Protection conferred by heterologous vaccination against tuberculosis is dependent on the ratio of CD4(+)/CD4(+) Foxp3(+) cells

CD4(+) Foxp3(+) regulatory T cells inhibit the production of interferon-?, which is the major mediator of protection against Mycobacterium tuberculosis infection. In this study, we evaluated whether the protection conferred by three different vaccines against tuberculosis was associated with the number of spleen and lung regulatory T cells. We observed that after homologous immunization with the 65 000 molecular weight heat-shock protein (hsp 65) DNA vaccine, there was a significantly higher number of spleen CD4(+) Foxp3(+) cells compared with non-immunized mice. Heterologous immunization using bacillus Calmette Guerin (BCG) to prime and DNA-hsp 65 to boost (BCG/DNA-hsp 65) or BCG to prime and culture filtrate proteins (CFP)-CpG to boost (BCG/CFP-CpG) induced a significantly higher ratio of spleen CD4(+)/CD4(+) Foxp3(+) cells compared with non-immunized mice. In addition, the protection conferred by either the BCG/DNA-hsp 65 or the BCG/CFP-CpG vaccines was significant compared with the DNA-hsp 65 vaccine. Despite the higher ratio of spleen CD4(+)/CD4(+) Foxp3(+) cells found in BCG/DNA-hsp 65-immunized or BCG/CFP-CpG-immunized mice, the lungs of both groups of mice were better preserved than those of DNA-hsp 65-immunized mice. These results confirm the protective efficacy of BCG/DNA-hsp 65 and BCG/CFP-CpG heterologous prime-boost vaccines and the DNA-hsp 65 homologous vaccine. Additionally, the prime-boost regimens assayed here represent a promising strategy for the development of new vaccines to protect against tuberculosis because they probably induce a proper ratio of CD4(+) and regulatory (CD4(+) Foxp3(+)) cells during the immunization regimen. In this study, this ratio was associated with a reduced number of regulatory cells and no injury to the lungs.

CD8(+) T-cell-mediated immunity against malaria: a novel heterologous prime-boost strategy

Evaluation of: Rodriguez D, Gonzalez-Aseguinolaza G, Rodriguez JR et al. Vaccine efficacy against malaria by the combination of porcine parvovirus-like particles and vaccinia virus vectors expressing CS of Plasmodium. PLoS ONE 7(4), e34445 (2012). Recently, a vaccine against malaria was successfully tested in a human Phase III trial. The efficacy of this vaccine formulation, based on the Plasmodium falciparum circumsporozoite protein, was approximately 50% and correlated with the presence of antibodies specific to the infective stages of the malaria parasites. Different strategies are being pursued to improve vaccine efficacy levels. One such strategy is the induction of specific cytotoxic T cells that can destroy the intracellular hepatocyte stages of the malaria parasite. In this study, a novel vaccination protocol was developed to elicit strong immune responses mediated by CD8(+) cytotoxic cells specific to the circumsporozoite protein. As proof-of-concept, the authors used the rodent malaria Plasmodium yoelii parasite. The vaccination strategy consisted of a heterologous prime-boost vaccination regimen involving porcine parvovirus-like particles for priming and the modified vaccinia virus Ankara for the booster immunization, both of which expressed the immunodominant CD8 epitope of the P. yoelii circumsporozoite protein. Results from this experimental model were extremely meaningful. This vaccination strategy led to a significant T-cell immune response mediated by CD8(+) multifunctional T effector and effector-memory cells. However, most importantly for the malaria vaccine development was the fact that following a sporozoite challenge, immunized mice eliminated more than 97% of the malaria parasites during the hepatocyte stages. These results confirm and extend a vast body of knowledge showing that a heterologous prime-boost vaccination strategy can elicit strong CD8(+) T-cell-mediated protective immunity and may increase the efficacy of malaria vaccines.

