Edith Suzarte

Center for Genetic Engineering and Biotechnology, La Habana, Ciudad de La Habana, Cuba

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Publications (17)29.19 Total impact

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    ABSTRACT: Previously, we reported the ability of the chimeric protein DIIIC-2 (domain III of the dengue envelope protein fused to the capsid protein of dengue-2 virus), to induce immunity and protection in mice, when it is highly aggregated with a non-defined oligodeoxynucleotide (ODN) and adjuvanted in alum. In this work, three different defined ODNs were studied as aggregating agents. Our results suggest that the nature of the ODN influences the capacity of protein DIIIC-2 to activate cell-mediated immunity in mice. Consequently, the ODN 39M was selected to perform further experiments in mice and nonhuman primates. Mice receiving the preparation 39M-DIIIC-2 were solidly protected against dengue virus (DENV) challenge. Moreover, monkeys immunized with the same preparation developed neutralizing antibodies, as measured by four different neutralization tests varying the virus strains and the cell lines used. Two of the immunized monkeys were completely protected against challenge, whereas the third animal had a single day of low-titer viremia. This is the first work describing the induction of short-term protection in monkeys by a formulation that is suitable for human use combining a recombinant protein from DENV with alum.Immunology and Cell Biology advance online publication, 2 September 2014; doi:10.1038/icb.2014.63.
    Immunology and Cell Biology 09/2014; · 3.93 Impact Factor
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    ABSTRACT: A dengue vaccine must induce protective immunity against the four serotypes of the virus. Our group has developed chimeric proteins consisting of the protein P64k from Neisseria meningitidis and the domain III from the four viral envelope proteins. In this study, the immunogenicity of a tetravalent vaccine formulation using aluminum hydroxide as adjuvant was evaluated in mice. After three doses, neutralizing antibody titers were detected against the four viral serotypes, the lowest seroconversion rate being against dengue virus serotype 4. One month after the last dose, immunized animals were challenged with infective virus, and partial but statistically significant protection was found to have been achieved. Based on these results, further studies in mice and non-human primates using this tetravalent formulation in a prime-boost strategy with attenuated viruses are strongly recommended.
    Microbiology and Immunology 04/2014; 58(4):219-26. · 1.55 Impact Factor
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    ABSTRACT: Dengue is currently one of the most important arthropod-borne diseases, causing up to 25,000 deaths annually. There is currently no vaccine to prevent dengue virus infection, which needs a tetravalent vaccine approach. In this work, we describe the cloning and expression in Escherichia coli of envelope domain III-capsid chimeric proteins (DIIIC) of the four dengue serotypes as a tetravalent dengue vaccine candidate that is potentially able to generate humoral and cellular immunity. The recombinant proteins were purified to more than 85 % purity and were recognized by anti-dengue mouse and human sera. Mass spectrometry analysis verified the identity of the proteins and the correct formation of the intracatenary disulfide bond in the domain III region. The chimeric DIIIC proteins were also serotype-specific, and in the presence of oligonucleotides, they formed aggregates that were visible by electron microscopy. These results support the future use of DIIIC recombinant chimeric proteins in preclinical studies in mice for assessing their immunogenicity and efficacy.
    Archives of Virology 01/2014; · 2.28 Impact Factor
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    ABSTRACT: The role of cellular immune response in dengue virus infection is not yet fully understood. Only few studies in murine models propose that CD8+ T-cells are associated with protection from infection and disease. At the light of recent reports about the protective role of CD8+ T-cells in humans and the no correlation between neutralizing antibodies and protection observed in several studies, a vaccine based on cell-mediated immunity constitute an attractive approach. Our group has developed a capsid-based vaccine as nucleocpasid-like particles from dengue-2 virus, which induced a protective CD4+ and CD8+ cell-mediated immunity in mice, without the contribution of neutralizing antibodies. Herein we evaluated the immunogenicity and protective efficacy of this molecule in monkeys. Neither IgG antibodies against the whole virus nor neutralizing antibodies were elicited after the antigen inoculation. However, animals developed a cell-mediated immunity, measured by gamma interferon secretion and cytotoxic capacity. Although only one out of three vaccinated animals was fully protected against viral challenge, a viral load reduction was observed in this group compared with the placebo one, suggesting that capsid could be the base on an attractive vaccine against dengue.
