Biotecnología Aplicada

El tratamiento basado en interferón como terapia adyuvante para los candidatos vacunales contra la infección crónica por el virus de la hepatitis C

Therapeutic vaccine candidates against hepatitis C virus infection have demonstrated immunogenicity, but none of them have been able to induce complete viral elimination. Reviewed evidence shows hepatitis C virus-specific T cell exhaustion as a major obstacle for the immune containment of the virus. The possibility that hepatitis C virus-specific T cells may be rescued from exhaustion by interferon-based treatment makes this therapy a promising candidate to combine with hepatitis C virus-specific therapeutic vaccine interventions. This review summarizes the main effects of interferon-based therapy on hepatitis C virus-specific cellular immune response in chronic patients and, focuses on its potential to favorably impact the performance of therapeutic vaccine approaches for sustained viral clarification.

Los candidatos vacunales terapéuticos contra el virus de la hepatitis C han demostrado ser inmunogénicos, pero no han sido capaces de inducir la eliminación viral completa. Las evidencias experimentales acumuladas indican que el agotamiento de las células T específicas contra este virus es un obstáculo fundamental, que debe ser salvado si se persigue el control de la infección por parte del sistema inmune. El potencial mostrado por los tratamientos basados en interferón para rescatar la funcionalidad de dichas células, convierte a esta terapia en un candidato promisorio para ser combinado con las estrategias vacunales contra el virus de la hepatitis C. La presente revisión se centra en los principales efectos que tienen las terapias basadas en interferón sobre la respuesta celular específica contra el virus de la hepatitis C, en los pacientes crónicos. Particularmente, se hace énfasis en su potencial para modificar favorablemente la eficacia de los enfoques vacunales terapéuticos para alcanzar una eliminación viral sostenida.

**Secuencia y estructura de la región control mitocondrial del roedor cubano Capromys pilorides (Rodentia: Capromyidae)**

The complete mitochondrial DNA (mtDNA) control region from Capromys pilorides, an autochthon Cuban rodent, was sequenced and compared to two other species of hystricognath caviomorph rodents, in order to know patterns of variation and to explore the existence of previously described domains and other elements in rodents. The results revealed that the complete D-loop region of this species is 1336 base pairs long. Our data were compatible with the proposal of three domains [extended terminal associated sequences (ETAS), central (CD), and conserved sequence blocks (CSB)] within the control region, as well as the subsequences ETAS1, ETAS2, CSB1, CSB2, and CSB3. Likewise, a repetitive DNA region between the subsequences CSB1 and CSB2 was observed. The most conserved domain in the mitochondrial control region was the CD domain followed by ETAS and CSB domains in that order. The comparative analysis on base composition and genetic distance support the rationale of using the mitochondrial control region as a source of useful markers for population genetic studies with application to the conservation of this and other related Cuban rodent species, some of them under severe extinction risk.

Con el objetivo de explorar los patrones de variación y la presencia de los dominios y subsecuencias se secuenció la región control mitocondrial (D-loop) completa de Capromys pilorides, un roedor autóctono cubano, y se comparó con la descripción de otros dos roedores hystricognath caviomorfos. Los resultados mostraron que la región control mitocondrial completa de esta especie tiene con 1336 pares de bases, y se verificó la presencia de los dominios y las secuencias extendidas asociadas a la terminación (ETAS), central (CD), y bloque de secuencias conservadas (CSB) y las subsecuencias ETAS1, ETAS2, CSB1, CSB2, y CSB3. A su vez, se observó una región de ADN repetitivo entre las subsecuencias CSB1 y CSB2. La región más conservada resultó ser la correspondiente al dominio CD, a la que siguen los dominios ETAS y CSB. El análisis comparativo de la composición de bases entre dominios y de la distancia genética, apoya el propósito de utilizar estas secuencias como fuentes de marcadores útiles para los estudios de genética poblacional, con aplicación a la conservación de esta y otras especies de roedores cubanos afines, algunas de ellas en severo riesgo de extinción.

Limitada variabilidad genética de la quinta introducción en Cuba de Litopenaeus vannamei estimada con el uso de marcadores microsatélites

The present work estimated the genetic variability and relatedness index of the fifth stock of Pacific white shrimp, Litopenaeus vannamei, imported into Cuba for farming purposes from the US Shrimp Improvement System (SIS). Genetic variability was estimated by genotyping 33 samples for four microsatellite loci: M1, Pvan 1815, Pvan 0040 and Pvan 1758. This stock had average expected and observed heterozygosities of 0.37 and 0.27 respectively; the lowest of all stocks previously introduced in Cuba. The above, together with the low amount of allelic variants detected for each microsatellite, was suggestive of low genetic variability. In addition, pairwise relatedness coefficients clustered around unity, indicating a high degree of consanguinity. Taken as a whole, the data suggests that this breeding stock should be crossed first with other individuals from a different source or with higher genetic variability.

