Development of a nasal vaccine for chronic hepatitis B infection that uses the ability of hepatitis B core antigen to stimulate a strong Th1 response against hepatitis B surface antigen.

There are estimated to be 350 million chronic carriers of hepatitis B infection worldwide. Patients with chronic hepatitis B are at risk of liver cirrhosis with associated mortality because of hepatocellular carcinoma and other complications. An important goal, therefore, is the development of an effective therapeutic vaccine against chronic hepatitis B virus (HBV). A major barrier to the development of such a vaccine is the impaired immune response to HBV antigens observed in the T cells of affected patients. One strategy to overcome these barriers is to activate mucosal T cells through the use of nasal vaccination because this may overcome the systemic immune downregulation that results from HBV infection. In addition, it may be beneficial to present additional HBV epitopes beyond those contained in the traditional hepatitis B surface antigen (HbsAg) vaccine, for example, by using the hepatitis B core antigen (HBcAg). This is advantageous because HBcAg has a unique ability to act as a potent Th1 adjuvant to HbsAg, while also serving as an immunogenic target. In this study we describe the effect of coadministration of HBsAg and HBcAg as part of a strategy to develop a more potent and effective HBV therapeutic vaccine.

Mitochondrial DNA variation in North American populations of Daphnia obtusa: continentalism or cryptic endemism?

The morphological stasis of many freshwater crustaceans has resulted in the prior delineation of cosmopolitan species and has been explained by their capacity for long-distance dispersal. This study examines the phylogeography of Daphnia obtusa, a cladoceran thought to be widespread in North America. However, sequence variation of the mitochondrial cytochrome c oxidase subunit I gene indicates that this taxon is composed of two morphologically cryptic species, designated D. obtusa NA1 and NA2. NA2 is restricted to the east, whereas NA1 is broadly distributed across the United States, and is subdivided into four phylogroups that show weak genetic differentiation over broad geographical areas, which likely reflects recent long-distance dispersal. The current distributions of the four phylogroups in NA1 can be explained by recent range expansion from different refugia following the last Pleistocene glacial advance. Interestingly, the mitochondrial phylogroups identified in this study do not correspond to lineages detected in a previous allozyme analysis. However, the latter groups are associated with a habitat shift suggesting that natural selection may have played a role in their divergence. The results of this and previous studies illustrate the complicated biogeographical history of freshwater cladocerans.

A phase I clinical trial of a multi-epitope polypeptide TAB9 combined with Montanide ISA 720 adjuvant in non-HIV-1 infected human volunteers.

A phase I clinical trial was performed to examine the safety and immunogenicity of a multi-epitope polypeptide comprising the central 15 amino acids of the V3 loop from six HIV-1 isolates. This protein called TAB9 was emulsified in Montanide ISA720 (Seppic, Paris) and administered intramuscularly at doses of 0, 0.2 and 1 mg to 24 healthy, HIV-1 seronegative adult males. Three immunisations were given at months 0, 1 and 6 in a randomised, double blind, placebo controlled clinical trial. The placebo was generally well tolerated. However, severe local reactions were observed in TAB9 vaccinated subjects after the second and third inoculations. Seven out of eight volunteers from the lower dose group showed moderate or severe local inflammation, while four out of eight subjects from the higher dose group developed granulomas and sterile abscesses. In general, the reactogenicity depended on the number of inoculations given and the dose of TAB9. Both doses were immunogenic, all immunised volunteers seroconverted and antibodies were broadly reactive against the V3 peptides included in the protein. All vaccine's sera reacted against gp120 in Western blot and 50% of them also neutralised at least one out of five laboratory isolates tested. No differences between doses were found. Anti TAB9 lymphoproliferative responses were observed, being more intense in the high dose group. Due to the strong local reactions that were found in this study, a change in the formulation will be required for further trials with this vaccine candidate in humans.

