Summary. Human visceral leishmaniasis (VL) is a major public health problem worldwide, leading to significant mortality rates if not properly treated and controlled. Precise identification of infected patients is essential to establish treatment and control measures. Although several VL serological diagnosis advances have been accomplished lately, mainly using recombinant antigens and immunochromatographic tests (ICTs), improvements may still be achieved using multiepitope chimeric proteins in different test platforms. Here, we reported on the evaluation of ELISA and an ICT developed with a new chimeric protein, named DTL-4, based on repetitive antigenic sequences, including those present in the A2 protein. Methods. A total of 1028 sera samples were used for the development and validation of ELISA (321 samples from L. infantum-infected patients, 62 samples from VL/AIDS coinfected patients, 236 samples from patients infected with other diseases, and 409 samples from healthy donors). A total of 520 sera samples were used to develop and validate ICT (249 samples from L. infantum-infected patients, 46 samples from VL/AIDS coinfected patients, 40 samples from patients infected with other diseases, and 185 samples from healthy donors). Findings. Using the validation sera panels, DTL-4-based ELISA displayed an overall sensitivity of 94.61% (95% CI: 89.94-97.28), a specificity of 99.41% (95% CI: 96.39-99.99), and an accuracy of 97.02% (95% CI: 94.61-98.38), while for ICT, sensitivity, specificity, and accuracy values corresponded to 91.98% (95% CI: 86.65-95.39), 100.00% (95% CI: 96.30-100.00), and 95.14% (95% CI: 91.62-97.15), respectively. When testing sera samples from VL/AIDS coinfected patients, DTL-4-ELISA displayed a sensitivity of 77.42% (95% CI: 65.48-86.16), a specificity of 99.41% (95% CI: 96.39-99.99), and an accuracy of 93.51% (95% CI: 89.49%-96.10%), while for DTL-4-ICT, sensitivity was 73.91% (95% CI: 59.74-84.40), specificity was 90.63% (95% CI: 81.02-95.63), and accuracy was 82.00% (95% CI: 73.63-90.91). Conclusion. DTL-4 is a promising candidate antigen for serodiagnosis of VL patients, including those with VL/AIDS coinfection, when incorporated into ELISA or ICT test formats.

Introduction:The world has been devastated by the spread of the new coronavirus. Thousands of lives are lost daily and the economic impact has been incalculable. Diagnosing quickly means one of the point to control the progress of the disease, as monitoring the antibody levels of disease development and vaccinated people. Thus, serological tests such as ELISA and immunochromatographic test are being developed and constantly improved. Producing recombinant antigens capable of being recognized with high sensitivity and specificity by antibodies in different stages of this disease, as infected, curate and vaccinated are the major challenge in the process of developing serological tests. Prokaryotic systems for expression of recombinant proteins are widely used because of their simplicity, short production time and low cost. However, eukaryotic systems are the only choice to conserve the structural features as post-translational modifications and native conformation, as insect and mammalian cells. In this context, the insect cells are simple, low cost, faster with significant expression protein level. The spike protein or fragments applied for Covid-19 diagnosis expressed in the eukaryotic system has been widely used. In Brazil, for most tests developed, this protein has being imported with high costs and long delivery times, which reinforces the need for national production.Objective: Express a fragment of the Spike protein from SARS-CoV-2 in insect cells transfected with recombinant baculovirus and evaluate its potential as an antigen for the development of serological diagnostic tests. Methodology:The Bac-to-BacR HBM TOPO Secreted Expression System (Invitrogen) was chosen to construct the system. Briefly a gene segment encoding the sequence of interest for the Spike protein wascloned into the pFastBac/HBM-TOPO plasmid (Invitrogen). Competent DH10Bac E. coli cells containing the baculovirus genome were transformed with plasmid construction for generating the recombinant viral genome. Then cultured Sf9 insect cells were transfected with recombinant bacmid purified for the generation of the recombinant vírus and expression of the protein of interest, expressed in fusion with six histidines for purification by affinity chromatography and with a signal peptide, for secretion in the culture médium. Purified protein samples were analyzed by 12,5 % SDS-PAGE and Western Blot. After purification, the protein was evaluated by ELISA for their ability to be recognized by antibodies in the serum of individuals infected with Sars-coV-2. Results:The protein was expressed in the infected cells as the recombinant virus and purified from the culture supernatant. In ELISA assays for IgG evaluation the preliminary results fixing 500 ng of protein per well and analyzing 16 positive and 16 negative samples showed 100% specificity and 73% sensitivity. Conclusion:The initial results led us to intensively explore the potential of the insect cell/baculovirus expression system for large-scale production of SARS-CoV-2 antigens for the develop...

Introduction: Human visceral leishmaniasis (HVL) ranks second in mortality rates among tropical infectious diseases. Therefore, there is an urgent need of a prophylactic vaccine for HVL. Among the antigens candidate, different studies show that Leishmania amastigote 2 (A2) protein is immunogenic and this protein is commercially available in a vaccine for canine visceral leishmaniasis. However, the presence of saponin as adjuvant makes its formulation improper to be used in humans. Thus, towards vaccination in humans, it was proposed to develop a recombinant chimeric protein with the presence (rDTL4_tag) and the absence (rDTL4) of the histidine tag. This antigen contains a fragment of A2 and, combined with specific adjuvants, may be used as a strategy for vaccination against HVL.Objective: Express and purify the rDTL4_tag and rDTL4 protein, as well as explore the vaccine potential of rDTL4_tag by testing it in combination with different immunological adjuvants already approved for human vaccination.Methodology: 1) The rDTL4_tag and rDTL4 proteins were expressed in E. coli BL21 (DE3) bacteria and purified, respectively, by affinity chromatography and by two ion exchange chromatographies in the Akta prime (GE) system. The purified fraction of the protein rDTL4_tag was loaded onto a ToxinEraserTM (GenScript) column to remove endotoxins. 2) Female Balb/c mice were immunized with rDTL4_tag antigen associated with different adjuvants (Alumem/CPG, Poly(I:C) or AddaVax). It should be noted that the formulation containing recombinant protein A2 (rA2) was established as a positive control and, as negative control, saline or adjuvants separetely. Therefore, the animals were evaluated for protection given against the challenge with 1x10 7 L. infantum promastigotes. After the challenge, the number of viable parasites per milligram of the infected organs was determined using limiting dilution tests. 3) For the assessment of immunogenicity, an ELISA test was used to measure the levels of total IgG, IgG1 and IgG2a specific for the recombinant proteins as well as to measure the levels of IFN-γ and IL-10 in the culture of splenocytes stimulated with rDTL4_tag and rA2. Results:The outcomes of the purification of rDTL4_tag and rDTL4 were satisfactory, presenting a high degree of purity.The immunized animals with the formulations containing rDTL4_tag showed stronger cellular immune response than the control groups, as revealed by the increased levels of IFN-γ. In addition, the rDTL4_tag protein associated with the Poly(I:C) adjuvant induced robust production of antigen-specific total IgG, IgG1 and IgG2a. Furthermore, this group still showed a superior protection against infection, as shown by the decrease of tissue parasitism. Conclusion:The success of the rDTL4_tag antigen makes it a promising candidate for vaccine formulation. Thus, the vaccination strategy explored reveals promising alternatives for the development of an effective vaccine against HVL, aiming at the transposition of the rDTL4 protein for clinical trials in humans.