Non-invasive urine-based ELISA using a recombinant Leishmania protein to diagnose tegumentary leishmaniasis.

The diagnosis of tegumentary leishmaniasis (TL) is hampered by variable sensitivity and/or specificity of the tests. Serological assays are suitable to diagnose visceral leishmaniasis (VL); however, they present low performance for the detection of TL cases. Additionally, blood collection to obtain patient serum represents a challenge, as it is an invasive and uncomfortable procedure, requiring laboratorial infrastructure and trained professionals. In this context, the present study proposed to evaluate patient urine to detect TL, given that this analyte has proven to be effective in ELISA experiments for the detection of VL cases. For this, a Leishmania protein called LiHyV, two specific B-cell epitopes derived from protein amino acid sequence, and a Leishmania antigenic extract (SLA) were used as antigens. A total of 215 paired urine and serum samples were evaluated, and results showed that, when serum was employed as an analyte, rLiHyV, Peptide1, Peptide2, and SLA presented a sensitivity of 85%, 29%, 58%, and 31%, respectively, and a specificity of 97.5%, 98%, 100%, and 97.5%, respectively, in the diagnosis of TL. When urine was used, rLiHyV, Peptide1, Peptide2, and SLA presented a sensitivity of 95%, 74%, 67%, and 52%, respectively, and a specificity of 100%, 99%, 98%, and 86%, respectively. In conclusion, preliminary data suggest that urine could be considered as an alternative biological sample for the detection of TL cases.

A recombinant chimeric antigen constructed with B-cell epitopes from Mycobacterium leprae hypothetical proteins is effective for the diagnosis of leprosy.

The diagnosis if leprosy is difficult, as it requires clinical expertise and sensitive laboratory tests. In this study, we develop a serological test for leprosy by using bioinformatics tools to identify specific B-cell epitopes from Mycobacterium leprae hypothetical proteins, which were used to construct a recombinant chimeric protein, M1. The synthetic peptides were obtained and showed good reactivity to detect leprosy patients, although the M1 chimera have showed sensitivity (Se) and specificity (Sp) values higher than 90.0% to diagnose both paucibacillary (PB) and multibacillary (MB) leprosy patients, but not those developing tegumentary or visceral leishmaniasis, tuberculosis, Chagas disease, malaria, histoplasmosis and aspergillosis, in ELISA experiments. Using sera from household contacts, values for Se and Sp were 100% and 65.3%, respectively. In conclusion, our proof-of-concept study has generated data that suggest that a new recombinant protein could be developed into a diagnostic antigen for leprosy.

Serodiagnosis of paucibacillary and multibacillary leprosy using a recombinant chimeric protein composed of specific B-cell epitopes derived from Mycobacterium leprae proteins.

Leprosy diagnosis is difficult due to the clinical similarity with other infectious diseases, and laboratory tests presents problems related to sensitivity and/or specificity. In this study, we used bioinformatics to assess Mycobacterium leprae proteins and formulated a chimeric protein that was tested as a diagnostic marker for the disease. The amino acid sequences from ML0008, ML0126, ML0308, ML1057, ML2028, ML2038, ML2498 proteins were evaluated, and the B-cell epitopes QASVAYPATSYADFRAHNHWWNGP, SLQRSISPNSYNTARVDP and QLLGQTADVAGAAKSGPVQPMGDRGSVSPVGQ were considered M. leprae-specific and used to construct the gene encoding the recombinant antigen. The gene was constructed, the recombinant protein was expressed, purified and tested in ELISA using 252 sera, which contained samples from multibacillary (MB) or paucibacillary (PB) leprosy patients, from their household contacts and healthy individuals, as well as from patients with Chagas disease, visceral and tegumentary leishmaniases (VL/TL), malaria, tuberculosis, and HIV. Sensitivity (Se) and specificity (Sp) for MB and PB samples compared to sera from both healthy subjects and individuals with cross-reactive diseases were 100%. The Se value for MB and PB samples compared to sera from household contacts was 100%, but Sp was 64%. In conclusion, data suggest that this protein could be considered in future studies for leprosy diagnosis.

Diagnosis of Canine Visceral Leishmaniasis by Flow Cytometry Serology using the rMELEISH Multiepitope Antigen Coupled in a Functional Bead.