[Impact of an anti-hepatitis B virus (HBV) immunization program in patients undergoing dialysis in Havana, Cuba]. / Impacto del Programa de inmunización anti-virus de hepatitis B (VHB) en pacientes sometidos a diálisis en La Habana, Cuba.

The follow-up of HBV markers in selected high infection risk populations, in patients from the hemodialysis and peritoneal dialysis services was used to assess the effectiveness of a special vaccination program. Viral infection markers were studied in prevalence cross sections of the whole population of patients, and also by recording the reports of clinical cases of hepatitis B occurred during that period in those groups of patients. The prevention program consisted of the vaccination of all patients negative to the viral markers and the indication of vaccination for the new cases during the period of the kidney disease, just before the start of the treatment at the hemodialysis unit; besides all the persons susceptible to infection that had already been included in the program, regardless of the stage of the disease. The results show the benefit of the vaccination in these patients, but it is more effective in the period before the treatment with dialysis where there is a lower possibility of being exposed to the virus and the immune system is still competent. Once the program was established, after a follow up o 6 years, there have been no reports of new cases of hepatitis B and the incidence of the disease has been declining.

Physico-chemical characterization of recombinant hepatitis B surface antigen by a multidimensional approach.

Initially, our work was directed to respond to the question: why hepatitis B surface antigen (HBsAg) produces a very broad peak in preparative size-exclusion chromatography (SEC). For this purpose, we used a multidimensional approach based on SEC fractionation of purified HBsAg followed by the individual analysis of SEC fractions by a battery of assays, such as SEC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, enzyme-linked immunosorbent assay and transmission electron microscopy. As a result, HBsAg particles were shown to be heterogeneous in terms of particle assembly. In order to elucidate the origin of HBsAg heterogeneity, we included here the denaturing SEC into a multidimensional approach. The data from denaturing SEC evidenced the fragmentation of protein monomers within the HBsAg particle that, probably, occurs during fermentation broth, rather than during in vitro HBsAg processing. The fractions isolated from widely separated regions of HBsAg peak differed in the extent of protein fragmentation, suggesting that the variable extent of protein degradation within HBsAg particles may be one of the factors responsible for broadening of the HBsAg peak in SEC.

Aggregation of recombinant hepatitis B surface antigen induced in vitro by oxidative stress.

In order to examine whether oxygen radicals could be responsible for aggregation of recombinant hepatitis B surface antigen (HBsAg) during its assembly in yeast, purified HBsAg was oxidized with ammonium peroxodisulphate (AP) and analyzed by non-denaturing and denaturing size exclusion chromatography, immunoassay and immunoelectron microscopy. As a result, peroxodisulphate radicals induced a reproducible aggregation of HBsAg. At 44 mM AP, the aggregation process took a few hours and the resulting structures were large, branched and non-antigenic. During more gentle oxidation with 9 mM AP and 20-80 microM Cu2+, a continuous structural modification to HBsAg delaying for tens of hours preceded the aggregation event. During this pre-aggregation period, peroxidation of HBsAg lipids and covalent cross-linking of S protein chains occurred that led a complete loss of antigenicity of oxidized particles. In contrast, yeast-derived HBsAg aggregate is decomposed to S monomers under reducing conditions and recognized by anti-HBsAg polyclonal and monoclonal antibodies, suggesting that is has been assembled in vivo from antigenic and reversibly cross-linked particles. Based on these observations, we conclude that oxidation, at least with respect to the specific molecular sites oxidized by AP, is not a primary event in HBsAg aggregate formation in vivo. Since oxidized HBsAg was shown to be irreversibly cross-linked and non-antigenic, there are no suitable techniques for detection HBsAg oxidation in biological samples. Hence, at present, the magnitude of the in-vivo oxidative damage to HBsAg cannot be evaluated and thus, whether the plasma-derived HBsAg undergoes radical-induced oxidation in the course of viral hepatitis remains to be established. If this occurs, this process is expected to contribute to low HBsAg levels in chronic hepatitis B carriers, failure of the currently available immunoassays to identify HBsAg-positive blood donors and inconsistency in the results provided by HBsAg- and anti-HBsAg-based tests in several recent reports.