Endophytic Methylobacterium extorquens expresses a heterologous beta-1,4-endoglucanase A (EglA) in Catharanthus roseus seedlings, a model host plant for Xylella fastidiosa

Based on the premise of symbiotic control, we genetically modified the citrus endophytic bacterium Methylobacterium extorquens, strain AR1.6/2, and evaluated its capacity to colonize a model plant and its interaction with Xylella fastidiosa, the causative agent of Citrus Variegated Chlorosis (CVC). AR1.6/2 was genetically transformed to express heterologous GFP (Green Fluorescent Protein) and an endoglucanase A (EglA), generating the strains ARGFP and AREglA, respectively. By fluorescence microscopy, it was shown that ARGFP was able to colonize xylem vessels of the Catharanthus roseus seedlings. Using scanning electron microscopy, it was observed that AREglA and X. fastidiosa may co-inhabit the C. roseus vessels. M. extorquens was observed in the xylem with the phytopathogen X. fastidiosa, and appeared to cause a decrease in biofilm formation. AREglA stimulated the production of resistance protein, catalase, in the inoculated plants. This paper reports the successful transformation of AR1.6/2 to generate two different strains with a different gene each, and also indicates that AREglA and X. fastidiosa could interact inside the host plant, suggesting a possible strategy for the symbiotic control of CVC disease. Our results provide an enhanced understanding of the M. extorquens-X. fastidiosa interaction, suggesting the application of AR1.6/2 as an agent of symbiotic control.

Protein secretion in Lactococcus lactis : an efficient way to increase the overall heterologous protein production

Anderson MIYOSHI, Daniela FREITAS, Luciana RIBEIRO, Jane E. GABRIEL, Sophie LECLERCQ, Maricê N. OLIVEIRA, and Valeria D. GUIMARÃES were recipients of a CAPES fellowship (project CAPESCOFECUB #319II). Luis BERMUDEZ and Sébastien NOUAILLE were recipients of a fellowship from the French Ministry of Education and Research. INRA and Région IledeFrance also financed L. BERMUDEZ and V. GUIMARAES. Cathy CHARLIER is recipient of a fellowship from INRA and Région Bretagne.

GUS expression in sweet oranges (Citrus sinensis L. Osbeck) driven by three different phloem-specific promoters

Huanglongbing (HLB) is associated with Candidatus Liberibacter spp., endogenous, sieve tube-restricted bacteria that are transmitted by citrus psyllid insect vectors. Transgenic expression in the phloem of specific genes that might affect Ca. Liberibacter spp. growth and development may be an adequate strategy to improve citrus resistance to HLB. To study specific phloem gene expression in citrus, we developed three different binary vector constructs with expression cassettes bearing the beta-glucuronidase (GUS) reporter gene (uidA) under the control of one of the three different promoters: Citrus phloem protein 2 (CsPP2), Arabidopsis thaliana phloem protein 2 (AtPP2), and Arabidopsis thaliana sucrose transporter 2 (AtSUC2). Transgenic lines of 'Hamlin', 'Pera', and 'Valencia' sweet oranges [Citrus sinensis (L.) Osbeck] were produced via Agrobacterium tumefaciens transformation. The epicotyl segments collected from in vitro germinated seedlings were used as explants. The gene nptII, which confers resistance to the antibiotic kanamycin, was used for selection. The transformation efficiency was expressed as the number of GUS-positive shoots over the total number of explants and varied from 1.54 to 6.08 % among the three cultivars and three constructs studied. Several lines of the three sweet orange cultivars analyzed using PCR and Southern blot analysis were genetically transformed with the three constructs evaluated. The histological GUS activity in the leaves indicates that the uidA gene was preferentially expressed in the phloem, which suggests that the use of the three promoters might be adequate for producing HLB-resistant transgenic sweet oranges. The results reported here conclusively demonstrate the preferential expression of GUS in the phloem driven by two heterologous and one homologous gene promoters. Key message The results reported here conclusively demonstrate the preferential expression of GUS in the phloem driven by two heterologous and one homologous gene promoters.

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.