BACKGROUND: Visceral leishmaniasis (VL) is a zoonotic disease, with dogs being the main reservoir of the Leishmania infantum parasite. OBJECTIVE: To develop a new flow cytometry test to diagnosis canine VL (CVL) diagnosis. METHODS: The current study addresses a new flow cytometry test using beads coupled to the multiepitope antigen rMELEISH. RESULTS: In the study set of samples a sensitivity (87.1%) and specificity (89.9%) was observed. Considering the dogs' clinical status, 20/20 (100.0%) of the symptomatic sera tested positive, while 19/22 (86.4%) of the oligosymptomatic and 16/20 (80.0%) of asymptomatic were positive. In the non-infected control, all samples (0/30) tested as negative. In the cross-reaction control, the test was more efficient in dogs infected with L. braziliensis (2/10) and Trypanosoma cruzi (0/10), than those with Babesia canis (4/10) and Ehrlichia canis (4/10). Dogs immunized with different vaccines (Leishmune, Leish-Tec®, or LBSap) did not present serological reactivity. CONCLUSION: The flow cytometry serology through coupling the antigen rMELEISH in functional beads showed high accuracy in diagnosing CVL.

Immunization with recombinant LiHyp1 protein plus adjuvant is protective against tegumentary leishmaniasis.

Tegumentary leishmaniasis (TL) is the main clinical manifestation of leishmaniasis, and it can cause the infected hosts to self-healing cutaneous lesions until mutilating scars in mucosal membranes, particularly in the nose and throat. The treatment against disease presents problems, and the diagnosis is hampered by variable sensitivity and/or specificity of the tests. In this context, the development of prophylactic vaccines could be considered as a strategy to control the disease. Previously, we showed that the recombinant LiHyp1 protein plus adjuvant protected mice from infection with Leishmania infantum, which causes visceral leishmaniasis. In the present study, we tested whether rLiHyp1 could induce protection against infection with L. amazonensis, a parasite species able to cause TL. We immunized BALB/c mice with rLiHyp1 plus saponin (rLiHyp1/S) or incorporated in micelles (rLiHyp1/M) as adjuvants and performed parasitological and immunological evaluations before and after infection. Results showed that after in vitro stimulation from spleen cell cultures using rLiHyp1 or a Leishmania antigenic extract (SLA), rLiHyp1/S and rLiHyp1/M groups developed a Th1-type immune response, which was characterized by high levels of IFN-γ, IL-2, TNF-α and IL-12 cytokines, nitrite, and IgG2a isotype antibodies when compared to values found in the control (saline, saponin, micelles alone) groups, which showed higher levels of anti-SLA IL-4, IL-10, and IgG1 antibodies before and after challenge. In addition, mice receiving rLiHyp1/S or rLiHyp1/M presented significant reductions in the lesion average diameter and parasite load in the infected tissue and internal organs. Blood samples were collected from healthy subjects and TL patients to obtain PBMC cultures, which were in vitro stimulated with rLiHyp1 or SLA, and results showed higher lymphoproliferation and IFN-γ production after stimulus using rLiHyp1, as compared to values found using SLA. These results suggest that rLiHyp1 plus adjuvant was protective against experimental TL and could also be considered for future studies as a vaccine candidate against human disease.

Efficacy of an Immunotherapy Combining Immunogenic Chimeric Protein Plus Adjuvant and Amphotericin B against Murine Visceral Leishmaniasis.

