Natural antibody response to Plasmodium falciparum merozoite antigens MSP5, MSP9 and EBA175 is associated to clinical protection in the Brazilian Amazon

BACKGROUND: Antibodies have an essential role in the acquired immune response against blood stage P. falciparum infection. Although several antigens have been identified as important antibody targets, it is still elusive which antigens have to be recognized for clinical protection. Herein, we analyzed antibodies from plasmas from symptomatic or asymptomatic individuals living in the same geographic area in the Western Amazon, measuring their recognition of multiple merozoite antigens. METHODS: Specific fragments of genes encoding merozoite proteins AMA1 and members of MSP and EBL families from circulating P. falciparum field isolates present in asymptomatic and symptomatic patients were amplified by PCR. After cloning and expression of different versions of the antigens as recombinant GST-fusion peptides, we tested the reactivity of patients' plasmas by ELISA and the presence of IgG subclasses in the most reactive plasmas. RESULTS: 11 out of 24 recombinant antigens were recognized by plasmas from either symptomatic or asymptomatic infections. Antibodies to MSP9 (X2(DF=1) = 9.26/p = 0.0047) and MSP5 (X2(DF=1) = 8.29/p = 0.0069) were more prevalent in asymptomatic individuals whereas the opposite was observed for MSP1 block 2-MAD20 (X2(DF=1) = 6.41/p = 0.0206, Fisher's exact test). Plasmas from asymptomatic individuals reacted more intensely against MSP4 (U = 210.5, p < 0.03), MSP5 (U = 212, p < 0.004), MSP9 (U = 189.5, p < 0.002) and EBA175 (U = 197, p < 0.014, Mann-Whitney's U test). IgG1 and IgG3 were predominant for all antigens, but some patients also presented with IgG2 and IgG4. The recognition of MSP5 (OR = 0.112, IC95% = 0.021-0.585) and MSP9 (OR = 0.125, IC95% = 0.030-0.529, cross tab analysis) predicted 8.9 and 8 times less chances, respectively, to present symptoms. Higher antibody levels against MSP5 and EBA175 were associated by odds ratios of 9.4 (IC95% = 1.29-69.25) and 5.7 (IC95% = 1.12-29.62, logistic regression), respectively, with an asymptomatic status. CONCLUSIONS: Merozoite antigens were targets of cytophilic antibodies and antibodies against MSP5, MSP9 and EBA175 were independently associated with decreased symptoms.

Oral immunization with attenuated Salmonella vaccine expressing Escherichia coli O157:H7 intimin gamma triggers both systemic and mucosal humoral immunity in mice

Human infections with EHEC such as O157:H7 have been a great concern for worldwide food-industry surveillance. This pathogen is commonly associated with bloody diarrhea that can evolve to the life-threatening hemolytic uremic syndrome. Animals are the natural reservoir where this pathogen remains asymptomatically, in steps of ingestion and colonization of the bowel. The bacterium is shed in the feces, contaminating the surroundings, including water and food that are directed for human consumption. A major player in this colonization process is intimin, an outer membrane adhesion molecule encoded by the E. coli attachment and effacement (eae) gene that has been shown to be essential for intimate bacterial attachment to eukaryotic host cells. In an attempt to reduce the colonization of animal reservoirs with EHEC O157:H7, we designed a vaccine model to induce an immune response against intimin gamma. The model is based on its recombinant expression in attenuated Salmonella, used as a suitable vaccine vector because of its recognized ability to deliver recombinant antigens and to elicit all forms of immunity: mucosal, systemic, and humoral responses. To test this model, mice were orally immunized with a S. enterica serovar Typhimurium strain carrying the pYA3137eaeA vector, and challenged with E. coli O157:H7. Here we show that immunization induced the production of high levels of specific IgG and IgA antibodies and promoted reduction in the fecal shedding of EHEC after challenge. The live recombinant vaccine reported herein may contribute to the efforts of reducing animal intestinal mucosa colonization.