Se estimó la variabilidad genética y el índice de parentesco entre lotes de camarón blanco del Pacífico, Litopenaeus vannamei, introducidos por quinta ocasión en Cuba, procedentes del Centro de mejora del camarón, de Estados Unidos (Shrimp Improving System: SIS), para su cultivo. La variabilidad genética se estimó mediante el genotipo de 33 muestras con cuatro loci microsatélites: M1, Pvan 1815, Pvan 0040 y Pvan 1758. El quinto lote de Litopenaeus vannamei tuvo los valores promedios de heterocigosidad esperada y observada, más bajos de todos los introducidos en Cuba: 0.37 y 0.27, respectivamente. Esto, unido a la poca cantidad de variantes alélicas para cada región microsatélite, indica una escasa variabilidad genética. Los valores del coeficiente de parentesco alrededor de la unidad expresan una alta consanguinidad. Todo ello sugiere el cruce de los individuos de esta introducción con otros de diferente origen o más variables genéticamente.

Estimación del riesgo de descendencia con aberraciones cromosómicas desbalanceadas en progenitores portadores de translocaciones

Reciprocal and Robertsonian translocations are structural chromosomal aberrations that can produce unbalanced gametes during meiosis. In some cases, these imbalances lead to an offspring with multiple malformations. The purpose of this study was to propose and evaluate a methodology to estimate the risk of live offspring with unbalanced chromosome aberrations (LOUCA) from parents carrying a reciprocal or Robertsonian translocation. The methodology is based on comparing the results from several widely known regression methods: multiple linear, logistic and Poisson regression. Predictive accuracy was evaluated on a database containing information on 41 families of translocations carriers from three Cuban provinces. The results yielded by the three models were quite consistent regarding variable selection (presence of chromosome 9, chromosome 21 and the existence of breaking points in the short arms of the chromosomes involved) and risk estimation. There was a 80% overlap between the classifications produced by the three methods.

Las translocaciones recíprocas y robertsonianas son aberraciones cromosómicas estructurales que durante la meiosis pueden originar gametos cromosómicamente desbalanceados. Estos desequilibrios pueden generar una progenie con múltiples malformaciones. El objetivo de este trabajo es proponer una metodología para estimar el riesgo de descendencia con aberraciones cromosómicas desbalanceadas compatibles con la vida, en progenitores portadores de alguna translocación recíproca o robertsoniana. La metodología combina los resultados de los métodos de regresión logística, regresión múltiple y regresión de Poisson. Por sus características, es teóricamente superior a otras variantes descritas en la literatura revisada. El riesgo se estima a partir de una base de datos que contiene información de 41 estudios de familias portadoras de translocaciones, de tres provincias de Cuba. Los resultados con los tres métodos aplicados son coherentes en la selección de las variables predictoras (presencia de cromosomas 9, cromosoma 21 y existencia de puntos de ruptura en los brazos cortos de los cromosomas involucrados) y en las estimaciones del riesgo. El 80% de las familias se clasificaron por estos métodos.

Detección de patógenos en cerdos con neumonía en las diferentes regiones de Cuba

Enzootic pneumonia (EP) is characterized by high morbidity and low mortality, and although pigs of all ages are susceptible to Mycoplasma hyopneumoniae infection, a variety of other respiratory pathogens plays an important role in the development of Porcine Respiratory Disease Complex (PRDC) like Actinobacillus pleuropneumoniae, Bordetella bronchiseptica, Pasteurella multocida, Streptococcus suis, influenza virus and porcine circovirus type 2 (PCV-2). A total of 280 pig lungs with typical EP lesions were processed by molecular techniques. M. hyopneumoniae and M. hyorhinis were detected as a result of bacteriological cultures. Streptococcus suis type II, P. multocida, Streptococcus spp. and S. equisimilis were found in lung tissue samples. These results are a contribution to the knowledge of the enzootic pneumonia and the factors should be kept in mind in future works for the control of this entity. This research sets the basis for more complete studies of EP molecular epidemiology in Cuba.