Trichoderma harzianum expressed sequence tags for identification of genes with putative roles in mycoparasitism against Fusarium solani

The plant pathogen Fusarium solani causes a disease root rot of common bean (Phaseolus vulgaris) resulting in great losses of yield in irrigated areas of the Southeast and Midwest regions of Brazil. Species of the genus Trichoderma have been used in the biological control of this pathogen as an alternative to chemical control. To gain new insights into the biocontrol mechanism used by Trichoderma harzianum against the phytopathogenic fungus, Fusarium solani, we performed a transcriptome analysis using expressed sequence tags (ESTs) and quantitative real-time PCR (RT-qPCR) approaches. A cDNA library from T. harzianum mycelium (isolate ALL42) grown on cell walls of F. solani (CWFS) was constructed and analyzed. A total of 2927 high quality sequences were selected from 3845 and 37.7% were identified as unique genes. The Gene Ontology analysis revealed that the majority of the annotated genes are involved in metabolic processes (80.9%), followed by cellular process (73.7%). We tested twenty genes that encode proteins with potential role in biological control. RT-qPCR analysis showed that none of these genes were expressed when T. harzianum was challenged with itself. These genes showed different patterns of expression during in vitro interaction between T. harzianum and F. solani. (C) 2012 Elsevier Inc. All rights reserved.

Functional characterization of sugarcane mustang domesticated transposases and comparative diversity in sugarcane, rice, maize and sorghum

Transposable elements (TEs) account for a large portion of plant genomes, particularly in grasses, in which they correspond to 50%-80% of the genomic content. TEs have recently been shown to be a source of new genes and new regulatory networks. The most striking contribution of TEs is referred as "molecular domestication", by which the element coding sequence loses its movement capacity and acquires cellular function. Recently, domesticated transposases known as mustang and derived from the Mutator element have been described in sugarcane. In order to improve our understanding of the function of these proteins, we identified mustang genes from Sorghum bicolor and Zea mays and performed a phenetic analysis to assess the diversity and evolutionary history of this gene family. This analysis identified orthologous groups and showed that mustang genes are highly conserved in grass genomes. We also explored the transcriptional activity of sugarcane mustang genes in heterologous and homologous systems. These genes were found to be ubiquitously transcribed, with shoot apical meristem having the highest expression levels, and were downregulated by phytohormones. Together, these findings suggest the possible involvement of mustang proteins in the maintenance of hormonal homeostasis.

Cytoplasmic and extracellular expression of pharmaceutical-grade mycobacterial 65-kDa heat shock protein in Lactococcus lactis

Lactic acid bacteria (LAB) are an attractive and safe alternative for the expression of heterologous proteins, as they are nonpathogenic and endotoxin-free organisms. Lactococcus lactis, the LAB model organism, has been extensively employed in the biotechnology field for large-scale production of heterologous proteins, and its use as a "cell factory" has been widely studied. We have been particularly interested in the use of L. lactis for production of heat shock proteins (HSPs), which reportedly play important roles in the initiation of innate and adaptive immune responses. However, this activity has been questioned, as LPS contamination appears to be responsible for most, if not all, immunostimulatory activity of HSPs. In order to study the effect of pure HSPs on the immune system, we constructed recombinant L. lactis strains able to produce and properly address the Mycobacterium leprae 65-kDa HSP (Hsp65) to the cytoplasm or to the extracellular medium, using a xylose-induced expression system. Approximately 7 mg/L recombinant Hsp65 was secreted. Degradation products related to lactococcal HtrA activity were not observed, and the Limulus amebocyte lysate assay demonstrated that the amount of LPS in the recombinant Hsp65 preparations was 10-100 times lower than the permitted levels established by the U. S. Food and Drug Administration. These new L. lactis strains will allow investigation of the effects of M. leprae Hsp65 without the interference of LPS; consequently, they have potential for a variety of biotechnological, medical and therapeutic applications.

Brazilian avian metapneumovirus subtypes A and B: experimental infection of broilers and evaluation of vaccine efficacy