Identification of Leishmania infantum chagasi proteins in urine of patients with visceral leishmaniasis: a promising antigen discovery approach of vaccine candidates

Visceral leishmaniasis (VL) is a serious lethal parasitic disease caused by Leishmania donovani in Asia and by Leishmania infantum chagasi in southern Europe and South America. VL is endemic in 47 countries with an annual incidence estimated to be 500 000 cases. This high incidence is due in part to the lack of an efficacious vaccine. Here, we introduce an innovative approach to directly identify parasite vaccine candidate antigens that are abundantly produced in vivo in humans with VL. We combined RP-HPLC and mass spectrometry and categorized three L. infantum chagasi proteins, presumably produced in spleen, liver and bone marrow lesions and excreted in the patients urine. Specifically, these proteins were the following: Li-isd1 (XP_001467866.1), Li-txn1 (XP_001466642.1) and Li-ntf2 (XP_001463738.1). Initial vaccine validation studies were performed with the rLi-ntf2 protein produced in Escherichia coli mixed with the adjuvant BpMPLA-SE. This formulation stimulated potent Th1 response in BALB/c mice. Compared to control animals, mice immunized with Li-ntf2+ BpMPLA-SE had a marked parasite burden reduction in spleens at 40 days post-challenge with virulent L. infantum chagasi. These results strongly support the proposed antigen discovery strategy of vaccine candidates to VL and opens novel possibilities for vaccine development to other serious infectious diseases.

Head-to-Head Comparison of Three Vaccination Strategies Based on DNA and Raw Insect-Derived Recombinant Proteins against Leishmania

Parasitic diseases plague billions of people among the poorest, killing millions annually, and causing additional millions of disability-adjusted life years lost. Leishmaniases affect more than 12 million people, with over 350 million people at risk. There is an urgent need for efficacious and cheap vaccines and treatments against visceral leishmaniasis (VL), its most severe form. Several vaccination strategies have been proposed but to date no head-to-head comparison was undertaken to assess which is the best in a clinical model of the disease. We simultaneously assayed three vaccination strategies against VL in the hamster model, using KMPII, TRYP, LACK, and PAPLE22 vaccine candidate antigens. Four groups of hamsters were immunized using the following approaches: 1) raw extracts of baculovirus-infected Trichoplusia ni larvae expressing individually one of the four recombinant proteins (PROT); 2) naked pVAX1 plasmids carrying the four genes individually (DNA); 3) a heterologous prime-boost (HPB) strategy involving DNA followed by PROT (DNA-PROT); and 4) a Control including empty pVAX1 plasmid followed by raw extract of wild-type baculovirus-infected T. ni larvae. Hamsters were challenged with L. infantum promastigotes and maintained for 20 weeks. While PROT vaccine was not protective, DNA vaccination achieved protection in spleen. Only DNA-PROT vaccination induced significant NO production by macrophages, accompanied by a significant parasitological protection in spleen and blood. Thus, the DNA-PROT strategy elicits strong immune responses and high parasitological protection in the clinical model of VL, better than its corresponding naked DNA or protein versions. Furthermore, we show that naked DNA coupled with raw recombinant proteins produced in insect larvae biofactories -the cheapest way of producing DNA-PROT vaccines-is a practical and cost-effective way for potential "off the shelf" supplying vaccines at very low prices for the protection against leishmaniases, and possibly against other parasitic diseases affecting the poorest of the poor.

Serological detection of Plasmodium vivax malaria using recombinant proteins corresponding to the 19-kDa C-terminal region of the merozoite surface protein-1

