Abstract Infection with hepatitis C virus (HCV), which is distributed worldwide, often becomes in chronic hepatitis, cirrhosis and hepatocellular carcinoma. The HCV genome is a single-stranded RNA molecule of positive polarity approximately 9.6 kb in length. This review summarizes the current knowledge of recent advances in the investigation of the molecular biology of HCV.

Resumen La infección por el virus de la hepatitis C (VHC) se distribuye en todo el mundo, frecuentemente se convierte en hepatitis crónica, cirrosis y hepatocarcinoma. El genoma del VHC es una molécula de ARN monocatenario, de polaridad positiva, de aproximadamente 9.6 kb de longitud. Esta revisión resume el conocimiento actual y los avances recientes en la investigación de la biología molecular del VHC.

Hepatitis C is a significant health problem worldwide, with incidence estimates around 160 million people and 500 000 annual deaths. The limited success of treatments in chronic genotype 1 hepatitis C virus (HCV) patients and the numerous and significant adverse effects of the therapeutic treatment with pegilated interferon and ribavirin have encouraged the development of different direct-acting antivirals (DAAs) with promising results. This was also stimulated by advances of the knowledge on virus cell cycle and the properties of its structural properties. However, DAAs are very expensive and some of those compounds have developed multiple adverse events, all these limiting their use in certain infected populations. Moreover, its use does not prevent from HCV reinfections. Hence, new treatments, such as therapeutic vaccines, have arisen as additional or combined therapies against chronic HCV infection.

La hepatitis C constituye un problema de salud mundial, con estimados de más de 160 millones de personas infectadas. Esta enfermedad es responsable de alrededor de 500 000 muertes anuales. El éxito limitado del tratamiento en pacientes infectados con el virus de la hepatitis C (VHC), genotipo 1, así como los numerosos e importantes efectos adversos de la terapia con interferón pegilado más ribavirina, unido a los avances en el conocimiento del ciclo de vida y de las características de las proteínas estructurales del virus, han estimulado el desarrollo de diferentes antivirales de acción directa (AAD), muy prometedores en sus efectos terapéuticos. Sin embargo, estos nuevos productos son extremadamente costosos y además algunos de ellos han presentado múltiples eventos adversos, lo que limita su empleo en determinadas poblaciones de individuos infectados, y no evitan la reinfección con el VHC. Por lo que se necesitan tratamientos con vacunas terapéuticas como un tratamiento adicional o alternativo para las infecciones crónicas por el VHC.

A scalable high-cell-density Escherichia coli culture method was established to obtain pharmaceutical grade plasmid DNA (pDNA), together with an optimized purification process. The effects of several components of the medium, such as carbon and nitrogen sources that ensure bacterial nutritional needs, were studied. The operation parameters, such as temperature, shaking and aeration, were set and the optimum values of cell growth and specific pDNA productivity in culture were determined. The subsequent purification process for pharmaceutical grade pDNA was implemented, by combining RNA precipitation with ammonium sulfate and two successive chromatographic steps consisting of size exclusion chromatography and reverse phase-high performance liquid chromatography. This work comprised the first report on the use of reverse phase to purify DNA for application in humans.

Se estableció un método de cultivo incrementado para obtención de ADN plasmídico en Escherichia coli a altas concentraciones celulares, y estudio el efecto de varios componentes del medio, como las fuentes de carbono y de nitrógeno para garantizar las necesidades nutricionales. Se estableció los parámetros de temperatura, agitación y aireación, y obtuvo los valores óptimos de crecimiento celular y productividad específica de ADN plasmídico en el cultivo. Se implementó un proceso productivo de ADN plasmídico de grado farmacéutico, combinando la precipitación de ARN con sulfato de amonio, y dos pasos cromatográficos sucesivos de cromatografía de exclusión molecular y de cromatografía líquida de alta presión en fase reversa. Este último proceso es la primera vez que se emplea en la literatura para la purificación de ADN para aplicación en humanos.

The congress Biotecnología Habana 2012 was held at the Convention Center in Havana, Cuba, March 5-8. Organized by the Center for Genetic Engineering and Biotechnology (CIGB), this edition was dedicated to medical applications of biotechnology, with pre-Congress specialized satellite symposia and full conference sessions. Here we present information regarding the symposium on Infectious diseases (covering pertussis, adjuvants, hepatitis B and C treatment and vaccines) and also that related to two of the three pre-congress satellite symposia on dengue and the human immunodeficiency virus.

