Biotecnología Aplicada

Therapies and clinical trials with vaccine candidates against HIV-1

Antiretroviral therapies combining three or more compounds frequently diminish the viral load (VL) in blood to undetectable levels (< 50 copies of RNA/mL), being considered as optimal. In contrast, more than 100 clinical studies with different vaccine candidates have barely achieved modest results and some studies have been discouraging. Therapies are, however, unable to eliminate viral infection. At the same time, they are a threat to the health of patients because of the accumulated toxicity derived from their prolonged use. Many researchers, therefore, believe that an effective (or even partially effective) vaccine might substitute therapies, eliminating the virus or at least controlling the VL through immune-mediated mechanisms. However, immune correlates for protection remain unknown requiring a strategy to evaluate the clinical effectiveness of vaccine candidates. Hence, the experience accumulated with therapies is highly valuable. This paper gives an update on some of the main results of antiretroviral therapies and therapeutic vaccination, giving recommendations in the field of vaccination against HIV-1.

Las terapias antirretrovirales que combinan tres o más compuestos, muchas veces reducen la carga viral en sangre a valores no detectables (< 50 copias de ARN/mL), por lo que se consideran muy efectivas. En contraste, más de 100 ensayos clínicos con diferentes candidatos vacunales contra el virus de inmunodeficiencia humana (VIH), solo han alcanzado modestos resultados y algunos estudios han sido decepcionantes. No obstante, las terapias no eliminan la infección viral, por lo que deben administrarse de por vida. Al mismo tiempo, el consumo prolongado eleva la toxicidad a niveles que ponen en riesgo la salud de los pacientes. Por eso, muchos investigadores creen que una vacuna efectiva (incluso parcialmente efectiva) podría sustituir tales terapias. Esa vacuna eliminaría el virus o, al menos, mantendría la carga viral controlada mediante mecanismos inmunes. Sin embargo, los correlatos inmunológicos de protección se desconocen. Por ello, es necesaria una estrategia que permita evaluar la efectividad clínica de los candidatos vacunales. En tal sentido, la experiencia con las terapias antirretrovirales es de gran valor. Se analizaron algunos de los principales resultados de estas terapias y de la vacunación anti-VIH, para emitir recomendaciones en este segundo campo.

Molecular diagnosis and control strategies for the relevant genetic diseases of cattle

The availability of the bovine genome sequence and the use of DNA markers have widened our understanding of a number of hereditary diseases in cattle, leading to the development of techniques for their early diagnosis. By isolating DNA from nucleated cell samples, followed by in vitro amplification techniques and digestion with restriction enzymes, it is now possible to detect the presence of lethal or mutant alleles for a specific phenotype. Such techniques are already being used for the study of genetic diseases of dairy cattle such as BLAD (Bovine leukocyte adhesion deficiency), CVM (Complex vertebral malformation), DUMPS (Deficiency of uridine-monophosphate synthase) and citrullinemia, among others, where the disorder is caused by the presence of a recessive allele in homozygosis. Although the frequency of these alleles in the population is usually very low, it can be easily increased if heterozygotic (carrier) sires are used during large-scale stockbreeding, ultimately resulting in significant economic losses; on the other hand, certifying the absence of such mutations in sires increases their value. Since there is a worldwide tendency towards the implementation of monitoring programs for hereditary diseases in cattle, it is important to update the technical personnel involved in cattle breeding (researchers and veterinary doctors) as well as farmers on the use and importance of DNA molecular markers in animal health.