Visceral leishmaniasis (VL) in the Americas is a chronic systemic disease caused by infection with Leishmania infantum parasites. The toxicity of antileishmanial drugs, long treatment course and limited efficacy are significant concerns that hamper adequate treatment against the disease. Studies have shown the promise of an immunotherapeutics approach, combining antileishmanial drugs to reduce the parasitism and vaccine immunogens to activate the host immune system. In the current study, we developed an immunotherapy using a recombinant T cell epitope-based chimeric protein, ChimT, previously shown to be protective against Leishmania infantum, with the adjuvant monophosphoryl lipid A (MPLA) and amphotericin B (AmpB) as the antileishmanial drug. BALB/c mice were infected with L. infantum stationary promastigotes and later they received saline or were treated with AmpB, MPLA, ChimT/Amp, ChimT/MPLA or ChimT/MPLA/AmpB. The combination of ChimT/MPLA/AmpB significantly reduced the parasite load in mouse organs (p < 0.05) and induced a Th1-type immune response, which was characterized by higher ratios of anti-ChimT and anti-parasite IgG2a:IgG1 antibodies, increased IFN-γ mRNA and IFN-γ and IL-12 cytokines and accompanied by lower levels of IL-4 and IL-10 cytokines, when compared to other treatments and controls (all p < 0.05). Organ toxicity was also lower with the ChimT/MPLA/AmpB immunotherapy, suggesting that the inclusion of the vaccine and adjuvant ameliorated the toxicity of AmpB to some degree. In addition, the ChimT vaccine alone stimulated in vitro murine macrophages to significantly kill three different internalized species of Leishmania parasites and to produce Th1-type cytokines into the culture supernatants. To conclude, our data suggest that the combination of ChimT/MPLA/AmpB could be considered for further studies as an immunotherapy for L. infantum infection.

Immunotherapy Using Immunogenic Mimotopes Selected by Phage Display plus Amphotericin B Inducing a Therapeutic Response in Mice Infected with Leishmania amazonensis.

Leishmania amazonensis can cause cutaneous and visceral clinical manifestations of leishmaniasis in infected hosts. Once the treatment against disease is toxic, presents high cost, and/or there is the emergence of parasite-resistant strains, alternative means through which to control the disease must be developed. In this context, immunotherapeutics combining known drugs with immunogens could be applied to control infections and allow hosts to recover from the disease. In this study, immunotherapeutics protocols associating mimotopes selected by phage display and amphotericin B (AmpB) were evaluated in L. amazonensis-infected mice. Immunogens, A4 and A8 phages, were administered alone or associated with AmpB. Other animals received saline, AmpB, a wild-type phage (WTP), or WTP/AmpB as controls. Evaluations performed one and thirty days after the application of immunotherapeutics showed that the A4/AmpB and A8/AmpB combinations induced the most polarized Th1-type immune responses, which reflected in significant reductions in the lesion's average diameter and in the parasite load in the infected tissue and distinct organs of the animals. In addition, the combination also reduced the drug toxicity, as compared to values found using it alone. In this context, preliminary data presented here suggest the potential to associate A4 and A8 phages with AmpB to be applied in future studies for treatment against leishmaniasis.

Recombinant endonuclease III protein from Leishmania infantum associated with Th1-type adjuvants is immunogenic and induces protection against visceral leishmaniasis.

Vaccination against visceral leishmaniasis (VL) should be considered as a safe and effective measure to disease control; however, few vaccines are available against canine VL and there is no an approved human vaccine. In this context, in the present study, we evaluated the endonuclease III (ENDO) protein, which was recently showed to be antigenic for human disease, as a vaccine candidate against Leishmania infantum infection. The recombinant protein (rENDO) was administered in BALB/c mice alone or associated with saponin (rENDO/Sap) or micelles (rENDO/Mic) as adjuvants. Controls received saline, saponin or empty micelles. Results showed that both rENDO/Sap and rENDO/Mic compositions induced higher levels of IFN-γ, IL-12, TNF-α, and GM-CSF cytokines, besides nitrite and IgG2a isotype antibodies, before and after challenge infection, which were related to both CD4+ and CD8+ T cell subtypes. The immunological results contributed to significant reductions in the parasite load found in the spleens, livers, bone marrows and draining lymph nodes of the vaccinated animals. In general, mice immunized with rENDO/Mic presented a slightly higher Th1-type cellular and humoral immune response, as compared to those receiving rENDO/Sap. In addition, saponin caused a slight to moderate inflammatory edema in their vaccinated footpads, which was not observed when micelles were used with rENDO. In addition, a preliminary analysis showed that the recombinant protein was immunogenic to human cells cultures, since PBMCs from treated VL patients and healthy subjects showed higher lymphoproliferation and IFN-γ production in the culture supernatants. In conclusion, data suggest that rENDO could be considered as a candidate to be evaluated in future studies as vaccine to protect against VL.

Patents Related to Pathogenic Human Coronaviruses.

