Theranostic applications of phage display to control leishmaniasis: selection of biomarkers for serodiagnostics, vaccination, and immunotherapy

AbstractPhage display is a high-throughput subtractive proteomic technology used for the generation and screening of large peptide and antibody libraries. It is based on the selection of phage-fused surface-exposed peptides that recognize specific ligands and demonstrate desired functionality for diagnostic and therapeutic purposes. Phage display has provided unmatched tools for controlling viral, bacterial, fungal, and parasitic infections, and allowed identification of new therapeutic targets to treat cancer, metabolic diseases, and other chronic conditions. This review presents recent advancements in serodiagnostics and prevention of leishmaniasis -an important tropical parasitic disease- achieved using phage display for the identification of novel antigens with improved sensitivity and specificity. Our focus is on theranostics of visceral leishmaniasis with the aim to develop biomarker candidates exhibiting both diagnostic and therapeutic potential to fight this important, yet neglected, tropical disease.

A high-copy T7 Escherichia coli expression vector for the production of recombinant proteins with a minimal N-terminal His-tagged fusion peptide

We report here the construction of a vector derived from pET3-His and pRSET plasmids for the expression and purification of recombinant proteins in Escherichia coli based on T7 phage RNA polymerase. The resulting pAE plasmid combined the advantages of both vectors: small size (pRSET), expression of a short 6XHis tag at N-terminus (pET3-His) and a high copy number of plasmid (pRSET). The small size of the vector (2.8 kb) and the high copy number/cell (200-250 copies) facilitate the subcloning and sequencing procedures when compared to the pET system (pET3-His, 4.6 kb and 40-50 copies) and also result in high level expression of recombinant proteins (20 mg purified protein/liter of culture). In addition, the vector pAE enables the expression of a fusion protein with a minimal amino-terminal hexa-histidine affinity tag (a tag of 9 amino acids using XhoI restriction enzyme for the 5'cloning site) as in the case of pET3-His plasmid and in contrast to proteins expressed by pRSET plasmids (a tag of 36 amino acids using BamHI restriction enzyme for the 5'cloning site). Thus, although proteins expressed by pRSET plasmids also have a hexa-histidine tag, the fusion peptide is much longer and may represent a problem for some recombinant proteins.

Progression of Salmonella Enteritidis phage type 4 strains in São Paulo State, Brazil

A total of 574 S. Enteritidis strains (383 from human sources and 191 from non-human sources) isolated between 1975-95, in São Paulo State, Brazil, were phagetyped. Among the strains isolated during the period of 1975-92, 80.9% of them belonged to phage type 8 (PT-8), but in 1993 strains of PT-4 accounted for 65.2% of all the S. Enteritidis isolates. In the following years, PT-4 strains accounted for 99.7% and 98.4% of phagetyped S. Enteritidis strains. The results obtained suggested that the current epidemic of S. Enteritidis in São Paulo State is clearly associated with the progression of PT-4 strains.

Rapid Characterization of S. mansoni Expression Library Clones of Potential Interest

A S. mansoni adult worm cDNA expression library was screened with sera from baboons in a early phase after infection. The clones that were positive with the early infection sera were examined for reactivity with pre-infection sera and heterologous infection sera. In order to discriminate a positive antibody reaction from the reactivity due to residual anti-E. coli antibodies, an unrelated cDNA clone was plated with the positive clone. The unrelated clone provided the negative background and the contrast necessary to discern a positive antibody reaction. In this way, we were able to eliminate selected clones that were positive with the pre-infection sera or heterologous infection sera. This characterization of the expression library clones enabled us to quickly target only clones with the desired pattern of antibody reactivity for sequencing, subcloning, and expressing

Phage types of Salmonella Enteritidis isolated from clinical and food samples, and from broiler carcasses in Southern Brazil

272 isolates of Salmonella Enteritidis (111 isolated from frozen broiler chicken carcasses, 126 from human food and other biological materials involved in food poisoning outbreaks and 35 from different poultry materials) were selected for phage typing. From these, 111 were phage typed, 57.65% being classified as phage type 4, 32.43% as phage type 4a, 3.60% as phage type 6a and 0.90% as phage type 7, whereas 5.40% samples were not phage typeable. The predominance of phage type 4 is in agreement with the results published worldwide, and reinforces the need for studies related to the epidemiological meaning of these findings.