El conocimiento del genoma bovino y la utilización de marcadores de ADN han permitido conocer el origen de algunas enfermedades hereditarias y desarrollar técnicas de diagnóstico precoz. Mediante el aislamiento de ADN a partir de muestras nucleadas y técnicas de amplificación in vitro y digestión con enzimas de restricción se puede diagnosticar si un animal es portador de un gen letal o mutante para determinadas características. En la actualidad es posible estudiar enfermedades hereditarias del ganado bovino lechero como la deficiencia de adhesión leucocitaria bovina (BLAD), la malformación vertebral compleja (CMV), la deficiencia de la enzima uridina monofosfato sintasa (DUMPs) y citrulinemia, entre otras, cuyos orígenes están relacionados con la presencia de alelos recesivos. Si bien la frecuencia de estos genes mutantes es muy baja, debe considerarse que la utilización a gran escala de reproductores portadores puede incrementarla y generar importantes pérdidas económicas; por otra parte, el conocimiento de que un reproductor está libre de tales mutaciones, incrementa su valor agregado. Es por ello, la tendencia mundial a la implementación de programas de vigilancia para enfermedades genéticas, de aquí la importancia de divulgar tanto entre investigadores y médicos como entre los productores la utilización e importancia de los marcadores moleculares de ADN en la sanidad animal.

**Evaluation of different formulations of a dengue-2 chimeric protein and outer membrane vesicles from Neisseria meningitidis in mice**

New generation vaccines, particularly those based on recombinant proteins, are generally less reactogenic than traditional live attenuated vaccines. Nevertheless, in terms of immunogenicity, they require potent adjuvants to reach a proper immune response in the recipients. We had previously evaluated the potential capacity of PD5 protein (a vaccine candidate against dengue-2, composed by the P64k protein of Neisseria meningitidis, and the domain III of the dengue Envelope protein), as a vaccine candidate with Freunds adjuvant. In this work, we evaluated the adjuvant capacity of the outer membrane vesicles (OMV) from N. meningitidis on the immunogenicity of the PD5 protein. As a result, after three doses in mice, the groups immunized with three different formulations of OMV elicited high titers of antiviral and neutralizing antibodies against dengue-2 with predominant IgG1 levels. Additionally, in the protection study, the most statistical difference was obtained in one of the three groups immunized with OMV, specifically with one formulation which favors the possible association between the protein and vesicles.

La nueva generación de vacunas, específicamente las basadas en proteínas recombinantes, son menos reactogénicas que las vacunas vivas atenuadas tradicionales. Sin embargo, en términos de inmunogenicidad, estas requieren de potentes adyuvantes para lograr la respuesta inmune adecuada. Nosotros previamente hemos evaluado la capacidad potencial de la proteína PD5 (candidato vacunal contra el virus dengue-2, compuesto por la proteína P64k de Neisseria meningitidis y el dominio III de la proteína de la Envoltura de dengue), como un candidato de vacuna con adyuvante de Freund. En el presente trabajo nosotros evaluamos la capacidad adyuvante de las vesículas de membrana externa (VME) de N. meningitidis sobre la inmunogenicidad de la proteína PD5. Como resultado, después de tres dosis en ratones, los grupos inmunizados con tres formulaciones diferentes de VME generaron altos títulos de anticuerpos antivirales y neutralizantes contra dengue-2 con niveles predominantes de anticuerpos IgG1. Adicionalmente, en el estudio de protección, la mayor diferencia estadística fue obtenida en uno de los tres grupos inmunizados con VME, específicamente en el cual se favorece la posible asociación entre la proteína y las vesículas.

A fermentation process for the production of P50 from Serratia marcescens

Serratia marcescens is an opportunistic gram-negative enteric bacterium isolated from the respiratory and urinary tracts in humans. Among the secreted S. marcescens extracellular proteins, the P50 protein is produced in large amounts and plays an important role in the pathogenesis of this organism. To produce this protein, a fermentation process was studied. First, in a 2³ factorial experimental design different culture supplement as tryptone, yeast extract and peptone, were studied. In 4 L bioreactors the influence of aeration rate and agitation speed over the P50 production were studied in a 3² experimental design. Finally, the optimal growth conditions were established (28 °C, 400 rpm and 0.5 vvm), at this scale, 9.0 ODu/mL, 62% level of P50 protein expression and 220 mg/L as the higher P50 volumetric production, were obtained.