BACKGROUND: Coronaviruses have caused outbreaks of respiratory disease since the beginning of the 21st century, representing a significant threat to public health. Together, the severe acute respiratory syndrome coronavirus (SARS-CoV), the respiratory syndrome coronavirus (MERS-CoV), and, more recently, the novel coronavirus (SARS-CoV-2) have caused a large number of deaths around the world. Thus, investments in research and the development of strategies aimed at diagnosing, treating, and preventing these infections are urgently needed. OBJECTIVE: The objective of this study was to analyze the patents that address pathogenic coronaviruses in Google Patents databases in the last year (2019-2020). METHODS: The search strategy was carried out in April 2020, based on the keywords "SARS", "SARS-CoV", "MERS", "MERS-CoV", "SARS-CoV-2" and "COVID-19. Out of the patents examined, 25 were selected for a short description in this study. RESULTS: A total of 191 patents were analyzed, 149 of which were related to SARS-CoV, and 29 and 12 were related to MERS-CoV and SARS- CoV2, respectively. The patents addressed the issues of diagnosis, therapeutic agents, prevention and control, along with other applications. CONCLUSION: Several promising strategies have been documented in intellectual property databases favoring the need for further studies on the pathogenesis and optimization of the diagnosis and therapeutic treatment for these emerging infections.

Recent Patents on the Industrial Application of Alpha-amylases.

BACKGROUND: Alpha-amylases are enzymes capable of degrading polysaccharides, such as starch and glycogen. Found in various organisms, such as fungi and bacteria, these enzymes have great biotechnological potential due to their insertion in several industrial sectors ranging from food to biofuels. OBJECTIVE: The aim of this study was to analyze patents deposited in intellectual property databases on alpha-amylases in the fields of food, beverages, detergents, animal feeds, biofuels, pharmaceuticals and textiles, with the search period being 5 years. METHODS: This study targeted the use of alpha-amylases in various industrial sectors, so searches were carried out on the intellectual property database Espacenet website (European Patent Office - EPO) which contains more than 90 million patents deposited in its database. RESULTS: During the search for patents filed in the last 5 years, 186 were found related to the use of alpha-amylases. These were disturbed as follows: 84 (biofuel), 41 (drinks), 16 (pharmaceuticals), 15 (detergents), 11 (food), 10 (animal feed), 9 (textiles). From the total number of patents found, we selected 6 from each area, except pharmaceutical products, to discuss and provide information on the application of this enzyme. CONCLUSION: This study demonstrated that the sectors of beverages and animal feed have preferences for thermostable alpha-amylases while sectors such as food, biofuels and textiles only regarded the importance of enzymatic efficiency. The detergent sector presented the greatest use of alpha-amylases which had distinct biochemical characteristics as solvent resistance and thermostability. The pharmaceutical sector was the one that presented less patents related to the application of alpha amylases. In addition, this work showed that China is the country with the highest patent registration for the use of alpha-amylases in the analyzed period.

Diagnostic markers selected by immunoproteomics and phage display applied for the serodiagnosis of canine leishmaniosis.

Canine leishmaniosis (CanL) is one of the most important parasitic diseases found in several countries worldwide. Dogs are considered important domestic reservoirs of the parasites, being relevant in the maintenance of transmission cycle of the disease between sandflies and humans. However, the prevalence of asymptomatic infection is considerably higher than that of apparent clinical illness in the infected animals; thus making promptly necessary to diagnose the infection in these animals, which could help to allow to the adoption of more efficient control measures against disease. Parasitological tests, which are considered as gold standard to demonstrate the infection and diagnose the disease, present problems related with their sensitivity. Also, the sample´s collect is considered invasive. As consequence, serological tests could be applied as an additional tool to detect the asymptomatic and symptomatic CanL. For this purpose, distinct recombinant antigens have been studied; however, problems in their sensitivity and/or specificity have been still registered. The present review focus in advances in the identification of new diagnostic targets applied for the CanL diagnose, represented here by recombinant single, combined or chimeric proteins, as well as by peptides that mimic epitopes (mimotopes); which were selected by means of immunoproteomics and phage display.

A Custom-Designed Recombinant Multiepitope Protein for Human Cytomegalovirus Diagnosis.