Characterization of a clone from an adult worm cDNA library selected with anti-Schistosoma mansoni human antibodies dissociated from immune complexes: a preliminary report

Considering the scarcity of defined antigens, actually useful and reliable for use in the field studies, we propose an alternative method for selection of cDNA clones with potential use in the diagnosis of schistosomiasis. Human antibodies specific to a protein fraction of 31/32 kDa (Sm31/32), dissociated from immune complexes, are used for screening of clones from an adult worm cDNA library. Partial sequencing of five clones, selected through this strategy, showed to be related to Schistosoma mansoni: two were identified as homologous to heat shock protein 70, one to glutathione S-transferase, one to homeodomain protein, and one to a previously described EST (expressed sequence tag) of S. mansoni. This last clone was the most consistently reactive during the screening process with the anti-Sm31/32 antibodies dissociated from the immune complexes. The complete sequence of this clone was obtained and the translation data yielded only one ORF (open reading frame) that code for a protein with 57 amino acids. Based on this amino acid sequence two peptides were chemically synthesized and evaluated separately against a pool of serum samples from schistosomiasis patients and non-schistosomiasis individuals. Both peptides showed strong reactivity only against the positive pool, suggesting that these peptides may be useful as antigens for the diagnosis of schistosomiasis mansoni.

The Role of B-Type Natriuretic Peptide in the Diagnosis of Congestive Heart Failure in Patients Presenting to an Emergency Department with Dyspnea

OBJECTIVE: To determine the utility of B-type natriuretic peptide (BNP) in the diagnosis of congestive heart failure (CHF) in patients presenting with dyspnea to an emergency department (ED). METHODS: Seventy patients presenting with dyspnea to an ED from April to July 2001 were included in the study. Mean age was 72±16 years and 33 (47%) were male. BNP was measured in all patients at the moment of admission to the ED. Emergency-care physicians, blinded to BNP values, were required to assign a probable initial diagnosis. A cardiologist retrospectively reviewed the data (blinded to BNP measurements) and assigned a definite diagnosis, which was considered the gold standard for assessing the diagnostic performance of BNP. RESULTS: The mean BNP concentration was higher in patients with CHF (n=36) than in those with other diagnoses (990±550 vs 80±67 pg/mL, p<0.0001). Patients with systolic dysfunction had higher BNP levels than those with preserved systolic function (1,180±641 vs 753±437 pg/mL, p=0.03). At a blood concentration of 200 pg/mL, BNP showed a sensitivity of 100%, specificity of 97.1%, positive predictive value of 97.3%, and negative predictive value of 100%. The application of BNP could have potentially corrected all 16 cases in which the diagnosis was missed by the emergency department physician. CONCLUSION: BNP measurement is a useful tool in the diagnosis of CHF in patients presenting to the ED with dyspnea.

Cellular immune response of humans to the circumsporozoite protein of Plasmodium vivax

The cellular immune response to the circumsporozoite (CS) protein of plasmodium vivax of individuals from malaria-endemic areas of Brazil was studied. We examined the in vitro proliferative response of the peripheral blood mononuclear cells (PBMC) of 22 individuals when stimulated with a CS recombinant protein (rPvCS-2) and two other synthetic peptides based on the sequenceof the P. vivax CS protein. Seven of the individuals from malaria-endemic area displayed an antigen specific in vitro proliferative responseto the recombinant protein PvCS-2 and one out of 6, proliferative response to the peptide 308-320. In contrast, none of the individuals displayed a proliferative reponse when stimulated with the D/A peptide which represent some of the repeated units present in this CS protein. Our study, therefore, provides evidence for the presence, withinthe major surface antigen of P. vivax sporozoites, of epitopes capble to induce proliferation of human PBMC.

The p53 gene expression and its developmental regulation in schistosomes

We have studied the gene expression, especially of the oncoproteins, and its regulation in schistosomes. Schistosomes have a complex life cycle with defined dimorphic lifestyle. The parasite are so far unique in biology in expressing oncogene products in their adult stage. In order to characterize the expression and developmental regulation, a lambda gt 11 cDNA library and lambda EMBL4 genomic DNA library of each growth stage of Schistosoma mansoni and S. japonicum was constructed, and was screened with various monoclonal antibodies against ongogene products. One positive plaque reacted to anti-p53 antibody (Ab-2, Oncogene Science, Inc.) was further analyzed. This fusion protein was about 120 KDa in molecular weights, and expressed as 1.4 Kb RNA in the adult stage. P53 gene is well-known as the negative regulator of the cell cicle, and the mutations in the gene are turning out to be the most common genetic alterations in human cancers. The comparison of the gene structure among species and stages were being conducted. Chromosome structures, C-band formation, and the results of in situ hybridization using the phage probe would be discussed.

Sequencing and identification of expressed Schistosoma mansoni genes by random selection of cDNA clones from a directional library

We have initiated a gene discovery program in Schistosoma mansoni based on the technique of Expressed Sequence Tags (ESTs), i.e. partial sequences of cDNAs obtained from single passes in automatic DNA sequencers. ESTs can be used to identify genese onf the basis of their homology whith sequences from other species deposited in DNA or protein databases. Trasncripts with sequences without matches in teh databases may represent novel parasite-specific genes. This approach has shown to be very efficient and in less than two years a broad range of novel genes has already been ascertained, more than doubling the number of known S. mansoni genes.