Abstract Background Serological tests to detect antibodies specific to Plasmodium vivax could be a valuable tool for epidemiological studies, for screening blood donors in areas where the malaria is not endemic and for diagnosis of infected individuals. Because P. vivax cannot be easily obtained in vitro, ELISA assays using total or semi-purified antigens are rarely used. Based on this limitation, we tested whether recombinant proteins representing the 19 kDa C-terminal region of the merozoite surface protein-1 of P. vivax (MSP119) could be useful for serological detection of malaria infection. Methods Three purified recombinant proteins produced in Escherichia coli (GST-MSP119, His6-MSP119 and His6-MSP119-PADRE) and one in Pichia pastoris (yMSP119-PADRE) were compared for their ability to bind to IgG antibodies of individuals with patent P. vivax infection. The method was tested with 200 serum samples collected from individuals living in the north of Brazil in areas endemic for malaria, 53 serum samples from individuals exposed to Plasmodium falciparum infection and 177 serum samples from individuals never exposed to malaria. Results Overall, the sensitivity of the ELISA assessed with sera from naturally infected individuals was 95%. The proportion of serum samples that reacted with recombinant proteins GST-MSP119, His6-MSP119, His6-MSP119-PADRE and yMSP119-PADRE was 90%, 93.5%, 93.5% and 93.5%, respectively. The specificity values of the ELISA determined with sera from healthy individuals and from individuals with other infectious diseases were 98.3% (GST-MSP119), 97.7% (His6-MSP119 and His6-MSP119-PADRE) or 100% (yMSP119-PADRE). Conclusions Our study demonstrated that for the Brazilian population, an ELISA using a recombinant protein of the MSP119 can be used as the basis for the development of a valuable serological assay for the detection of P. vivax malaria.

Evaluation of the acquired immune responses to Plasmodium vivax VIR variant antigens in individuals living in malaria-endemic areas of Brazil

Abstract Background The naturally-acquired immune response to Plasmodium vivax variant antigens (VIR) was evaluated in individuals exposed to malaria and living in different endemic areas for malaria in the north of Brazil. Methods Seven recombinant proteins representing four vir subfamilies (A, B, C, and E) obtained from a single patient from the Amazon Region were expressed in Escherichia coli as soluble glutathione S-transferase fusion proteins. The different recombinant proteins were compared by ELISA with regard to the recognition by IgM, IgG, and IgG subclass of antibodies from 200 individuals with patent infection. Results The frequency of individuals that presented antibodies anti-VIR (IgM plus IgG) during the infection was 49%. The frequencies of individuals that presented IgM or IgG antibodies anti-VIR were 29.6% or 26.0%, respectively. The prevalence of IgG antibodies against recombinant VIR proteins was significantly lower than the prevalence of antibodies against the recombinant proteins representing two surface antigens of merozoites of P. vivax: AMA-1 and MSP119 (57.0% and 90.5%, respectively). The cellular immune response to VIR antigens was evaluated by in vitro proliferative assays in mononuclear cells of the individuals recently exposed to P. vivax. No significant proliferative response to these antigens was observed when comparing malaria-exposed to non-exposed individuals. Conclusion This study provides evidence that there is a low frequency of individuals responding to each VIR antigens in endemic areas of Brazil. This fact may explain the host susceptibility to new episodes of the disease.

Production and characterization of poly-(3-hydroxybutyrate) from recombinant Escherichia coli grown on cheap renewable carbon substrates

Although the biopolymer poly-(3-hydroxybutyrate), P[3HB], presents physicochemical properties that make it an alternative material to conventional plastics, its biotechnological production is quite expensive. As carbon substrates contribute greatly to P[3HB] production cost, the utilization of a cheaper carbon substrate and less demanding micro-organisms should decrease its cost. In the present study a 23 factorial experimental design was applied, aiming to evaluate the effects of using hydrolysed corn starch (HCS) and soybean oil (SBO) as carbon substrates, and cheese whey (CW) supplementation in the mineral medium (MM) on the responses, cell dried weigh (DCW), percentage P[3HB] and mass P[3HB] by recombinant Escherichia coli strains JM101 and DH10B, containing the P[3HB] synthase genes from Cupriavidus necator (ex-Ralstonia eutropha). The analysis of effects indicated that the substrates and the supplement and their interactions had positive effect on CDW. Statistically generated equations showed that, at the highest concentrations of HCS, SO and CW, theoretically it should be possible to produce about 2 g L(1) DCW, accumulating 50% P[3HB], in both strains. To complement this study, the strain that presented the best results was cultivated in MM added to HCS, SBO and CW ( in best composition observed) and complex medium (CM) to compare the obtained P[3HB] in terms of physicochemical parameters. The obtained results showed that the P[3HB] production in MM (1.29 g L(-1)) was approximately 20% lower than in CM (1.63 g L(-1)); however, this difference can be compensated by the lower cost of the MM achieved by the use of cheap renewable carbon sources. Moreover, using differential scanning calorimetry and thermogravimetry analyses, it was observed that the polymer produced in MM was the one which presented physicochemical properties (Tg and Tf) that were more similar to those found in the literature for P[3HB].