BACKGROUND: The Human Cytomegalovirus (HCMV) has infected more than 90% of the world population and its prevalence can be related to the individuals geographical and socialeconomic status. Serological tests based on ELISA are pivotal for HCMV diagnosis. Due to the lack of standardization in the production/purification of antigens from viral preparations, ELISA tests are based on several recombinant proteins or peptides. As an alternative, multiepitope proteins may be employed. OBJECTIVE: In this work, we developed a recombinant multiepitope protein (rMEHCMV) for HCMV diagnosis based on conserved and immunodominant epitopes derived from tegument (pp150, pp65 and pp28), glycoprotein gB (pp38) and DNA polymerase subunit (pp52) of HCMV. METHODS: The rMEHCMV gene was synthesized de novo and overexpressed in Escherichia coli cells. The recombinant protein was purified to homogeneity using a Ni-NTA column. Biophysical analysis of recombinant protein was performed by circular dichroism. A preliminary biological activity test was performed using 12 positive human sera samples by using an in-house IgG ELISA. The following patents database were consulted: Espacenet, Google Patents and the National Institute of Intellectual Property (INPI, Brazil). RESULTS: The recombinant multiepitope protein was successfully expressed in E. coli. The structural data obtained by circular dichroism spectroscopy showed that rMEHCMV is structurally disordered. An in-house IgG ELISA test with rMEHCMV was successfully used to recognized IgG from human serum samples. CONCLUSION: Together, our results show that rMEHCMV should be considered as a potential antigenic target for HCMV diagnosis.

Recent Patents Applications in Red Biotechnology: A Mini-Review.

BACKGROUND: The different fields of biotechnology can be classified by colors, as a "rainbow" methodology. In this sense, the red biotechnology, focused on the preservation of health, has been outstanding in helping to solve this challenge through the provision of technologies, including diagnostic kits, molecular diagnostics, vaccines, innovations in cancer research, therapeutic antibodies and stem cells. OBJECTIVE: The main goal of this work is to highlight the different areas within the red Biotechnology. In this sense, we revised some patents regarding red biotechnology as examples to cover this subject. METHODS: A literature search of patents was performed from the followings Patents Database: INPI, USPTO, Esp@cenet, WIPO and Google Patents. RESULTS: Our analysis showed the following numbers from patents found: cancer research (8), diagnosis kit (9), vaccines (8), stem cells (9) and therapeutic antibodies (5), where the United States is the leader for most filled patents in Red Biotechnology. CONCLUSION: This mini-review has provided an update of some patents on Recent Patents in Red Biotechnology. As far as we know, this is the first mini-review report on Red Biotechnology based on patents.

Expression and Biochemical Characterization of a Yersinia intermedia Phytase Expressed in Escherichia coli.

BACKGROUND: Phytases are enzymes capable of degrading phytic acid and used in animal feed supplementation in order to improve digestibility through the release of minerals such as phosphorus. OBJECTIVE: The main goal of this study was to express and characterize a Yersinia intermedia phytase expressed in Escherichia coli cells. METHODS: The Y. intermedia phytase gene was synthesized and overexpressed in Escherichia coli cells. The phytase recombinante (rPHY) was purified to homogeneity using a Ni-NTA column. The biochemical and biophysical properties of the rPHY were measured in order to fully characterize the recombinant enzyme. The following patents database were consulted: Espacenet, USPTO, LATIPAT, Patent Scope, WIPO and Google Patents. RESULTS: The results showed that the rPHY is active at 37-40ºC and presented an optimal pH and temperature of 8.0 and 40°C, respectively. The phytase rPHY was activated by Cu2+ ion and showed resistance to trypsin and pepsin, retaining 55% of the activity at the ratio of 0.02. Furthermore, the dissociation constant (Kd = 1.1150 ± 0.0087 mM), as estimated by a fluorescence binding assay, suggests a medium affinity of the enzyme with the substrate. CONCLUSION: The results of this article can be considered as innovative and for this reason, they were protected by Intellectual Property Law in Brazil. Take together, the biochemical properties of the rPHY could be useful in future for its industrial application of this enzyme as an additive in the monogastric feed.

Immunogenicity and protective efficacy of a new Leishmania hypothetical protein applied as a DNA vaccine or in a recombinant form against Leishmania infantum infection.