Aggregation of recombinant hepatitis B surface antigen in Pichia pastoris.

The combination of immunoaffinity and size-exclusion chromatography (SEC) is a powerful tool to analyze multiprotein particle assembly. This approach was used to investigate the source of aggregation of recombinant hepatitis B surface antigen (HBsAg) detected in purified material. As HBsAg aggregation does not originate in the stresses, such as the concentration of HBsAg solutions, temperature and chaotropic agents, it is less probable that the HBsAg aggregate is produced during the process. To test whether aggregation takes place in vivo, crude yeast extract containing the expressed HBsAg was fractioned on a Sephacryl S-400 column just after cell disruption, and each fraction immunopurified individually. As a result, the HBsAg aggregate was isolated from a fraction corresponding to the elution of large particle aggregates only, not native HBsAg particles. It was biologically active, which demonstrates aggregate formation by specific assembly of partially or wholly folded HBsAg intermediates.

Evidence for the denaturation of recombinant hepatitis B surface antigen on aluminium hydroxide gel.

Despite the complexity of the subject of protein-alum interactions, a valuable information can be obtained by analyzing the adsorbed and desorbed protein by common physico-chemical methods. In the present work, to approach the adsorption of hepatitis B surface antigen (HBsAg) on alum, the experimental data were supported by complementary analyses of the adsorbed protein by immunoelectron microscopy and the desorbed protein by denaturing size-exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. First, the depletion of HBsAg was investigated. The aspects assessed were the conditions, recovery and chromatographic performance of the desorbed protein. The results obtained strongly suggested the loss of particulate structure of HBsAg after adsorption on alum. This conclusion was further reinforced by direct immunoelectron microscopic visualization of HBsAg in the adsorbed state.

Adsorption-desorption of recombinant hepatitis B surface antigen (r-HBsAg) from P. pastoris on a diatomaceous earth matrix: Optimization of parameters for purification.

Recombinant hepatitis B surface antigen (r-HBsAg) produced in yeast is adsorbed on a diatomaceous earth matrix for purification purposes. A pH dependence in the adsorption-elution behavior was found. The capacity of celite (Hyflo Super Cei) for adsorbing r-HBsAg increased with decreasing pH. Nonspecific proteins were also adsorbed, but a low pH dependence was found. Elution from the matrix was performed using a basic pH buffer, in which r-HBsAg is more specifically adsorbed/desorbed than contaminant proteins, permitting the purification of the r-HBsAg. A pH of 4.0 was used for adsorption and pH 8.2 was used for desorption. The described protocol allows a purification factor between three- and fivefold with respect to contaminant proteins and sixfold with respect to contaminant DNA. Finally, the adsorption step was successfully scaled-up for production purposes.

Purificación de ß-galactosidasa recombinante y su evaluación como enzima marcadora en un sistema inmunoenzimático con anticuerpos monoclonales / Purification of recombinant ß-galactosidase and its evaluation as label enzyme in an immunoenzymatic assay with monoclonal antibodies

La enzima ß-galactosidasa producida por vía recombinante en E. Coli fue purificada por cromatografía de intercambio iónico y filtración por gel, lográndose una preparación de enzima de más del 95 % de pureza. Su capacidad como enzima marcadora en inmunoensayo enzimático sobre fase sólida (ELISA), fue probada comparándose con peroxidasa, la cual ha sido ampliamente usada en estos ensayos. Se realizaron conjugados con anticuerpo monoclonal anti-rotavirus para un sistema de detección de rotavirus, utilizando el agente heterobifuncional SPDP. Se obtuvieron niveles similares de sensibilidad para los conjugados realizados con peroxidasa y ß-galactosidasa; igualmente no se observaron diferencias significativas en el parámetro unión específica/unión no específica. En el presente trabajo mostramos los resultados obtenidos en la purificación de la enzima, su seguimiento por actividad enzimática y su uso como enzima marcadora en inmunoensayo enzimático

