Development of an antibody against EtpA from enterotoxigenic Escherichia coli and evaluation of its use for bacterial isolation using magnetic beads.

The enterotoxigenic Escherichia coli (ETEC) strain is one of the most frequent causative agents of childhood diarrhea and travelers' diarrhea in low-and middle-income countries. Among the virulence factors secreted by ETEC, the exoprotein EtpA has been described as an important. In the present study, a new detection tool for enterotoxigenic E. coli bacteria using the EtpA protein was developed. Initially, antigenic sequences of the EtpA protein were selected via in silico prediction. A chimeric recombinant protein, corresponding to the selected regions, was expressed in an E. coli host, purified and used for the immunization of mice. The specific recognition of anti-EtpA IgG antibodies generated was evaluated using flow cytometry. The tests demonstrated that the antibodiesdeveloped were able to recognize the native EtpA protein. By coupling these antibodies to magnetic beads for the capture and detection of ETEC isolates, cytometric analyses showed an increase in sensitivity, specificity and the effectiveness of the method of separation and detection of these pathogens. This is the first report of the use of this methodology for ETEC separation. Future trials may indicate their potential use for isolating these and other pathogens in clinical samples, thus accelerating the diagnosis and treatment of diseases.

Improvement of Bacillus subtilis Spore Enumeration and Label Analysis in Flow Cytometry.

The spores of Bacillus subtilis have already been proposed for different biotechnological and immunological applications; however, there is an increasing need for the development of methodologies that improve the detection of antigens immobilized on the surface of spores together with their quantification. Flow cytometry-based analyses have been previously proposed as fast, reliable, and specific approaches for detecting labeled cells of B. subtilis. Herein, we propose the use of flow cytometry to evaluate the display efficiency of a fluorescent antibody (FA) on the surface of the spore and quantify the number of spores using counting beads. For this, we used ethidium bromide as a DNA marker and an allophycocyanin (APC)-labeled antibody, which was coupled to the spores, as a surface marker. The quantification of spores was performed using counting beads since this technique demonstrates high accuracy in the detection of cells. The labeled spores were analyzed using a flow cytometer, which confirmed the coupling. As a result, it was demonstrated that DNA labeling improved the accuracy of quantification by flow cytometry, for the detection of germinated spores. It was observed that ethidium bromide was not able to label dormant spores; however, this technique provides a more precise determination of the number of spores with fluorescent protein coupled to their surface, thus helping in the development of studies that focus on the use of spores as a biotechnological platform in different applications.

Plasmodium falciparum merozoite surface protein 3 as a vaccine candidate: a brief review.

Despite the many efforts of researchers around the world, there is currently no effective vaccine for malaria. Numerous studies have been developed to find vaccine antigens that are immunogenic and safe. Among antigen candidates, Plasmodium falciparum merozoite surface protein 3 (MSP3) has stood out in a number of these studies for its ability to induce a consistent and protective immune response, also being safe for use in humans. This review presents the main studies that explored MSP3 as a vaccine candidate over the last few decades. MSP3 formulations were tested in animals and humans and the most advanced candidate formulations are MSP3-LSP, a combination of MSP3 and LSP1, and GMZ2 (a vaccine based on the recombinant protein fusion GLURP and MSP3) which is currently being tested in phase II clinical studies. This brief review highlights the history and the main formulations of MSP3-based vaccines approaches against P. falciparum .

Bacillus subtilis spores as delivery system for nasal Plasmodium falciparum circumsporozoite surface protein immunization in a murine model.

Malaria remains a widespread public health problem in tropical and subtropical regions around the world, and there is still no vaccine available for full protection. In recent years, it has been observed that spores of Bacillus subtillis can act as a vaccine carrier and adjuvant, promoting an elevated humoral response after co-administration with antigens either coupled or integrated to their surface. In our study, B. subtillis spores from the KO7 strain were used to couple the recombinant CSP protein of P. falciparum (rPfCSP), and the nasal humoral-induced immune response in Balb/C mice was evaluated. Our results demonstrate that the spores coupled to rPfCSP increase the immunogenicity of the antigen, which induces high levels of serum IgG, and with balanced Th1/Th2 immune response, being detected antibodies in serum samples for 250 days. Therefore, the use of B. subtilis spores appears to be promising for use as an adjuvant in a vaccine formulation.

Plasmodium falciparum merozoite surface protein 3 as a vaccine candidate: a brief review

ABSTRACT Despite the many efforts of researchers around the world, there is currently no effective vaccine for malaria. Numerous studies have been developed to find vaccine antigens that are immunogenic and safe. Among antigen candidates, Plasmodium falciparum merozoite surface protein 3 (MSP3) has stood out in a number of these studies for its ability to induce a consistent and protective immune response, also being safe for use in humans. This review presents the main studies that explored MSP3 as a vaccine candidate over the last few decades. MSP3 formulations were tested in animals and humans and the most advanced candidate formulations are MSP3-LSP, a combination of MSP3 and LSP1, and GMZ2 (a vaccine based on the recombinant protein fusion GLURP and MSP3) which is currently being tested in phase II clinical studies. This brief review highlights the history and the main formulations of MSP3-based vaccines approaches against P. falciparum .

