Publications (8)22.55 Total impact
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Article: Purified and highly aggregated chimeric protein DIIIC-2 induces a functional immune response in mice against dengue 2 virus.
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ABSTRACT: It was previously reported that DIIIC-2 (a fusion protein composed of domain III of the envelope protein and the capsid protein from dengue 2 virus), as an aggregate antigen from a partially purified preparation, induced a functional protective immune response against dengue 2 virus in the mouse encephalitis model. In the present work, a purification procedure was developed for DIIIC-2, and soluble and aggregated fractions of the purified protein were characterized and evaluated in mice. The purification process rendered a protein preparation of 91 % purity, and the remaining 9 % consisted of fragments and aggregates of the same recombinant protein. After the in vitro aggregation process, upon addition of oligodeoxynucleotides, 80 % of the protein formed aggregates, whereas 20 % remained as soluble protein. An immunological evaluation revealed the proper immunogenicity of the aggregated purified protein in terms of induction of antiviral and neutralizing antibodies, cell-mediated immunity and protection upon dengue 2 virus challenge in the mouse encephalitis model. Based on these results, we can assert that the purified protein DIIIC-2 is functional and could be used for further scalable steps and preclinical studies in non-human primates.Archives of Virology 09/2012; · 2.11 Impact Factor -
Article: TLP01, an mshA mutant of Vibrio cholerae O139 as vaccine candidate against cholera.
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ABSTRACT: No commercially live vaccine against cholera caused by Vibrio cholerae O139 serogroup is available and it is currently needed. Virulent O139 strain CRC266 was genetically modified by firstly deleting multiple copies of the filamentous phage CTXφ, further tagging by insertion of the endoglucanase A coding gene from Clostridium thermocellum into the hemagglutinin/protease gene and finally deleting the mshA gene, just to improve the vaccine biosafety. One of the derived strains designated as TLP01 showed full attenuation and good colonizing capacity in the infant mouse cholera model, as well as highly immunogenic properties in the adult rabbit and rat models. Since TLP01 lacks MSHA fimbriae, it is refractory to infection with another filamentous phage VGJφ and therefore protected of acquiring CTXφ from a recombinant hybrid VGJφ/CTXφ. This strategy could reduce the possibilities of stable reversion to virulence out of the human gut. Furthermore, this vaccine strain was impaired to produce biofilms under certain culture conditions, which might have implications for the strain survival in natural settings contributing to vaccine biosafety as well. The above results has encouraged us to consider TLP01 as a live attenuated vaccine strain having an adequate performance in animal models, in terms of attenuation and immunogenicity, so that it fulfills the requirements to be evaluated in human volunteers.Microbes and Infection 04/2012; 14(11):968-78. · 3.10 Impact Factor -
Article: Anaerobic growth promotes synthesis of colonization factors encoded at the Vibrio pathogenicity island in Vibrio cholerae El Tor.
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ABSTRACT: Pathogenesis of the facultative anaerobe Vibrio cholerae takes place at the gut under low oxygen concentrations. To identify proteins which change their expression level in response to oxygen availability, proteomes of V. cholerae El Tor C7258 grown in aerobiosis, microaerobiosis and anaerobiosis were compared by two-dimensional electrophoresis. Twenty-six differentially expressed proteins were identified which are involved in several processes including iron acquisition, alanine metabolism, purine synthesis, energy metabolism and stress response. Moreover, two proteins implicated in exopolysaccharide synthesis and biofilm formation were produced at higher levels under microaerobiosis and anaerobiosis, which suggests a role of oxygen deprivation in biofilm development in V. cholerae. In addition, six proteins encoded at the Vibrio pathogenicity island attained the highest expression levels under anaerobiosis, and five of them are required for colonization: three correspond to toxin-coregulated pilus biogenesis components, one to soluble colonization factor TcpF and one to accessory colonization factor A. Thus, anaerobiosis promotes synthesis of colonization factors in V. cholerae El Tor, suggesting that it may be a key in vivo signal for early stages of the pathogenic process of V. cholerae.Research in Microbiology 11/2008; 160(1):48-56. · 2.76 Impact Factor -
Article: El Tor and Calcutta CTXPhi precursors coexisting with intact CTXPhi copies in Vibrio cholerae O139 isolates.