Purificación de ß-galactosidasa recombinante y su evaluación como enzima marcadora en un sistema inmunoenzimático con anticuerpos monoclonales / Purification of recombinant ß-galactosidase and its evaluation as label enzyme in an immunoenzymatic assay with monoclonal antibodies

La enzima ß-galactosidasa producida por vía recombinante en E. Coli fue purificada por cromatografía de intercambio iónico y filtración por gel, lográndose una preparación de enzima de más del 95

de pureza. Su capacidad como enzima marcadora en inmunoensayo enzimático sobre fase sólida (ELISA), fue probada comparándose con peroxidasa, la cual ha sido ampliamente usada en estos ensayos. Se realizaron conjugados con anticuerpo monoclonal anti-rotavirus para un sistema de detección de rotavirus, utilizando el agente heterobifuncional SPDP. Se obtuvieron niveles similares de sensibilidad para los conjugados realizados con peroxidasa y ß-galactosidasa; igualmente no se observaron diferencias significativas en el parámetro unión específica/unión no específica. En el presente trabajo mostramos los resultados obtenidos en la purificación de la enzima, su seguimiento por actividad enzimática y su uso como enzima marcadora en inmunoensayo enzimático

Uso del interferón leucocitario durante una epidemia de dengue hemorrágico (virus tipo II) en Cuba / Use of leukocyte interferon during an epidemic outbreak of haemorragic dengue (Type II) in Cuba

Durante una epidemia de fiebre del dengue hemorrágico en Cuba, el interferón leucocitario se utilizó en dos diseños experimentales. Por una parte, el interferón disminuyó significativamente el número de complicaciones y muertes cuando se aplicó por vía intramuscular, a razón de 5 . 104 U/Kg de peso corporal durante tres días, en 165 niños comparados con un grupo control de 160. En otro estudio, el uso del interferón leucocitario por vía intramuscular (1,5 . 106 U diariamente durante tres días) en un grupo tomado al azar, de 9 pacientes adultos comparados con un grupo control de ocho casos mostró una tendencia hacia resultados positivos de acuerdo con las diferencias significativas encontradas en parámetros de laboratorio. No se encontró sistemáticamente la toxicidad debida al interferón

Uso del interferón leucocitario durante una epidemia de dengue hemorrágico (virus tipo II) en Cuba / Use of leukocyte interferon during an epidemic outbreak of haemorragic dengue (Type II) in Cuba

Durante una epidemia de fiebre del dengue hemorrágico en Cuba, el interferón leucocitario se utilizó en dos diseños experimentales. Por una parte, el interferón disminuyó significativamente el número de complicaciones y muertes cuando se aplicó por vía intramuscular, a razón de 5 . 104 U/Kg de peso corporal durante tres días, en 165 niños comparados con un grupo control de 160. En otro estudio, el uso del interferón leucocitario por vía intramuscular (1,5 . 106 U diariamente durante tres días) en un grupo tomado al azar, de 9 pacientes adultos comparados con un grupo control de ocho casos mostró una tendencia hacia resultados positivos de acuerdo con las diferencias significativas encontradas en parámetros de laboratorio. No se encontró sistemáticamente la toxicidad debida al interferón

Hexosaminidase C in Tay-Sachs and Sandhoff disease.

1. Hexosaminidase C has been purified from human placenta. Complete separation from hexosaminidases A and B was achieved. 2. The following properties of hexosaminidase C differ from those of the A and B isozymes. Presence in the supernatant rather than the lysosomes, neutral pH optimum, higher molecular weight, lack of activity on beta-N-acetylgalactosamine derivatives, and lack of immunological relationship. 3. Hexosaminidase C is active in patients deficient in hexosaminidases A and B and can be recognized by its characteristic electrophoretic mobility. It is concluded that the genetic origin of hexosaminidase C is probably different from that of hexosaminidases A and B.