Vaccination is one the most important strategies for the prevention of visceral leishmaniasis (VL). In the current study, a new Leishmania hypothetical protein, LiHyP, which was previously showed as antigenic in an immunoproteomic search in canine VL, was evaluated regarding its immunogenicity and protective efficacy against Leishmania infantum infection. The effects of the immunization using LiHyP were evaluated when administered as a DNA plasmid (DNA LiHyP) or recombinant protein (rLiHyP) associated with saponin. The immunity elicited by both vaccination regimens reduced the parasitism in liver, spleen, bone marrow and draining lymph nodes, being associated with high levels of IFN-γ, IL-12, GM-CSF, and specific IgG2a antibody, besides low production of IL-4, IL-10, and protein and parasite-specific IgG1 antibodies. CD4+ T cells contributed more significantly to IFN-γ production in the rLiHyP/saponin group, while CD8+ T cells were more important in the production of this cytokine in the DNA LiHyP group. In addition, increased IFN-γ secretion, along with low levels of IL-10, were found when PBMCs from treated VL subject and healthy individuals were stimulated with the recombinant protein. In conclusion, when administered either as a DNA plasmid or recombinant protein, LiHyP can direct the immune response towards a Th1 immune profile, protecting animals against L. infantum infection; therefore, it can be seen as a promising immunogen against human VL.

Vaccination with a CD4+ and CD8+ T-cell epitopes-based recombinant chimeric protein derived from Leishmania infantum proteins confers protective immunity against visceral leishmaniasis.

Vaccination seems to be the best approach to control visceral leishmaniasis (VL). Resistance against infection is based on the development of a Th1 immune response characterized by the production of interferons-γ (IFN-γ), interleukin-12 (IL-12), granulocyte-macrophage-colony-stimulating factor (GM-CSF), and tumor necrosis factor-α (TNF-α), among others. A number of antigens have been tested as potential targets against the disease; few of them are able to stimulate human immune cells. In the present study, 1 prediction of MHC class I and II molecules-specific epitopes in the amino acid sequences of 3 Leishmania proteins: 1 hypothetical, prohibitin, and small glutamine-rich tetratricopeptide repeat-containing proteins, was performed using bioinformatics tools, and a T-cell epitopes-based recombinant chimeric protein was constructed, synthetized and purified to be evaluated in invitro and in vivo experiments. The purified protein was tested regarding its immunogenicity in peripheral blood mononuclear cells (PBMCs) from healthy subjects and VL patients, as well as to its immunogenicity and protective efficacy in a murine model against Leishmania infantum infection. Results showed a Th1 response based on high IFN-γ and low IL-10 levels derived from in chimera-stimulated PBMCs in both healthy subjects and VL patients. In addition, chimera and/or saponin-immunized mice presented significantly lower parasite burden in distinct evaluated organs, when compared to the controls, besides higher levels of IFN-γ, IL-2, IL-12, and GM-CSF, and an IgG2a isotype-based humoral response. In addition, the CD4+ and CD8+ T-cell subtypes contributed to IFN-γ production in the protected animals. The results showed the immunogenicity in human cells and the protective efficacy against L. infantum in a murine model, and well indicate that this recombinant chimera can be considered as a promising strategy to be used against human disease.

Serological diagnosis and prognostic of tegumentary and visceral leishmaniasis using a conserved Leishmania hypothetical protein.

New candidates for serological markers against leishmaniasis are required to be identified, since the presence of high titers of anti-Leishmania antibodies remain detected in sera of treated and cured patients, when current antigens have being employed. In this study, the diagnostic performance of a conserved Leishmania hypothetical protein was evaluated against a human and canine serological panel. The serological follow-up of the patients was also evaluated, using this recombinant antigen (rLiHyS) in ELISA assays. In the results, high sensitivity and specificity values were found when rLiHyS was used in the serological tests, while when the recombinant A2 (rA2) protein or an antigenic Leishmania preparation were used as controls, low sensitivity and specificity were found. Regarding the serological follow-up of the patients, significant reductions in the anti-rLiHyS antibody levels were found and, one year after the treatments, the anti-protein IgG production was similar to this found in the non-infected groups, reflecting a drop of the anti-rLiHyS antibody production. In conclusion, the present study shows for the first time a new recombinant antigen used to identify tegumentary and visceral leishmaniasis, as well as being able to serologically distinguish treated and cured patients from those developing active disease.