Analysis of the protective potential of antigens released by Leishmania (Viannia) shawi promastigotes

Leishmania (Viannia) shawi causes cutaneous lesions in humans. Parasite antigens conferring significant protection against American tegumentar leishmaniosis (ATL) might be important for the development of effective vaccine. Therefore, this work evaluates the protective effect of antigenic fractions released by L. shawi. Antigens released by promastigotes to culture medium were concentrated and isolated by SDS-PAGE. The three main fractions LsPass1 (>75 kDa), LsPass2 (75-50 kDa) and LsPass3 (<50 kDa) were electro-eluted according with their molecular mass. Immunized BALB/c mice were challenged with L. shawi promastigotes and the course of infection monitored during 5 weeks. LsPass1-challenged mice showed no protection, however, a strong degree of protection associated to smaller lesions and high expression of IFN-gamma and TNF-alpha by CD4(+) T, CD8(+) T and double negative CD4CD8 cells was achieved in LsPass3-challenged mice. Furthermore, LsPass2-challenged mice showed an intermediated degree of protection associated to high levels of IFN-gamma, IL-4 and IL-10 mRNA. In spite of increased expression of IFN-gamma and TNF-alpha, high amounts of IL-4 and IL-10 mRNA were also detected in LsPass3-challenged mice indicating a possible contribution of these cytokines for the persistence of a residual number of parasites that may be important in inducing long-lasting immunity. Therefore, LsPass3 seems to be an interesting alternative that should be considered in the development of an effective vaccine against ATL.

A novel protein refolding protocol for the solubilization and purification of recombinant peptidoglycan-associated lipoprotein from Xylella fastidiosa overexpressed in Escherichia coil

Xylella fastidiosa is a Gram-negative xylem-limited plant pathogenic bacterium responsible for several economically important crop diseases. Here, we present a novel and efficient protein refolding protocol for the solubilization and purification of recombinant X. fastidiosa peptidoglycan-associated lipoprotein (XfPal). Pal is an outer membrane protein that plays important roles in maintaining the integrity of the cell envelope and in bacterial pathogenicity. Because Pal has a highly hydrophobic N-terminal domain, the heterologous expression studies necessary for structural and functional protein characterization are laborious once the recombinant protein is present in inclusion bodies. Our protocol based on the denaturation of the XfPal-enriched inclusion bodies with 8 M urea followed by buffer-exchange steps via dialysis proved effective for the solubilization and subsequent purification of XfPal, allowing us to obtain a large amount of relatively pure and folded protein. In addition, XfPal was biochemically and functionally characterized. The method for purification reported herein is valuable for further research on the three-dimensional structure and function of Pal and other outer membrane proteins and can contribute to a better understanding of the role of these proteins in bacterial pathogenicity, especially with regard to the plant pathogen X. fastidiosa. (C) 2012 Elsevier Inc. All rights reserved.