La neumonía enzoótica (EP) se caracteriza por una alta morbilidad y baja mortalidad, y aunque los cerdos de todas las edades son susceptibles a la infección por Mycoplasma hyopneumoniae, existe una variedad de otros patógenos respiratorios que desempeñan un papel importante en el desarrollo del complejo respiratorio porcino (PRCD, siglas en inglés) como Actinobacillus pleuropneumoniae, Bordetella bronchiseptica, Pasteurella multocida, Streptococcus suis, virus de la influenza y el circovirus porcino tipo 2 (PCV-2). Un total de 280 pulmones de cerdos con lesiones típicas EP fueron procesados mediante técnicas moleculares detectándose M. hyopneumoniae y M. hyorhinis, además como resultado de cultivos bacteriológicos se detectaron Streptococcus suis tipo II, P. multocida, Streptococcus spp. y S. equisimilis a partir de muestras de tejido pulmonar. Estos resultados son un aporte al conocimiento de la neumonía enzoótica y los factores que deben tenerse en cuenta en futuros trabajos para el control de esta entidad. Esta investigación establece las bases para estudios más completos de la EP de epidemiología molecular en Cuba.

Transferencia rápida de los marcos abiertos de lectura entre vectores del sistema de dos híbridos de levadura y vectores de ensayo de complementación de proteínas mediante clonaje por recombinación homóloga en bacterias

A common rate-limiting step in many high throughput proteome scale analyses is the cloning of predicted open reading frames (ORFs) into technique-specific vectors. For example, methodologies for the detection of protein interactions such as Yeast-two-hybrid (Y2H) assay require validation using alternative methods as Protein Fragment Complementation Assays (PCA) or pulldown experiments. Various experimental alternatives for rapid homologous recombination gene transfer in vivo between the Y2H and PCA vectors were evaluated. Two sets of universal primers sharing an overlap of 31 b homology between inserts and acceptor vectors were designed and PCR performance conditions were tested. Cotransformation of PCR products with the digested acceptor vector in E. coli strain allowed homologous recombination cloning. The method was proved to be effective for cloning 5 ORFs with sizes ranging from 0.294 to 1.2 kb. The proposed method allows the quick transfer of any open reading frames between the Y2H and PCA assay vector systems by using a universal set of primers. It doesn't depend on the presence of specific restriction sites in the acceptor vector or causes changes in the open reading frame. This system will be useful for routine validation of protein interactions. We also report here the feasibility of DH10B strain for homologous recombination cloning.

Un paso limitante en muchos análisis del proteoma a alto flujo es la clonación de losmarcos abiertos de lectura (ORF) en los vectores específicos para cada técnica. Por ejemplo, las metodologías para la detección de interacciones entre proteínas tales como el ensayo de dos híbridos de levadura (Y2H) requieren una validación utilizando métodos alternativos, como por ejemplo los ensayos de complementación de fragmentos de la proteína (PCA) o experimentos de precipitación de proteinas. Diversas alternativas experimentales para una rápida transferencia mediante la recombinación homóloga de genes in vivo entre los vectores Y2H y PCA fueron evaluadas. Dos juegos de cebadores universales con una superposición de homología entre 31 b de los insertos y los vectores aceptadores fueron diseñados y las condiciones de amplificación de la reacción de PCR fueron evaluadas. Los productos de PCR fueron cotransformados con el vector aceptor en cepas de E. coli que permiten la clonación mediante recombinación homóloga. El método demostró ser eficaz para la clonación de cinco ORFs con tamaños que oscilan entre 0.294 a 1.2 kb. El método propuesto permite la rápida transferencia de los marcos abiertos de lectura entre los vectores de los sistemas de Y2H y PCA mediante el uso de un conjunto universal de los cebadores. El método de clonaje no depende de la presencia de determinadas sitios de restricción en el vector aceptor y no provoca cambios en el marco abierto de lectura. Este sistema será útil para la validación de rutina de las interacciones de proteínas. También mostramos la viabilidad de la cepa DH10B para la clonación por recombinación homóloga.

NASVAC: una vacuna terapéutica con potencialidades para mejorar la calidad de vida del paciente con hepatitis B crónica

Desarrollo de Ambliseius largoensis como agente de control biológico del ácaro blanco (Polyphagotarsenonemus latus)

The broad mites, Polyphagotarsonemus latus (Banks) (Acari: Tarsonemidae) is a cosmopolitan pest of many important crops as potatoes, beans, peppers, and citrus which lives in tropical, subtropical regions and greenhouses worldwide. Acording to its biological and ethological characteristics its control is complex by traditional methods of plant protection. For this reason, the use of the predatory mites of the Phytoseiidae family is an adequate and efficient management alternative. The present paper comprises the characterization of the potentiality of Amblyseius largoensis (Muma)(Acari: Phtyoseiidae) as biological control agent of P. latus integrating, for the first time, the predator's biological characteristics; feeding behavior and the numeric and functional responses; development of methodologies for mass rearing of the biological agent and parameters for quality control processes; the study of pest behavior in the protected production of Cuban hybrids of pepper, adapting the sampling methodology and determining the thresholds to estimate the population levels in these conditions. The compatibility of this predator with the principal chemicals and biological products used in the protected production and their effectiveness as biological control agent of P. latus in different conditions were evaluated. The results obtained demonstrated that A. largoensis is a new biological alternative for the management of broad mite populations in the protected production of peppers, which could be used in other agroecosystems; those that P. latus constitutes a problem, prior validation.