El congreso Biotecnología Habana 2012 se celebró del 5 al 8 de marzo en La Habana, Cuba. Auspiciado por el Centro de Ingeniería Genética y Biotecnología (CIGB), esta edición se dedicó a las aplicaciones médicas de la biotecnología, con simposios satélites precongreso y conferencias plenarias. Este reporte expone información del simposio sobre enfermedades infecciosas (que incluyó temáticas relativas a pertusis, adyuvantes, vacunas y tratamiento de las hepatitis virales B y C), y dos de los simposios satélites precongreso referidos a dengue y virus de la inmunodeficiencia humana.

The hepatitis C virus (HCV) infects over 170 million people worldwide and is a leading cause of chronic hepatitis and severe forms of liver damage as cirrhosis and hepatocellular carcinoma. There is no vaccine available against this pathogen and the current therapeutic option, based on the combination of pegylated interferon plus Ribavirin, is expensive, produces undesirable side effects, and is effective in approximately half of the patients treated. HCV establishes a complex and not completely understood interaction with the host. In addition to its variability and interference with the immune system function, the HCV life cycle is closely associated with lipid metabolism and this relationship contributes to viral persistence. The present review analyzes the current state of the art in this association and the disturbances generated, mainly expressed as intracellular lipid accumulation in hepatocytes and increased oxidative stress with negative consequences in the immune response. Moreover, the potential impact on the development of vaccines and more effective therapeutic interventions against this virus, in the context of the disorders in lipid metabolism, is discussed. Finally, perspectives for rational intervention, taking into account the dependence of HCV to lipid metabolism, and potential targets, are evaluated.

El virus de la hepatitis C (VHC) infecta a más de 170 millones de personas globalmente y es la causa principal de hepatitis crónica y formas graves de daño hepático, como cirrosis y carcinoma hepatocelular. No existe vacuna disponible contra este patógeno y la terapia actual que se basa en la combinación de interferón pegilado más Rivabirina provoca efectos secundarios y solo es efectiva en aproximadamente la mitad de los pacientes tratados. El VHC establece una compleja interacción con el hospedero que aún no ha sido completamente caracterizada. El ciclo de vida del VHC se relaciona estrechamente con el metabolismo lipídico, lo que junto a su variabilidad genética e interferencia con el funcionamiento del sistema inmune contribuye a la persistencia viral. En esta revisión se analiza el estado del arte de tal interacción, así como las alteraciones que provoca, fundamentalmente la acumulación de lípidos en los hepatocitos y el incremento del estrés oxidativo, con la consiguiente afectación a la respuesta inmune. Además, se discute el impacto potencial para el desarrollo de vacunas e intervenciones terapéuticas contra el VHC en el contexto de un metabolismo lipídico alterado. También se abordan las perspectivas para una intervención racional de la infección, teniendo en cuenta la dependencia del VHC en el metabolismo lipídico y los blancos potenciales de tales procedimientos.

In the present work, immunogenicity of recombinant in vitro assembled hepatitis C virus core particles, HCcAg.120-VLPs, either alone or in combination with different adjuvants was evaluated in BALB/c mice. HCcAg.120-VLPs induced high titers of anti-HCcAg.120 antibodies and virus-specific cellular immune responses. Particularly, HCcAg.120-VLPs induced specific delayed type hypersensitivity, and generated a predominant T helper 1 cytokine pro file in immunized mice. In addition, HCcAg.120-VLPs prime splenocytes proliferate in vitro against different HCcAg.120-specific peptides, depending on either the immunization route or the adjuvant used. Remarkably, immunization with HCcAg.120-VLPs/Montanide ISA888 formulation resulted in a significant control of vaccinia virus titer in mice after challenge with a recombinant vaccinia virus expressing HCV core protein, vvCore. Animals immunized with this formulation had a marked increase in the number of IFN-γ producing spleen cells, after stimulation with P815 cells infected with vvCore. These results suggest the use of recombinant HCV core particles as components of therapeutic or preventive vaccine candidates against HCV.

Humoral and cellular immune responses are currently induced against hepatitis C virus (HCV) core following vaccination with core-encoding plasmids. However, the anti-core antibody response is frequently weak or transient. In this paper, we evaluated the effect of different additives and DNA-protein combinations on the anti-core antibody response. BALB/c mice were intramuscularly injected with an expression plasmid (pIDKCo), encoding a C-terminal truncated variant of the HCV core protein, alone or combined with CaCl2, PEG 6000, Freund's adjuvant, sonicated calf thymus DNA and a recombinant core protein (Co.120). Mixture of pIDKCo with PEG 6000 and Freund's adjuvant accelerated the development of the anti-core Ab response. Combination with PEG 6000 also induced a bias to IgG2a subclass predominance among anti-core antibodies. The kinetics, IgG2a/IgG1 ratio and epitope specificity of the anti-core antibody response elicited by Co.120 alone or combined with pIDKCo was different regarding that induced by the pIDKCo alone. Our data indicate that the antibody response induced following DNA immunization can be modified by formulation strategies.