Cysticercosis in experimentally and naturally infected pigs: Parasitological and immunological diagnosis

Silva M. R. M., Uyhara C.N.S., Silva F. H., Espindola N.M., Poleti M. D., Vaz A.J., Meirelles F. V. & Maia A. A. M. 2012. Cysticercosis in experimentally and naturally infected pigs: Parasitological and immunological diagnosis. Pesquisa Veterinaria Brasileira 32(4): 297-302. Departamento de Ciencias Basicas, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de Sao Paulo, Av. Duque de Caxias Norte 225, Pirassununga, SP 13635-900, Brazil. E-mail: antomaia@usp.br. Our objective was to evaluate the diagnosis of swine cysticercosis by examining "ante mortem" (inspection of the tongue), "post mortem" (inspection and detailed necropsy) and ELISA for research in serum of antibodies (Ab-ELISA) and antigens (Ag-ELISA). Seven (7) pigs were experimentally infected orally with eggs of Taenia solium and another 10 were naturally infected. In the pigs experimentally infected, inspection of the tongue was negative in all animals, in the routine inspection detailed necropsy and cysticercis were identified in all of them. In pigs with heavy natural infection, inspection of the tongue identified cysticerci in two (20%), while at inspection with necropsy the parasites were identified in large quantities in all animals. In ELISA for antibody search (Ab-ELISA) TS-14 recombinant protein was used, and in search for antigen (Ag-ELISA) a monoclonal antibody against this protein. In animals experimentally infected, blood was collected weekly for 140 days. The Ab-ELISA identified an increase in titers of antibody to cysticerci 21 days after infection, and at the end of the experimental period six animals (86%) were positive to the test. The search for circulating antigens (Ag-ELISA) was positive in two pigs 28 to 91 days after infection. All naturally infected pigs were positive for Ag-ELISA and Ab-ELISA. The search for antibodies and antigens by ELISA in serum from 30 pigs of a local farm and without history of cysticercosis was negative. Thus, the use of TS-14 antigen in ELISA test (Ab-ELISA) can be useful for the diagnosis of cysticercosis in pigs with low infection.

Generation and Biological Properties of a Recombinant Dodecahedron Containing the Short Fiber Protein of the Human Adenovirus 41

Objective: In order to gain further insight into the function of the enteric adenovirus short fiber (SF), we have constructed a recombinant dodecahedron containing the SF protein of HAdV-41 and the HAdV-3 penton base. Methods: Recombinant baculoviruses expressing the HAdV-41 SF protein and HAdV-3 penton base were cloned and amplified in Sf9 insect cells. Recombinant dodecahedra were expressed by coinfection of High Five (TM) cells with both baculoviruses, 72 h post-infection. Cell lysate was centrifuged on sucrose density gradient and the purified recombinant dodecahedra were recovered. Results: Analysis by negative staining electron microscopy demonstrated that chimeric dodecahedra made of the HAdV-3 penton base and decorated with the HAdV-41 SF were successfully generated. Next, recombinant dodecahedra were digested with pepsin and analyzed by Western blot. A 'site-specific' proteolysis of the HAdV-41 SF was observed, while the HAdV-3 penton base core was completely digested. Conclusion: These results show that, in vitro, the HAdV-41 SF likely undergoes proteolysis in the gastrointestinal tract, its natural environment, which may facilitate the recognition of receptors in intestinal cells. The results obtained in the present study may be the basis for the development of gene therapy vectors towards the intestinal epithelium, as well as orally administered vaccine vectors, but also for the HAdV-41 SF partner identification. Copyright (C) 2011 S. Karger AG, Basel

A Recombinant Protein Based on Trypanosoma cruzi P21 Enhances Phagocytosis

Background: P21 is a secreted protein expressed in all developmental stages of Trypanosoma cruzi. The aim of this study was to determine the effect of the recombinant protein based on P21 (P21-His(6)) on inflammatory macrophages during phagocytosis. Findings: Our results showed that P21-His(6) acts as a phagocytosis inducer by binding to CXCR4 chemokine receptor and activating actin polymerization in a way dependent on the PI3-kinase signaling pathway. Conclusions: Thus, our results shed light on the notion that native P21 is a component related to T. cruzi evasion from the immune response and that CXCR4 may be involved in phagocytosis. P21-His(6) represents an important experimental control tool to study phagocytosis signaling pathways of different intracellular parasites and particles.