Serratia marcescens es una enterobacteria gram-negativa oportunista aislada de las vías respiratorias y el tracto urinario en humanos. Entre las proteínas extracelulares segregadas de S. marcescens, esta la proteína P50 la que se produce en mayores cantidades y desempeña un papel importante en la patogénesis de este microorganismo. Para producir esta proteína se ha estudiado un proceso de fermentación. En primer lugar, en un diseño experimental, factorial de 2³, se estudiaron cultivos suplementados con triptona, extracto de levadura y peptona. En bioreactores de 4L se estudio la influencia de la aireación y la velocidad de agitación sobre la producción de la P50 en un diseño experimental 3². Por último, se establecieron las condiciones óptimas de crecimiento (28 °C, 400 rpm y 0.5 vvm), a esta escala, se obtuvo el 9.0 ODu/mL, 62% de nivel de expresión de la proteína P50 y 220 mg/L como la mayor producción volumétrica de P50.

Stability of standard curves of hepatitis C virus transcripts used for viral quantification

The World Health Organization currently estimates the prevalence of hepatitis C virus (HCV) infections to be around 3%, representing approximately 170 million of infected people worldwide. An important step in the confirmatory diagnosis of HCV is the detection of its ribonucleic acid (RNA) in human serum or plasma, using nucleic acid amplification methods. Likewise, the measurement of HCV RNA viral loads by these methodologies is important for monitoring the therapeutic efficacy of antiviral drugs and monitor disease progression in HCV-infected individuals. The aim of this work was to study the stability of external curves built with different concentrations of an HCV transcript, which are used as components of a competitive Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) assay for the quantification of HCV RNA in human serum or plasma samples. The curves were studied for a period of 18 months by quantification at each time point against a fresh external curve, using an internal standard diluted in lysis buffer. RNA was extracted from each point of the curve and amplified by competitive RT-PCR, using a fluorimetric detection scheme based on the use of separate, specific probes for the HCV amplicons or the internal quantification standard. The results obtained in this work demonstrated that the RNA from each point of the quantification curve remained stable for all studied time points, allowing the use of ready-to-use curves, prepared in lysis buffer containing the internal quantification standard, for HCV viral load assays.

La Organización Mundial de la Salud estima que la prevalencia del virus de la hepatitis C (VHC) es del 3%, lo que representa aproximadamente 170 millones de personas infectadas en el mundo. Para la confirmación del diagnóstico del VHC, es importante la detección del ácido ribonucleico (ARN) del virus en el suero o plasma humano, por métodos de amplificación de ácidos nucleicos. La determinación cuantitativa del ARN viral circulante permite predecir la respuesta al tratamiento y monitorear la terapia antiviral. El objetivo de este trabajo fue estudiar la estabilidad de curvas de cuantificación construidas a partir de un transcripto de VHC, como componentes de un ensayo de Reacción en cadena de la polimerasa con transcripción reversa (RT-PCR) competitivo utilizado para la cuantificación del ARN del VHC en muestras de suero o plasma humano. Se estudiaron las curvas hasta los 18 meses, y en cada tiempo se cuantificaron contra una curva preparada en el momento, utilizando un estándar de cuantificación interno diluido en un tampón de lisis. A partir de cada punto de la curva se extrajo el ARN y se amplificó mediante RT-PCR competitiva y detección fluorimétrica, con el empleo de sondas específicas para los amplicones del VHC y del estándar de cuantificación interno. Los resultados de este análisis mostraron que en cada punto de la curva, el ARN fue estable en todos los tiempos estudiados. Ello posibilita preparar curvas en el tampón de lisis que contiene el estándar de cuantificación interno, de manera que se puedan usar en ensayos de determinación de la carga viral del VHC.

Modelation of growth kinetics of mammalian cells in perfusion culture

Specific equations describing the behavior of cell growth, substrate use and product formation in stirred tank fermentors and cell bank-scale perfusion cultures (30 L working volume) were developed from basic mass balance equations. A third-order polynomial equation was statistically fitted to the restricted cell passage through the screen (0) obtained from experimentally run data to model the behavior of the culture with time, since a clear description of the hydrodynamics of the system has not yet been developed. The results of the simulation of the operation process with the VisSim software application using these equations agreed with the experimental data available. The model was used to analyze the influence of cell concentration, specific growth rate and specific product formation rate on the process during years 2000 and 2001, comparing the results obtained for both periods.