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ABSTRACT: Restriction fragment length polymorphism analyses of the array of CTXPhi prophages in strains CRC262 and CRC266 of Vibrio cholerae O139 revealed the presence of copies of complete CTXPhi and pre-CTXPhi prophages coexisting at a single chromosomal locus in each strain. Restriction pattern and comparative nucleotide sequence analysis revealed pre-CTXPhi precursors of both the El Tor and Calcutta lineages. Thus, we hypothesize that two precursor variants independently acquired cholera toxin genes and gave rise to the current El Tor and Calcutta CTXPhi prophages. We discuss the implications of these results in terms of the evolution and origin of the current diversity of CTXPhi prophages.Research in Microbiology 04/2008; 159(2):81-7. · 2.76 Impact Factor -
Article: Estudio de los genes VCA0260 y VCA0261 de Vibrio cholerae y de la proteína única que codifican
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ABSTRACT: The sequence of the genome of Vibrio cholerae N16961 is deposited in a database of The Institute for Genomic Research (TIGR). In that database, genes VCA0260 and VCA0261 are annotated as two independent and overlapped genes, which are predicted to encode two hypothetical proteins of 11.9 kDa and 7.3 kDa of molecular weight, respectively. In a previous proteomic study of V. cholerae C7258 cells, one spot up-regulated under anaerobic culture revealed a correspondence with predicted polypeptides from genes VCA0260 and VCA0261. This finding suggested that genes VCA0260 and VCA0261 constitute a single gene encoding a single protein. Here, it is described PCR amplifications, cloning and nucleotide sequencing of regions spanning both genes from V. cholerae strains C7258 and N16961. Nucleotide sequencing of both regions showed the existence of a guanine nucleotide preceding the stop codon of VCA0261, which is absent in the published sequence. The presence of this guanine changes the reading frame of ORF VCA0261 to the same as VCA0260, resulting in a predicted larger ORF coding for a protein of 18.7 kDa of molecular weight. Production of this protein was co-rroborated by expression, purification and sequencing by mass spectrometry of the peptide synthesized from cloned regions in Escherichia coli. Thus, VCA0260 and VCA0261 genes constitute a single gene (VCA0261-VCA260) encoding a single protein. A mutant of V. cholerae C7258 with the gene VCA0261-VCA0260 deleted from its chromosome was constructed. In the presence of copper, growth of the VCA0261-VCA0260 mutant was affected compared to the growth of wild type strain, suggesting that the protein coded by VCA0261-VCA0260 gene is involved in copper homeostasis in V. cholerae. On the other hand, this protein does not seem to play an important role during the colonization process in the presence of 10 µmol/L of copper, in an infant mouse model of cholera colonizationCNIC journal. 02/2007; 38(02):124-131. -
Article: The vaccine candidate Vibrio cholerae 638 is protective against cholera in healthy volunteers.
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ABSTRACT: Vibrio cholerae 638 is a living candidate cholera vaccine strain attenuated by deletion of the CTXPhi prophage from C7258 (O1, El Tor Ogawa) and by insertion of the Clostridium thermocellum endoglucanase A gene into the hemagglutinin/protease coding sequence. This vaccine candidate was previously found to be well tolerated and immunogenic in volunteers. This article reports a randomized, double-blind, placebo-controlled trial conducted to test short-term protection conferred by 638 against subsequent V. cholerae infection and disease in volunteers in Cuba. A total of 45 subjects were enrolled and assigned to receive vaccine or placebo. The vaccine contained 10(9) CFU of freshly harvested 638 buffered with 1.3% NaHCO(3), while the placebo was buffer alone. After vaccine but not after placebo intake, 96% of volunteers had at least a fourfold increase in vibriocidal antibody titers, and 50% showed a doubling of at least the lipopolysaccharide-specific immunoglobulin A titers in serum. At 1 month after vaccination, five volunteers from the vaccine group and five from the placebo group underwent an exploratory challenge study with 10(9) CFU of DeltaCTXPhi attenuated mutant strain V. cholerae 81. Only two volunteers from the vaccine group shed strain 81 in their feces, but none of them experienced diarrhea; in the placebo group, all volunteers excreted the challenge strain, and three had reactogenic diarrhea. An additional 12 vaccinees and 9 placebo recipients underwent challenge with 7 x 10(5) CFU of virulent strain V. cholerae 3008 freshly harvested from a brain heart infusion agar plate and buffered with 1.3% NaHCO(3). Three volunteers (25%) from the vaccine group and all from the placebo group shed the challenge agent in their feces. None of the 12 vaccinees but 7 volunteers from the placebo group had diarrhea, and 2 of the latter exhibited severe cholera (>5,000 g of diarrheal stool). These results indicate that at 1 month after ingestion of a single oral dose (10(9) CFU) of strain 638, volunteers remained protected against cholera infection and disease provoked by the wild-type challenge agent V. cholerae 3008. We recommend that additional vaccine lots of 638 be prepared under good manufacturing practices for further evaluation.Infection and Immunity 05/2005; 73(5):3018-24. · 4.16 Impact Factor -
Article: Novel type of specialized transduction for CTX phi or its satellite phage RS1 mediated by filamentous phage VGJ phi in Vibrio cholerae.