    Virology. 01/2014; s 456–457:70–76.
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    ABSTRACT: Live attenuated viruses are the most advanced candidates against a dengue infection. They have been demonstrated to be immunogenic in preclinical and clinical studies. However, due to their replicative capacity, they require several doses to achieve the balanced immune response against the four serotypes. The use of a suitable combination using nonreplicative immunogens, without viral interference, can help to induce such a balanced response. This work dealt with the proof of concept of the heterologous prime-boost strategy combining in the same schedule in non-human primates of a live virus and the recombinant proteins containing the domain III of the viral envelope. These combina-tions may result in condensed immunization schedules for humans, thus reducing the number of doses with attenu-ated virus and the dose time spacing. In both studies, the humoral and cellular immune responses after the boost dose with each recombinant protein were evaluated. In the second study, additionally, the possibility of shortening the schedule was assessed, an advantage related with this prime-boost strategy. The boost effect was demonstrated by the neutralizing antibodies induced after recombinant protein immunizations. Additionally, it was confi rmed that these neutralizing antibodies were long lasting, also the animals were able to mount a specifi c cellular immune response after the boost. This study won the Annual Award of the Academy of Sciences of Cuba in 2012. RESUMEN Prueba de concepto en primates no humanos de estrategia de inmunización contra dengue con virus del serotipo 2 y re-estimulación con proteínas recombinantes que portan el dominio III de la proteína de la envoltura viral. Los virus vivos atenuados son los candidatos vacunales más avanzados contra la infección por dengue. Sus propiedades inmunogénicas se han demostrado en estudios preclínicos y clínicos. Sin embargo, por su cualidad replicativa es necesario administrarlos en varias dosis para alcanzar el equilibrio entre las respuestas inmunológicas contra los cuatro serotipos del virus dengue. Las combinaciones adecuadas de inmunógenos no replicativos sin interferencia viral pudiera ayudar a alcanzar esta respuesta. En este trabajo se describe la prueba de concepto de la estrategia de inducción-potenciación de la respuesta inmune en un mismo esquema en pri-mates no humanos tras la inmunización: primero con un virus vivo (dengue serotipo 2) y después con proteínas recombinantes que contenían el dominio III de la envoltura viral.
    Biotecnologia Aplicada 01/2013; 30(2):150-152.
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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; · 2.28 Impact Factor
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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. · 2.92 Impact Factor
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    ABSTRACT: Copper is an essential trace element but is toxic if in excess. Under acidic anaerobic conditions of gastrointestinal tract, this element is more toxic. Copper homeostasis in V. cholerae has not been studied deeply previously. We were interested in elucidating copper tolerance systems used by V. cholerae in different culture conditions. Single mutants of V. cholerae C7258 in genes VCA0260, VCA0261, VC2215 and VC2216 as well as double mutants in genes VC2215/VC2216 and VCA061-260/VC2216 and a triple mutant in genes VC2215/VC2216/VCA0261-0260 were constructed and characterized respect to copper sensitivity under aerobic and anaerobic conditions. The main copper resistance system in V. cholerae is constituted by the cation-carrier ATPase CopA, codified by VC2215, which functions under aerobiosis and anaerobiosis. The gene VC2216, which codifies for a conserved hypothetical protein, does not play an important role in copper resistance under aerobiosis. Under anaerobiosis, its contribution to this phenotype is only important if CopA is functional. The previously independent genes VCA0260 and VCA0261, codifying for two hypothetical proteins, constitute a single ORF that codifies for a single protein. Contribution of this gene to copper tolerance is important under aerobiosis and at high copper concentrations, but under anaerobiosis its participation in copper resistance is only evident if CopA is no-functional. Thus, copper tolerance systems in V. cholerae include the product of genes VC2215, VC2216 and VCA0261-0260, which play different functions under various growth conditions.