Santos M.B., Martini M.C., Ferreira H.L., Silva L.H.A., Fellipe P.A., Spilki F.R. & Arns C.W. 2012. Brazilian avian metapneumovirus subtypes A and B: experimental infection of broilers and evaluation of vaccine efficacy. Pesquisa Veterinaria Brasileira 32(12):1257-1262. Laboratorio de Virologia, Instituto de Biologia, Universidade Estadual de Campinas, Rua Monteiro Lobato s/n, Cx. Postal 6109, Campinas, SP 13083-970, Brazil. E-mail: arns@unicamp.br Avian metapneumovirus (aMPV) is a respiratory pathogen associated with the swollen head syndrome (SHS) in chickens. In Brazil, live aMPV vaccines are currently used, but subtypes A and, mainly subtype B (aMPV/A and aMPV/B) are still circulating. This study was conducted to characterize two Brazilian aMPV isolates (A and B subtypes) of chicken origin. A challenge trial to explore the replication ability of the Brazilian subtypes A and B in chickens was performed. Subsequently, virological protection provided from an aMPV/B vaccine against the same isolates was analyzed. Upon challenge experiment, it was shown by virus isolation and real time PCR that aMPV/B could be detected longer and in higher amounts than aMPV/A. For the protection study, 18 one-day-old chicks were vaccinated and challenged at 21 days of age. Using virus isolation and real time PCR, no aMPV/A was detected in the vaccinated chickens, whereas one vaccinated chicken challenged with the aMPV/B isolate was positive. The results showed that aMPV/B vaccine provided a complete heterologous virological protection, although homologous protection was not complete in one chicken. Although only one aMPV/B positive chicken was detected after homologous vaccination, replication in vaccinated animals might allow the emergence of escape mutants.

Enzyme activity in the small intestine of goat kids during the period of passive immunity acquisition

Enzyme activity of protein and carbohydrate degradation in small intestinal mucosa was investigated in goat kids fed with lyophilized bovine and goat colostrum. At 0,7 and 14 h of life 15 male newborns received 5% of body weight of lyophilized bovine colostrum and 14 goat colostrum, both with 55 mg/mL of IgG. Duodenum, jejunum and ileum samples were collected at 18,36 and 96 h of life. Three animals were sampled at birth, without colostrum intake. Activity of aminopeptidase N and A, dipeptidil peptidase IV, lactase, maltase and sucrase was determined as one international unit per gram of tissue. Intracellular enzymatic activity of acid phosphatase was observed by histochemistry in tissue section. Only the activity of aminopeptidase A in the ileum was affected by treatment, with a greater value for LBC than for GC (P < 0.05). The aminopeptidase N activity was the highest at 36 h in the duodenum (P < 0.05) and lowest at 96 h in the jejunum (P < 0.05). Dipeptidil peptidase IV activity was highest at 36 h in the duodenum (P < 0.05), lowest at 96 h in the jejunum (P < 0.05) and higher at 36 h than at 96 h in the ileum (P < 0.05). Aminopeptidase A activity in the ileum was highest at 36 h (P < 0.05), followed by 18 and 96 h of life (P < 0.05). Lactase activity in the duodenum increased from 18 to 36 h and from 36 to 96 h in the jejunum (P < 0.05). Maltase activity increased only in the duodenum from 18 to 96 h (P < 0.05). Sucrase activity in the jejunum decreased from 18 to 36 h and from 36 to 96 h in the ileum (P < 0.05). At birth, activity of most enzymes was similar to that at later times (P < 0.05). Histochemistry analyses showed a higher frequency of lysosomes with acid phosphatase activity in the duodenum, especially at 36 h of life. In the jejunum, the presence of lysosomes with acid phosphatase activity was the highest at 96 h, followed by 36 and 18 h of life. In the ileum, all samples showed low presence of lysosomes with acid phosphatase activity. These results indicate that lyophilized bovine colostrum, as a heterologous source of antibodies or nutrients, is a possible alternative management tool for goats. The present work also suggests that in the first 4 days of life, enzyme activity in the intestinal epithelium of goats is still not fully stimulated, which is an important characteristic for these animals that depend on macromolecule absorption to acquire passive protection after birth. (C) 2012 Elsevier B.V. All rights reserved.

