province in 2012 and 2013 using cluster sampling. Filter blood spot papers were collected from all participants. Antibodies against M.RCAg-1 were detected using indirect ELISA. The Mann-Whitney test and Spearman's rank correlation test were performed to analyze antibody data. P. falciparum malaria transmission intensity was estimated using a catalytic conversion model based on the maximum likelihood of generating a community seroconversion rate (SCR). RESULTS: M.RCAg-1 was well-recognized by the naturally acquired anti-malaria antibodies in P. falciparum patients and had very limited cross-reactivity with P. vivax infection. The total amount of IgG antibodies was decreased with the decrease in parasitemia after taking medication and lasted several weeks. In a population survey, the antibody levels were higher in residents living close to the China-Myanmar border than those living in non-epidemic areas (P < 0.0001), but no significant difference was observed between residents from Hainan and non-epidemic areas. The calculated SCR was 0.0128 for Jieyangka, 0.004 for Susuzhai, 0.0047 for Qiushan, and 0.043 for Kayahe. The estimated exposure rate obtained from the anti-M.RCAg-1 antibody level correlated with traditional measures of transmission intensity derived from altitude. CONCLUSION: Our study demonstrates that M.RCAg-1 is potentially useful as a serological indicator of exposure to P. falciparum malaria, especially for malaria surveillance in low transmission areas.

Asunto(s)

RESUMEN

The aim of the study was to evaluate the usefulness of 3 chimeric Toxoplasma gondii antigens, P35-MAG1, MIC1-ROP1 and MAG1-ROP1, in the serodiagnosis of an acute toxoplasmosis in humans. Proteins were produced as fusion proteins containing His tags ends and then further purified by metal affinity chromatography. Their application for the diagnosis of recently acquired T. gondii infection was tested in IgG and IgM enzyme-linked immunosorbent assays (ELISAs). At 100%, 77.3%, and 86.4%, respectively, the reactivity of the IgG ELISA using P35-MAG1, MIC1-ROP1, and MAG1-ROP1 for sera from patients where acute toxoplasmosis was suspected was significantly higher than for the samples from people with a chronic infection, at 26.2%, 36.1%, and 32.8%, respectively. Moreover, P35-MAG1, MIC1-ROP1, and MAG1-ROP1 detected IgM antibodies with a reactivity at 81.8%, 72.7%, and 59.1%, respectively. The results presented in the article show that, particularly, P35-MAG1 may be useful in the preliminary detection of recent T. gondii infection.

Asunto(s)

RESUMEN

Helicobacter pylori neutrophil-activating protein (NAP) is a toll-like receptor 2 (TLR2) agonist and potent immunomodulator inducing Th1-type immune response. Here we present data about characterization of the humoral immune response against NAP-tagged antigens, encoded by attenuated measles virus (MV) vector platform, in MV infection susceptible type I interferon receptor knockout and human CD46 transgenic (Ifnarko-CD46Ge) mice. Immunogenicity of MV expressing a full-length human immunoglobulin lambda light chain (MV-lambda) was compared to that of MV expressing lambda-NAP chimeric protein (MV-lambda-NAP). MV-lambda-NAP immunized Ifnarko-CD46Ge mice developed significantly higher (6-20-fold) anti-lambda ELISA titers as compared to the MV-lambda-immunized control animal group, indicating that covalently-linked NAP co-expression significantly enhanced lambda immunogenicity. In contrast, ELISA titers against MV antigens were not significantly different between the animals vaccinated with MV-lambda or MV-lambda-NAP. NAP-tagged antigen expression did not affect development of protective anti-measles immunity. Both MV-lambda and MV-lambda-NAP-immunized groups showed strong virus neutralization serum titers in plaque reduction microneutralization test. These results demonstrated that MV-encoded lambda-NAP is highly immunogenic as compared to the unmodified full-length lambda chain. Boost of immune response to poor immunogens using live vectors expressing NAP-tagged chimeric antigens is an attractive approach with potential application in immunoprophylaxis of infectious diseases and cancer immunotherapy.

Asunto(s)