Partiendo de las ecuaciones básicas del balance de masa, se llegó a las ecuaciones particulares que describen el comportamiento del crecimiento celular, del consumo de sustrato y de la formación de producto en los fermentadores de tanque agitado y cultivo en perfusión a escala de banco (30 L de volumen de trabajo). En función de los datos reales de las corridas, se desarrolló el ajuste estadístico de un polinomio de tercer orden, al factor de paso de las células a través de la membrana del filtro rotatorio (spinfilter (0)), para describir su comportamiento en función del tiempo, ya que no se tiene una idea clara del comportamiento hidrodinámico del sistema, el cual será desarrollado en trabajos futuros. Con las ecuaciones desarrolladas y utilizando el programa VisSim, se simuló el proceso de operación, y se obtuvo una semejanza con los datos reales. En dicho modelo se analizaron la influencia de la concentración celular, la velocidad de crecimiento específica y la velocidad de formación específica de producto, durante el proceso, en los años 2000 y 2001; y se compararon ambos años.

Evaluating sterilizing filtration for API-HBsAg

The sterilizing filtration procedure for the active pharmaceutical ingredient of the Cuban anti-hepatitis B vaccine (HBsAg API) was evaluated by using cellulose nitrate type 113 membranes and Sartobran P capsules (Sartorius, Germany). The study of sterilization processes using saturated steam for two loads, one for the flat membranes within their respective carcasses and the other for auxiliary materials, demonstrated that the measuring points reached an F0 > 15 minutes, guaranteeing an appropriate sterilization. The physical integrity of both filtration media was maintained after the filtration process, indicating a successful operation. The extractables were studied by Fourier transformed infrared spectroscopy (FTIRS) and reverse phase high performance liquid chromatography (RP-HPLC), showing the lack of membrane-derived contaminants. The bacterial retention test was carried out at industrial scale simulating the operations used for the buffer and HBsAg API. Both filtration media were able to remove a microbiological load of ≥ 10(7) c.f.u./cm² of Brevundimonas diminuta (ATCC 19146), ensuring filtrate sterility. These results indicate that the API HBsAg sterilization procedure is safe and reliable.

Se evaluó el procedimiento de filtración esterilizante del ingrediente farmacéutico activo de la vacuna cubana antihepatitis B recombinante (IFA-HBsAg), usando membranas planas 113 y cápsulas Sartobran P (Sartorius, Alemania) como medio filtrante. El estudio de los procesos de esterilización con vapor saturado para dos cargas: una para membranas planas en sus carcazas, y otra para materiales auxiliares, demostró que los puntos medidos alcanzan un F0 > 15 minutos, lo que garantizó una correcta esterilización. La integridad física de ambos medios filtrantes se mantuvo antes y después del proceso de filtración, lo que indicó una operación exitosa. El estudio de extractables por espectroscopia infrarroja con transformada de Fourier (EIRTF) y cromatografía líquida de alta resolución en fase inversa (CLAR-FI), mostró la ausencia de compuestos contaminantes provenientes de las membranas. La prueba de retención bacteriana a escala industrial, simulando las operaciones con tampón e IFA-HBsAg, reveló que ambos medios filtrantes pueden retener una carga microbiológica de ≥ 10(7) u.f.c./cm² de Brevundimona diminuta, lo que garantiza la esterilidad del material filtrado. Estos resultados indican la garantía y seguridad del proceso de esterilización del IFA-HBsAg.

