Immunoinformatic approaches for ErpY-LemA chimeric protein design for use in leptospirosis control.

AIMS: Currently, immunoinformatic approaches have shown promise in rapidly and cost-effectively identifying new antigens from the Leptospira proteome. Chimeric multiepitope proteins offer a strategy with significant potential for implementation in serology and the design of effective vaccines. METHODS AND RESULTS: In this study, we detail the immunoinformatic analyses and design of a new recombinant chimeric protein constructed with epitopes identified from the sequences of ErpY-like and LemA proteins, previously identified as potential antigens for controlling leptospirosis. We expressed the chimeric protein using Escherichia coli heterologous systems, evaluated its antigenicity using serum from naturally infected patients, and its immunogenicity in mice as an animal model, with Freund as an adjuvant. The resulting recombinant chimeric protein, named rErpY-LemA, was successfully expressed and purified using a prokaryotic system, with an expected mass of 35 kDa. Serologic assays using serum samples from naturally infected patients demonstrated recognition of the chimera protein by antibodies present in sera. Animals immunized with the chimera exhibited a significant IgG antibody response from the 7th day (P<0.001), persisting until day 49 of experimentation, with a titer of 1:12,800 (P<0.05). Notably, significant production of IgA, IgM, and IgG subclasses was observed in animals immunized with the chimera. CONCLUSIONS: These results highlight the promising role of immunoinformatics in rapidly identifying antigens and the potential of chimeric multiepitope proteins in developing effective strategies for leptospirosis control.

Advancing serologic diagnosis: assessing the efficacy of rErpY-like protein in human leptospirosis detection.

Leptospirosis is a globally distributed infectious disease caused by pathogenic spirochetes of the Leptospira genus, often overlooked. It is estimated that the disease affects approximately one million people annually, resulting in more than 58,900 deaths. The gold standard for serodiagnosis of leptospirosis is the Microscopic Agglutination Test (MAT). However, the limitations of this technique necessitate the exploration of alternative diagnostic methods. In this study, we evaluated the ErpY-like recombinant protein (rErpY-like) in the development of a serologic diagnostic assay for human leptospirosis. Eighty-six human sera samples, characterized by MAT, underwent evaluation through indirect IgM-ELISA and IgG-ELISA. The sensitivity and specificity values obtained from IgM-ELISA were 60% and 76%, respectively, while those from IgG-ELISA were 96.4% and 100%, respectively. The use of the rErpY-like protein in both IgM-ELISA and IgG-ELISA proves to be a sensitive and specific method for antibody detection. This could potentially serve as a valuable alternative tool in the diagnosis of human leptospirosis.

Antibodies anti-rFilF protein has anti-biofilm activity against carbapenem-resistant Acinetobacter baumannii.

Acinetobacter baumannii is an opportunistic bacterium that causes infection in several sites. Carbapenem-resistant A. baumannii strains (CRAb) lead the World Health Organization's list of 12 pathogens considered a priority for developing new antimicrobials. The pathogenicity of A. baumannii is related to the different virulence factors employed in the colonization of biotic and abiotic surfaces, biofilm formation and multidrug resistance. We analyze the outer membrane protein FilF from A. baumannii in silico and produce it in recombinant form (rFilF). rFilF protein was successfully expressed in Escherichia coli BL21 Star in an insoluble form. Immunization with rFilF induced significant anti-rFilF IgG antibody production in mice, detected by indirect enzyme-linked immunosorbent assay, since the first evaluation until 49th. On the last experimentation day, the predominant immunoglobulin found was IgG1 followed by IgG2a, IgG2b, IgM, IgG3, and IgA. We observe that interleukins 4 and 10 show significant production after the 28th day of experimentation in mice immunized with rFilF. Anti-rFilF pAbs were able to inhibit biofilm formation in nine CRAb strains evaluated, and in the standard strain ATCC® 19606. These results demonstrate the anti-biofilm activity of anti-rFilF antibodies, promising in the development of a non-antibiotic approach based on the control of CRAb strains.

In silico analyses and design of chimeric proteins containing epitopes of Bartonella henselae antigens for the control of cat scratch disease.

Bartonella henselae is a Gram-negative bacterium that causes cat scratch disease (CSD), as well as bacteremia, endocarditis, and other clinical presentations. CSD remains one of the most common infections caused by bacteria in the genus Bartonella, and it is transmitted to humans through a scratch or cat bite. Vaccination and more efficient diagnostic methods would represent a promising and sustainable alternative measure for CSD control in humans and animals. Here, we described the in silico analyses and design of three recombinant chimeric proteins (rC1, rC2, and rC3), for use in the control of CSD. The chimeras were constructed with epitopes identified from the sequences of the GroEL, 17 kDa, P26, BadA, Pap31, OMP 89, and OMP 43, previously described as the most important B. henselae antigens. The rC1, rC2, and rC3 were expressed and purified using a heterologous system based on Escherichia coli and reacted with antibodies present in the sera of humans naturally infected. The chimeric proteins were used to immunize mice using Freund adjuvant, and the humoral immune response was evaluated. Animals immunized with rC1 and rC3 showed a significant IgG antibodies response from the 28th day (P < 0.05), and the animals immunized with the rC2 from the 35th day (P < 0.05) remained until the 56th day of experimentation, with a titer of 1:3200 (P < 0.05), 1:1600 (P < 0.05) and 1:1600 (P < 0.05) from rC1, rC2, and rC3, respectively. Significant production of IgA and IgG1 isotype was detected in animals immunized with rC1 and rC2 proteins. Additionally, analysis using 13 serum samples from naturally infected patients showed that the proteins are recognized by antibodies present in sera, reinforcing the possibility of using these chimeras for CSD control. KEY POINTS: • The recombinant chimeras were expressed in Escherichia coli with 37 kDa (rC1), 35 kDa (rC2), and 38 kDa (rC3). • Animals immunized with rC1, rC2, and rC3 showed significant antibody response. • The chimeras were recognized by the sera of naturally infected patients.

A hypothetical adhesin protein induces anti-biofilm antibodies against multi-drug resistant Acinetobacter baumannii.

The increase in multidrug-resistant (MDR) Acinetobacter baumannii strains in hospital environments has generated great concern around the world. Biofilm is one of the forms of bacterial adaptation that is increasingly leading to antimicrobial resistance and therapeutic failure. The search for alternative therapeutic strategies, especially non-antibiotic-based, is urgently needed. In this study, we produce polyclonal antibodies (pAbs) in murine models against recombinant CAM87009.1 antigen, an A. baumannii fimbriae protein. The pAbs produced were isotyped and anti-biofilm activity evaluated in the A. baumannii ATCC® 19606 standard strain and nine MDR clinical isolates. All clinical isolates were analyzed for the presence of the cam87009.1 gene using the PCR technique, and one of the isolates did not have the gene in its genome. After four intraperitoneal immunizations (days 0, 14, 21, and 28) of mice with rCAM87009.1 and Freund's adjuvant, a significant antibody titer was detected by indirect enzyme-linked immunosorbent assay (ELISA) since the first immunization (1:6400), and the level increased until the 4th immunization (1:819,200). IgM, IgA, IgG1, IgG2a, IgG2b, and IgG3 isotypes were identified in the serum of immunized mice (P < 0.001). The anti-rCAM87009.1 pAb was able to inhibit biofilm formation in 80 % of the strains evaluated in this study, including the ATCC® 19606 strain. The rCAM87009.1 proves to be a promising target in the development of alternative strategies to control biofilm-forming in A. baumannii MDR strains.