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ABSTRACT: The main virulence factor of Vibrio cholerae, the cholera toxin, is encoded by the ctxAB operon, which is contained in the genome of the lysogenic filamentous phage CTX phi. This phage transmits ctxAB genes between V. cholerae bacterial populations that express toxin-coregulated pilus (TCP), the CTX phi receptor. In investigating new forms of ctxAB transmission, we found that V. cholerae filamentous phage VGJ phi, which uses the mannose-sensitive hemagglutinin (MSHA) pilus as a receptor, transmits CTX phi or its satellite phage RS1 by an efficient and highly specific TCP-independent mechanism. This is a novel type of specialized transduction consisting in the site-specific cointegration of VGJ phi and CTX phi (or RS1) replicative forms to produce a single hybrid molecule, which generates a single-stranded DNA hybrid genome that is packaged into hybrid viral particles designated HybP phi (for the VGJ phi/CTX phi hybrid) and HybRS phi (for the VGJ phi/RS1 hybrid). The hybrid phages replicate by using the VGJ phi replicating functions and use the VGJ phi capsid, retaining the ability to infect via MSHA. The hybrid phages infect most tested strains more efficiently than CTX phi, even under in vitro optimal conditions for TCP expression. Infection and lysogenization with HybP phi revert the V. cholerae live attenuated vaccine strain 1333 to virulence. Our results reinforce that TCP is not indispensable for the acquisition of CTX phi. Thus, we discuss an alternative to the current accepted evolutionary model for the emergence of new toxigenic strains of V. cholerae and the importance of our findings for the development of an environmentally safer live attenuated cholera vaccine.Journal of Bacteriology 01/2004; 185(24):7231-40. · 3.83 Impact Factor -
Article: VGJ phi, a novel filamentous phage of Vibrio cholerae, integrates into the same chromosomal site as CTX phi.
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ABSTRACT: We describe a novel filamentous phage, designated VGJ phi, isolated from strain SG25-1 of Vibrio cholerae O139, which infects all O1 (classical and El Tor) and O139 strains tested. The sequence of the 7,542 nucleotides of the phage genome reveals that VGJ phi has a distinctive region of 775 nucleotides and a conserved region with an overall genomic organization similar to that of previously characterized filamentous phages, such as CTX phi of V. cholerae and Ff phages of Escherichia coli. The conserved region carries 10 open reading frames (ORFs) coding for products homologous to previously reported peptides of other filamentous phages, and the distinctive region carries one ORF whose product is not homologous to any known peptide. VGJ phi, like other filamentous phages, uses a type IV pilus to infect V. cholerae; in this case, the pilus is the mannose-sensitive hemagglutinin. VGJ phi-infected V. cholerae overexpresses the product of one ORF of the phage (ORF112), which is similar to single-stranded DNA binding proteins of other filamentous phages. Once inside a cell, VGJ phi is able to integrate its genome into the same chromosomal attB site as CTX phi, entering into a lysogenic state. Additionally, we found an attP structure in VGJ phi, which is also conserved in several lysogenic filamentous phages from different bacterial hosts. Finally, since different filamentous phages seem to integrate into the bacterial dif locus by a general mechanism, we propose a model in which repeated integration events with different phages might have contributed to the evolution of the CTX chromosomal region in V. cholerae El Tor.Journal of Bacteriology 11/2003; 185(19):5685-96. · 3.83 Impact Factor
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Institutions
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2003–2012
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Centro Nacional de Investigaciones Cientificas
Havana, Provincia de La Habana, Cuba
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