Identificación de una nueva tripsina de Spodoptera frugiperda que participa en un mecanismo de defensa contra la toxina CryCa1 de Bacillus thruringiensis

Both, insecticidal formulations based on the entomopathogenic bacterium Bacillus thuringiensis (Bt) and transgenic plants expressing Bt Cry toxins, have been threatened by the potential appearance of insect resistance in major crop pests. With the aim of identifying genes and mechanisms triggered against a Bt Cry1-class toxin in the midgut of fall armyworm Spodoptera frugiperda, the major pest of maize in Cuba, subtractive cDNA libraries of the molecular interaction insect-Bt Cry1Ca1 toxin were constructed. Among those genes specifically regulated in response to the intoxication, one coding for a new trypsin-like serin proteinase (SfT6) was identified. Gene function analysis using RNA interference showed SfT6 plays a crucial role for the Cry1Ca1 toxicity against S. frugiperda; gene expression suppression caused a reduction of the proteolytic processing of Cry1Ca1 by the larval midgut juice and a reduced susceptibility of insects in bioassays. Our study represents the first report on S. frugiperda midgut genes differentially-expressed in response to Cry1Ca1 intoxication. Besides, we have identified and cloned the full-length cDNA sequence of a novel serine proteinase whose regulation is linked to the natural process of insect adaptation to Bt in order to survive the pathogenic process.

Conocimientos novedosos sobre la influencia de la proporción entre las proteínas estructurales del virus de la hepatitis C para la inducción de una respuesta inmune protectora en un modelo de reto con virus sustituto

The incomplete definition of immunologic parameters that correlates with the protection against HCV infection has limited the development of a vaccine against this pathogen. Several studies advocate for the induction of strong cellular immune response against viral antigens, as well as neutralizing antibodies against HCV E2 envelope protein, as a success criteria. However, the influence of several antigens against each others in a protein preparation and the impact on the immunogenicity, have not been explored yet. In the present study a factorial design was used to generate several HCV vaccine preparations with the structural core, E1 and E2 proteins as antigens. An optimal antigen composition was chosen based on the lymphoproliferative response against HCV as well as protection against challenge with a surrogate virus in BALBC/c mice. The protein ratio (1:160:160), with 0.1 µg of Co.120-16.7 µg of E1.340-16.7 µg E2.680 (Co-E1-E2) was selected as the optimal composition to induce a functional immune response in mice. Additionally, strong humoral immune response against the 412-438 amino acid region from HCV E2 protein was detected when African green monkeys were immunized with Co-E1-E2. This region includes a conserved epitope, involved in T cell lymphoproliferative response against Co.120 and E2.680 proteins as well as in HCV neutralization. The results evidenced the relevance of proportion between HCV structural antigens in vaccine preparations for eliciting successful immune response.

Desarrollo de plataforma de adyuvación para vacunas preventivas y terapeúticas basada en partículas semejantes a virus: demostración de efecto en modelos animales y humanos

Developing an effective vaccine entails: 1) proper antigenic presentation to the immune system, inducing a response of adequate intensity 2) a long duration for said response 3) the ability to steer this response towards the immune system pattern most suited for the elimination of the pathogen. Among the current crop of adjuvants, only aluminum-based gels and some oil emulsions are compliant with established regulatory and safety requirements and, not surprisingly, have received extensive use. The present work describes the development of a technological framework for the inclusion of virus-like particles (VLP) as adjuvants in vaccine preparations, allowing the induction of a functional immune response in humans that achieves seroconversion and protective antibody levels without the need for additional adjuvants. This technological framework comprises both the production and formulation of antigens as virus-like particles and the use of these VLP as adjuvants for soluble antigens, obtaining high levels of induction of the Th1 cell response which, in combination with the humoral response, have been shown to confer a fully functional immunoprotective status in animal models. Some of the novel aspects of the present work are the development of a common methodology for the obtention and formulation of different VLP; the demonstration that VLP-containing formulations are able to bias response towards a Th1 pattern; the finding that VLP can be used as adjuvants stimulating a fully functional response in humans to soluble antigens; the safety of VLP-adjuvanted formulations and, lastly, the induction of a functional, protective response in animal models and humans.