Circumsporozoite Surface Protein-based malaria vaccines: a review.

Malaria represents a serious public health problem, presenting with high rates of incidence, morbidity and mortality in tropical and subtropical regions of the world. According to the World Health Organization, in 2018 there were 228 million cases and 405 thousand deaths caused by this disease in the world, affecting mainly children and pregnant women in Africa. Despite the programs carried out to control this disease, drug resistance and invertebrate vector resistance to insecticides have generated difficulties. An efficient vaccine against malaria would be a strategy with a high impact on the eradication and control of this disease. Researches aimed at developing vaccines have focused on antigens of high importance for the survival of the parasite such as the Circumsporozoite Surface Protein, involved in the pre-erythrocytic cycle during parasites invasion in hepatocytes. Currently, RTS'S is the most promising vaccine for malaria and was constructed using CSP; its performance was evaluated using two types of adjuvants: AS01 and AS02. The purpose of this review was to provide a bibliographic survey of historical researches that led to the development of RTS'S and its performance analysis over the decade. The search for new adjuvants to be associated with this antigen seems to be a way to obtain higher percentages of protection for a future malaria vaccine.

HTLV-2 infection in Manaus, Brazil: first description of HTLV-2c subtype in an urban area of the Western Amazon region

Abstract INTRODUCTION: We investigated the prevalence of human T-cell lymphotropic virus types 1 and 2 (HTLV-1/2) infection in patients with hematological diseases from the western Amazon region of Brazil. METHODS: Samples from 306 patients were submitted for the molecular diagnosis of HTLV-1/2 infection by real time PCR (qPCR), with amplification, sequencing, and phylogenetic analysis of the long terminal repeat (LTR) region. RESULTS: A 29-year-old male carrier of sickle cell anemia with a history of multiple blood transfusions was diagnosed with the HTLV-2c subtype. CONCLUSIONS: This study describes the first known occurrence of HTLV-2c in the urban area of Brazil's western Amazon region.

HTLV-2 infection in Manaus, Brazil: first description of HTLV-2c subtype in an urban area of the Western Amazon region.

INTRODUCTION: We investigated the prevalence of human T-cell lymphotropic virus types 1 and 2 (HTLV-1/2) infection in patients with hematological diseases from the western Amazon region of Brazil. METHODS: Samples from 306 patients were submitted for the molecular diagnosis of HTLV-1/2 infection by real time PCR (qPCR), with amplification, sequencing, and phylogenetic analysis of the long terminal repeat (LTR) region. RESULTS: A 29-year-old male carrier of sickle cell anemia with a history of multiple blood transfusions was diagnosed with the HTLV-2c subtype. CONCLUSIONS: This study describes the first known occurrence of HTLV-2c in the urban area of Brazil's western Amazon region.

A simple, ex vivo phagocytosis assay of Plasmodium vivax merozoites by flow cytometry.

As phagocytosis is the first line of defense against malaria, we developed a phagocytosis assay with Plasmodium vivax (P. vivax) merozoites that can be applied to evaluate vaccine candidates. Briefly, after leukocyte removal with loosely packed cellulose powder in a syringe, P. vivax trophozoites matured to the merozoite-rich schizont stages in the presence of the E64 protease inhibitor. The Percoll gradient-enriched schizonts were chemically disrupted to release merozoites that were submitted to merozoite opsonin-dependent phagocytosis in two phagocytic lines with human and mouse antibodies against the N- and C-terminus of P. vivax Merozoite Surface Protein-1 (Nterm-PvMSP1 and MSP119). The resulting assay is simple and efficient for use as a routine phagocytic assay for the evaluation of merozoite stage vaccine candidates.

Chicken eggs as a surveillance tool for malaria and leishmaniasis vector presence.

INTRODUCTION: Malaria and leishmaniases are transmitted by vectors during blood-feeding. Vector-infected animals develop antibodies against the vector's saliva. This study evaluated IgY antibody detection in the chicken eggs exposed to bites from Migonemyia migonei, Lutzomyia longipalpis and Anopheles aquasalis. METHODS: We used ELISA to quantify the antibody levels in the sera and exposed chicken eggs. RESULTS: High IgY levels were observed following immunization; furthermore, higher reactivity was observed in the eggs and species-specific immune response was observed post final immunization. CONCLUSIONS: Chicken eggs can be used as sentinels to surveil vector saliva antibodies.