    Revista CENIC. Ciencias Biológicas. 01/2010; 41:1-13.
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    ABSTRACT: The Gram-negative bacterium Bordetella pertussis is the etiologic agent of Whooping cough. The disease is caused by the effect of an array of virulence factors expressed by the bacterium, which are controlled by the bvg system. One of the most important virulence factors is the pertussis toxin, for this reason its inactivated form is used as the main component of acellular vaccines. Pertussis toxin has six polypeptides encoded in a single operon, forming an A-B structure. The S1 polypeptide is the enzimaticaly active subunit, which catalyzes the ADP-ribose transference from NAD to the á subunits of the G proteins in eucariotic cells, triggering some biological effects such as histamine sensitization, enhancement of insulin and both suppressive and stimulatory immunologic effects. The present study deals with the procedure for generating Bordetella pertussis strains that express high levels of genetically inactivated pertussis toxin. For this purpose the aminoacids Arg9 and Glu129 was replaced by Lys and Gly, respectively, in the subunit S1. The mutant pertussis toxin operon was cloned into a wide host-range vector, regulated by an early promoter (fhaB). The resulting clones could be used as expression systems for the production of acellular vaccines in Cuba.
    Revista CENIC. Ciencias Biológicas. 01/2010; 41(2):115-120.
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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. · 2.89 Impact Factor
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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. · 2.89 Impact Factor
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    ABSTRACT: OmpU is an integral outer membrane protein belonging to the porine family, which is present in the outer membrane of bacteria of the genus Vibrio . This manuscript describes the construction of a recombinant plasmid coding for OmpU protein fused to a myc-hexa-histidine domain (myc-hexa-his) of 2 kDa. The plasmid encoded recombinant protein OmpU-myc-hexa-his, is expressed at high levels in the E. coli Top 10 strain, which is accumulated insoluble in inclusion bodies inside the cell. The recombinant protein is recovered in 7 mol/L urea from insoluble inclusion bodies. The denatured form was purified by immobilized metal-chelate affinity chromatography. SDS-PAGE analysis of the purified fractions detected a band of approximately 40 kDa, which is in correspondence with the molecular mass reported for the wild type monomer of OmpU (38 kDa) in Vibrio cholerae strains coupled to the recombinant c-myc-hexa-his domain. The recombinant protein reacted with anti-OmpU y anti-hexa-his monoclonal antibodies in western blotting. The detection of comigrating bands with these two antibodies indicates the presence of OmpU protein fused to hexa-hystidine tag in the same peptide. The protocol described allowed obtaining this protein in a single chromatographic step and increased the purity up to 90 %, yielding on average a 23 % of the initial amount. Consequently, a host -vector system is available to express this protein
    01/2008; 39(2):109-114.
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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 colonization
    CNIC journal. 02/2007; 38(02):124-131.
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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. · 4.07 Impact Factor
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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. · 3.19 Impact Factor
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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. · 3.19 Impact Factor
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    ABSTRACT: Murine monoclonal antibodies (MAbs) against Vibrio cholerae toxin co-regulated pilus (TCP) were generated using conventional hybridoma procedures. Four hybridomas were obtained and two characterized. Hybridomas 10E10E1 and 4D6F9 secreted antibodies of the IgG2a and IgG1 isotypes, respectively, that reacted with a 24-kDa antigen corresponding to the product of the El Tor tcpA gene fused to a six Histidine tail. Additionally, MAbs produced by 4D6F9 selectively recognized the major pilin subunit (TcpA) of El Tor and O139 vibrios in western immunoblot, while MAbs from 10E10E1 also cross-reacted with classical TcpA. Furthermore, vibrios expressing TCP on their surface selectively inhibited binding of the antibodies secreted by both hybridomas to TcpA-coated microtiter plates. Thus, the MAbs reported in this work detected the structural subunit of the pilus either denatured or assembled on the bacterial surface.
    Hybridoma and Hybridomics 11/2003; 22(5):315-20.