Bladder expression of CD cell surface antigens and cell-type-specific transcriptomes

Many cell types have no known functional attributes. In the bladder and prostate, basal epithelial and stromal cells appear similar in cytomorphology and share several cell surface markers. Their total gene expression (transcriptome) should provide a clear measure of the extent to which they are alike functionally. Since urologic stromal cells are known to mediate organ-specific tissue formation, these cells in cancers might exhibit aberrant gene expression affecting their function. For transcriptomes, cluster designation (CD) antigens have been identified for cell sorting. The sorted cell populations can be analyzed by DNA microarrays. Various bladder cell types have unique complements of CD molecules. CD9(+) urothelial, CD104(+) basal and CD13(+) stromal cells of the lamina propria were therefore analyzed, as were CD9(+) cancer and CD13(+) cancer-associated stromal cells. The transcriptome datasets were compared by principal components analysis for relatedness between cell types; those with similarity in gene expression indicated similar function. Although bladder and prostate basal cells shared CD markers such as CD104, CD44 and CD49f, they differed in overall gene expression. Basal cells also lacked stem cell gene expression. The bladder luminal and stromal transcriptomes were distinct from their prostate counterparts. In bladder cancer, not only the urothelial but also the stromal cells showed gene expression alteration. The cancer process in both might thus involve defective stromal signaling. These cell-type transcriptomes provide a means to monitor in vitro models in which various CD-isolated cell types can be combined to study bladder differentiation and bladder tumor development based on cell-cell interaction.

Expression of cancer-testis antigens MAGE-A4 and MAGE-C1 in oral squamous cell carcinoma

Background Tumor markers are genes or their products expressed exclusively or preferentially in tumor cells and cancer-testis antigens (CTAs) form a group of genes with a typical expression pattern expressed in a variety of malignant neoplasms. CTAs are considered potential targets for cancer vaccines. It is possible that the CTA MAGE-A4 (melanoma antigen) and MAGE-C1 are expressed in carcinoma of the oral cavity and are related with survival. Methods This study involved immunohistochemical analysis of 23 patients with oral squamous cell carcinoma (SCC) and was carried out using antibodies for MAGE-A4 and MAGE-C1. Fisher's exact test and log-rank test were used to evaluate the results. Results The expression of the MAGE-A4 and MAGE-C1 were 56.5% and 47.8% without statistical difference in studied variables and survival. Conclusion The expression of at least 1 CTA was present in 78.3% of the patients, however, without correlation with clinicopathologic variables and survival. (c) 2011 Wiley Periodicals, Inc. Head Neck, 2012

Immobilization of a recombinant endo-1,5-arabinanase secreted by Aspergillus nidulans strain A773

An endo-1,5-arabinanase (abnA) encoding gene from Aspergillus niveus was identified, cloned and successfully expressed in Aspergillus nidulans strain A773. Based on amino acid sequence comparison, the 34-kDa enzyme could be assigned to CAZy GH family 43. Characterization of purified recombinant endo-1,5-arabinanase (AbnA) revealed that it is active at a wide pH range (pH 4.0-7.0) and an optimum temperature at 70 degrees C. The immobilization of the AbnA was performed via covalent binding onto agarose-modified supports: glyoxyl iminodiacetic acid-Ni2+, glyoxyl amine, glyoxyl (4% and 10%) and cyanogen bromide activated sepharose. The yield of immobilization was similar on glyoxyl amine and glyoxyl (96%), and higher than glyoxyl iminodiacetic acid-Ni2+ (43%) support. The thermal inactivation of these immobilized preparations showed that the stability of the AbnA immobilized on glyoxyl 4 and 10% was improved by 4.0 and 10.3-fold factor at 70 degrees C. The half-life of glyoxyl 4% derivative at 60 degrees C was >48 h (pH 5), 9 h (pH 7) and 88 min (pH 9). The major hydrolysis product of debranched arabinan or arabinopentaose by glyoxyl agarose-immobilized AbnA was arabinobiose. (C) 2012 Elsevier B.V. All rights reserved.