A simple, ex vivo phagocytosis assay of Plasmodium vivax merozoites by flow cytometry

As phagocytosis is the first line of defense against malaria, we developed a phagocytosis assay with Plasmodium vivax (P. vivax) merozoites that can be applied to evaluate vaccine candidates. Briefly, after leukocyte removal with loosely packed cellulose powder in a syringe, P. vivax trophozoites matured to the merozoite-rich schizont stages in the presence of the E64 protease inhibitor. The Percoll gradient-enriched schizonts were chemically disrupted to release merozoites that were submitted to merozoite opsonin-dependent phagocytosis in two phagocytic lines with human and mouse antibodies against the N- and C-terminus of P. vivax Merozoite Surface Protein-1 (Nterm-PvMSP1 and MSP119). The resulting assay is simple and efficient for use as a routine phagocytic assay for the evaluation of merozoite stage vaccine candidates.

Chicken eggs as a surveillance tool for malaria and leishmaniasis vector presence

Abstract INTRODUCTION: Malaria and leishmaniases are transmitted by vectors during blood-feeding. Vector-infected animals develop antibodies against the vector's saliva. This study evaluated IgY antibody detection in the chicken eggs exposed to bites from Migonemyia migonei, Lutzomyia longipalpis and Anopheles aquasalis. METHODS: We used ELISA to quantify the antibody levels in the sera and exposed chicken eggs. RESULTS: High IgY levels were observed following immunization; furthermore, higher reactivity was observed in the eggs and species-specific immune response was observed post final immunization. CONCLUSIONS: Chicken eggs can be used as sentinels to surveil vector saliva antibodies.

Genetic diversity of MSP1 Block 2 of Plasmodium vivax isolates from Manaus (central Brazilian Amazon).

The diversity of MSP1 in both Plasmodium falciparum and P. vivax is presumed be associated to parasite immune evasion. In this study, we assessed genetic diversity of the most variable domain of vaccine candidate N-terminal PvMSP1 (Block 2) in field isolates of Manaus. Forty-seven blood samples the polymorphism of PvMSP1 Block 2 generates four fragment sizes. In twenty-eight of them, sequencing indicated seven haplotypes of PvMSP1 Block 2 circulating among field isolates. Evidence of striking exchanges was observed with two stretches flanking the repeat region and two predicted recombination sites were described. Single nucleotide polymorphisms determined with concurrent infections per patient indicated that nonsynonymous substitutions occurred preferentially in the repeat-rich regions which also were predicted as B-cell epitopes. The comprehensive understanding of the genetic diversity of the promising Block 2 associated with clinical immunity and a reduced risk of infection by Plasmodium vivax would be important for the rationale of malaria vaccine designs.

A novel polyclonal antibody-based sandwich ELISA for detection of Plasmodium vivax developed from two lactate dehydrogenase protein segments.

BACKGROUND: Immunoassays for Plasmodium detection are, presently, most frequently based on monoclonal antibodies (MAbs); Polyclonal antibodies (PAbs), which are cheaper to develop and manufacture, are much less frequently used. In the present study we describe a sandwich ELISA assay which is capable of detecting P. vivax Lactate Dehydrogenase (LDH) in clinical blood samples, without cross reacting with those infected with P. falciparum. METHODS: Two recombinant proteins were produced from different regions of the P. vivax LDH gene. Two sandwich ELISA assay were then designed: One which uses mouse anti-LDH 1-43aa PAbs as primary antibodies ("Test 1") and another which uses anti-LDH 35-305aa PAbs ("Test 2") as the primary antibodies. Rabbit anti-LDH 1-43aa PAbs were used as capture antibodies in both ELISA assays. Blood samples taken from P. vivax and P. falciparum infected patients (confirmed by light microscopy) were analysed using both tests. RESULTS: "Test 2" performed better at detecting microscopy-positive blood samples when compared to "Test 1", identifying 131 of 154 positive samples (85%); 85 positives (55%) were identified using "test 1". "Test 1" produced one false positive sample (from the 20 malaria-free control) blood samples; "test 2" produced none. Kappa coefficient analysis of the results produced a value of 0.267 when microscope-positive blood smears were compared with "test 1", but 0.734 when microscope-positive blood smears were compared with the results from "test 2". Positive predictive value (PPV) and negative predictive value (NPV) were observed to be 98% and 22% respectively, for "Test 1", and 99% and 45%, for "test 2". No cross reactivity was detected with P. falciparum positive blood samples (n = 15) with either test assay. CONCLUSION: Both tests detected P. vivax infected blood and showed no evidence of cross-reacting with P. falciparum. Further studies will need to be conducted to establish the full potential of this technique for malaria diagnostics. As well as representing a promising new cost-effective novel technique for P. vivax diagnosis and research, the method for developing this assay also highlights the potential for PAb-based strategies for diagnostics in general.