Angiotensin-(1-7) decreases LPS-induced inflammatory response in macrophages

It has been previously shown that besides its classical role in blood pressure control the reninangiotensin system, mainly by action of angiotensin II on the AT1 receptor, exerts pro-inflammatory effects such as by inducing the production of cytokines. More recently, alternative pathways to this system were described, such as binding of angiotensin-(17) to receptor Mas, which was shown to counteract some of the effects evoked by activation of the angiotensin IIAT1 receptor axis. Here, by means of different molecular approaches we investigated the role of angiotensin-(17) in modulating inflammatory responses triggered in mouse peritoneal macrophages. Our results show that receptor Mas transcripts were up-regulated by eightfold in LPS-induced macrophages. Interestingly, macrophage stimulation with angiotensin-(17), following to LPS exposure, evoked an attenuation in expression of TNF-a and IL-6 pro-inflammatory cytokines; where this event was abolished when the receptor Mas selective antagonist A779 was also included. We then used heterologous expression of the receptor Mas in HEK293T cells to search for the molecular mechanisms underlying the angiotensin-(17)-mediated anti-inflammatory responses by a kinase array; what suggested the involvement of the Src kinase family. In LPS-induced macrophages, this finding was corroborated using the PP2 compound, a specific Src kinase inhibitor; and also by Western blotting when we observed that Ang-(17) attenuated the phosphorylation levels of Lyn, a member of the Src kinase family. Our findings bring evidence for an anti-inflammatory role for angiotensin-(17) at the cellular level, as well as show that its probable mechanism of action includes the modulation of Src kinases activities. J. Cell. Physiol. 227: 21172122, 2012. (C) 2011 Wiley Periodicals, Inc.

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.

In silico and in vivo analysis reveal a novel gene in Saccharomyces cerevisiae trehalose metabolism

Abstract Background The ability to respond rapidly to fluctuations in environmental changes is decisive for cell survival. Under these conditions trehalose has an essential protective function and its concentration increases in response to enhanced expression of trehalose synthase genes, TPS1, TPS2, TPS3 and TSL1. Intriguingly, the NTH1 gene, which encodes neutral trehalase, is highly expressed at the same time. We have previously shown that trehalase remains in its inactive non-phosphorylated form by the action of an endogenous inhibitor. Recently, a comprehensive two-hybrid analysis revealed a 41-kDa protein encoded by the YLR270w ORF, which interacts with NTH1p. Results In this work we investigate the correlation of this Trehalase Associated Protein, in trehalase activity regulation. The neutral trehalase activity in the ylr270w mutant strain was about 4-fold higher than in the control strain. After in vitro activation by PKA the ylr270w mutant total trehalase activity increased 3-fold when compared to a control strain. The expression of the NTH1 gene promoter fused to the heterologous reporter lacZ gene was evaluated. The mutant strain lacking YLR270w exhibited a 2-fold increase in the NTH1-lacZ basal expression when compared to the wild type strain. Conclusions These results strongly indicate a central role for Ylr270p in inhibiting trehalase activity, as well as in the regulation of its expression preventing a wasteful futile cycle of synthesis-degradation of trehalose.

Promoter regions of Plasmodium vivax are poorly or not recognized by Plasmodium falciparum

Abstract Background Heterologous promoter analysis in Plasmodium has revealed the existence of conserved cis regulatory elements as promoters from different species can drive expression of reporter genes in heterologous transfection assays. Here, the functional characterization of different Plasmodium vivax promoters in Plasmodium falciparum using luciferase as the reporter gene is presented. Methods Luciferase reporter plasmids harboring the upstream regions of the msp1, dhfr, and vir3 genes as well as the full-length intergenic regions of the vir23/24 and ef-1α genes of P. vivax were constructed and transiently transfected in P. falciparum. Results Only the constructs with the full-length intergenic regions of the vir23/24 and ef-1α genes were recognized by the P. falciparum transcription machinery albeit to values approximately two orders of magnitude lower than those reported by luc plasmids harbouring promoter regions from P. falciparum and Plasmodium berghei. A bioinformatics approach allowed the identification of a motif (GCATAT) in the ef-1α intergenic region that is conserved in five Plasmodium species but is degenerate (GCANAN) in P. vivax. Mutations of this motif in the P. berghei ef-1α promoter region decreased reporter expression indicating it is active in gene expression in Plasmodium. Conclusion Together, this data indicates that promoter regions of P. vivax are poorly or not recognized by the P. falciparum transcription machinery suggesting the existence of P. vivax-specific transcription regulatory elements.