Report of the V Conference of IAS on HIV pathogenesis, treatment and prevention of HIV

GM3 ganglioside: a novel target for the therapy against melanoma

Malignant melanoma is a tumor with a steeply increasing incidence and scarce therapeutic options once metastatic. Currently, no vaccine is widely commercially available for melanoma treatment or prevention. The overexpression of GM3 ganglioside in murine and human melanomas and its important role in tumor progression makes this self antigen a potential target for preventive immunotherapy of this neoplasm. Previously, we have shown that preventive vaccination with GM3/VSSP induced a specific antitumor response; and elicited the rejection of syngeneic GM3- positive melanoma cells in immunized mice. In the present paper, we published the induction of a potent antitumor effect of this vaccine administered in a minimal residual disease B16 melanoma model. These findings propose the GM3/VSSP vaccine as a therapy designed to elicit and/or boost antitumor immunity in patients with minimal residual disease after surgery; thereby preventing or prolonging the time to recurrence. This is an important issue of the clinical setting because patients with stage II melanoma were reported to have 60% chance of survival 5 years after surgery. In addition, we examined the mechanisms by which this immunogen confers tumor protection. Surprisingly, in spite of the glycolipidic nature of this antigen, we have found that induction of anti-GM3 IgG antibodies and tumor- specific IFN γ secreting CD8+ T cells correlated with tumor protection. As a result, these findings demonstrate, for the first time, the direct involvement of the cellular immune response in the anti-tumor protection induced by a ganglioside-based vaccine.

Virus-like particles of the Rabbit Hemorrhagic Disease Virus obtained in yeast are able to induce protective immunity against classical strains and a viral subtype circulating in Cuba

Four epizootics caused by rabbit hemorrhagic disease virus (RHDV) have been recorded in Cuba from 1993 to 2005. Each time, thousands of animals have died or have been slaughtered to avoid the spread of the disease. Cell culture systems allowing the in vitro replication of RHDV are not available to date. Moreover, the amount of the recombinant capsid protein (VP60) obtained in heterologous expression systems does not commonly exceed a few tens of milligrams per liter of culture. In this paper, we report the expression of VP60 in two strains of the Pichia pastoris yeast with the highest expression levels obtained so far. VP60 was glycosylated, associated to the yeast cell disruption pellet at a concentration of 1.5 g/L in the first case, or soluble in the intracellular fraction at approximately 300 mg/L, following a different cloning strategy. These recombinant variants showed similar antigenic properties to those of the native protein, as determined by monoclonal antibodies. The soluble VP60 showed a higher number of protective epitopes, due to the formation of multimers that were similar in size and structure to the native RHDV capsids. Both antigens induced potent RHDV-specific immune responses in experimental animals. The antibodies produced were able to inhibit the in vitro hemagglutination of a viral strain isolated during the last outbreak in Cuba. A molecular and antigenic characterization of this strain was also carried out and led to its classification as a member of the highly pathogenic RHDVa subtype. Both recombinant antigens induced a specific, protective and long-lasting immune response against classical strains and also against the RHDVa subtype.

Tilapia somatotropin polypeptides: potent enhancers of fish growth and innate immunity

Growth hormone (GH) is a single chain polypeptide of approximately 22 kDa, produced by the pituitary gland and with pleiotropic functions among vertebrates. It mainly regulates body growth, being also involved in reproduction, immunity and osmoregulation in teleost fish. In order to obtain the tilapia (Oreochromis hornorum) growth hormone (tiGH) in Pichia pastoris cells, its gene was cloned into expression vectors in both with and without a heterologous secretion signal. The tiGH, obtained either intracellularly or extracellularly in P. pastoris cells, was characterized showing its production as associated to the cellular rupture precipitate with an approximate molecular weight of 22 kDa; or being secreted with an approximate molecular weight of 18 kDa, respectively. The mass spectrometry analysis of the recombinant protein obtained in the culture supernatant corroborated the identity of the protein as tiGH but lacking 46 aminoacids of its carboxyl terminal sequence. The tiGH biological activity of P. pastoris intact cells producing this protein was carried out in tilapia larvae (Oreochromis sp.), showing, for the first time, that it is possible to stimulate fish growth by immersion baths with recombinant tiGH-producing yeast. On the contrary what was previously postulated for mammals, the evaluation of P. pastoris cells expressing the truncated variant of tiGH demonstrated that this protein is also able to stimulate growth and immune system in fish. This is the first report of a biologically active, truncated GH variant in fish.