Biofactories for the Production of Recombinant Phytases and their Application in the Animal Feed Industry.

BACKGROUND: Phytases are enzymes capable of degrading phytic acid and are used in animal feed supplementation in order to improve digestibility through the release of minerals such as phosphorus. Recent inventions show interest in production and optimization of recombinant phytases with biochemical and physicochemical characteristics promising for animal feed industry. METHODS: This review article is focused on relevant patents of promising phytases, together with the commonly used expression systems for their production and tools currently employed to generate new phytases. We revised all patents related to recombinant phytases and their application in the animal feed industry. The following patents databases were consulted: European Patent Office (Espacenet), the United States Patent and Trademark Office (USPTO), the United States Latin America Patents (LATIPAT), Patent scope -Search International and National Patent Collections (WIPO) and Google Patents. RESULTS: In this review, information was collected from recent publications, including 38 patents related production systems for different recombinant phytases and their application in the animal feed industry. CONCLUSION: We showed that important recombinant phytases were successfully produced in different expression systems. In addition, this work highlights certain biotechnological tools such as mutagenesis for generation of novel enzymes with biochemical properties of use in the animal feed industry.

Recombinant prohibitin protein of Leishmania infantum acts as a vaccine candidate and diagnostic marker against visceral leishmaniasis.

Visceral leishmaniasis (VL) represents a serious public health problem, as Leishmania infantum is one of main disease causative agents in the Americas. In a previous immunoproteomic study, the prohibitin (PHB) protein was identified in L. infantum promastigote and amastigote extracts by antibodies in asymptomatic and symptomatic VL dog sera. This protein was found to be highly conserved between different Leishmania spp., but it presented a low identity with amino acid sequences of other organisms. The aim of the present study was to evaluate the cellular response induced by the recombinant PHB (rPHB) protein in BALB/c mice, as well as in PBMCs purified from untreated and treated VL patients, as well as to evaluate its protective efficacy against an infection by L. infantum promastigotes. Our data showed that there was a Th1 cellular response to rPHB, based on high levels of IFN-γ, IL-12, and GM-CSF in the immunized animals, as well as a proliferative response specific to the protein and higher IFN-γ levels induced in PBMCs from individuals who had recovered from the disease. The protection was represented by significant reductions in the parasite load in the animals' spleen, liver, bone marrow, and draining lymph nodes, as compared to results found in the control groups. In addition, an anti-rPHB serology, using a canine and human serological panel, showed a high performance of this protein when diagnosing VL based on high sensitivity and specificity values, as compared to results found for the rA2 antigen and the soluble Leishmania antigenic extract. Our data suggest that PHB has a potential application for the diagnosis of canine and human VL through antibody detection, as well as an application as a vaccine candidate to protect against disease.

Antigenicity, immunogenicity and protective efficacy of a conserved Leishmania hypothetical protein against visceral leishmaniasis.

In this study, a Leishmania hypothetical protein, LiHyS, was evaluated regarding its antigenicity, immunogenicity and protective efficacy against visceral leishmaniasis (VL). Regarding antigenicity, immunoblottings and an enzyme-linked immunosorbent assay using human and canine sera showed high sensitivity and specificity values for the recombinant protein (rLiHyS) in the diagnosis of VL. When evaluating the immunogenicity of LiHyS, which is possibly located in the parasite's flagellar pocket, proliferative assays using peripheral blood mononuclear cells from healthy subjects or VL patients showed a high proliferative index in both individuals, when compared to the results obtained using rA2 or unstimulated cultures. Later, rLiHyS/saponin was inoculated in BALB/c mice, which were then challenged with Leishmania infantum promastigotes. The vaccine induced an interferon-γ, interleukin (IL)-12 and granulocyte-macrophage colony-stimulating factor production, which was maintained after infection and which was associated with high nitrite and IgG2a antibody levels, as well as low IL-4 and IL-10 production. Significant reductions in the parasite load in liver, spleen, bone marrow and draining lymph nodes were found in these animals. In this context, the present study shows that the rLiHyS has the capacity to be evaluated as a diagnostic marker or vaccine candidate against VL.