Development of an APC-targeted multivalent E2-based vaccine against Bovine Viral Diarrhea Virus types 1 and 2.

The aim of this study was to develop and test a multivalent subunit vaccine against Bovine Viral Diarrhea Virus (BVDV) based on the E2 virus glycoprotein belonging to genotypes 1a, 1b and 2a, immunopotentiated by targeting these antigens to antigen-presenting cells. The E2 antigens were expressed in insect cells by a baculovirus vector as fusion proteins with a single chain antibody, named APCH I, which recognizes the ß-chain of the MHC Class II antigen. The three chimeric proteins were evaluated for their immunogenicity in a guinea pig model as well as in colostrum-deprived calves. Once the immune response in experimentally vaccinated calves was evaluated, immunized animals were challenged with type 1b or type 2b BVDV in order to study the protection conferred by the experimental vaccine. The recombinant APCH I-tE21a-1b-2a vaccine was immunogenic both in guinea pigs and calves, inducing neutralizing antibodies. After BVDV type 1b and type 2 challenge of vaccinated calves in a proof of concept, the type 1b virus could not be isolated in any animal; meanwhile it was detected in all challenged non-vaccinated control animals. However, the type 2 BVDV was isolated to a lesser extent compared to unvaccinated animals challenged with type 2 BVDV. Clinical signs associated to BVDV, hyperthermia and leukopenia were reduced with respect to controls in all vaccinated calves. Given these results, this multivalent vaccine holds promise for a safe and effective tool to control BVDV in herds.

Expression and characterization of the gD protein of HSV-2 fused to the tetramerization domain of the transcription factor p53.

The highly immunogenic glycoprotein D (gD) of herpes simplex virus type 2 (HSV-2) is a very important element for entry of this virus into host cells. These characteristics have made this protein a very interesting HSV-2 subunit vaccine candidate. Despite efforts to prevent genital herpes using gD-based subunit vaccines, to date, clinical trials using this antigen have failed. Therefore, using a small animal model, we sought to determine if a tetramerized truncated form of gD subunit vaccine, produced by recombinant baculovirus infected insect larvae, would elicit better protection against genital herpes than a monomeric gD-2 subunit vaccine. Three out of 5 mice immunized with the tetramerized antigen produced in a baculovirus expression vector system, survived a lethal challenge with a wild type HSV-2 strain (for more than 3 weeks after challenge). In contrast, all the mice (5) immunized with the truncated protein, produced by the same methodology, died within 2 weeks after challenge. These results suggest that multimerization (increasing the structural complexity) of the truncated gD antigen might be more likely protective than the monomer form. Also the use of an alternative cost-efficient eukaryotic expression system is described.

Comparison of strategies for the production of FMDV empty capsids using the baculovirus vector system.

Recombinant FMDV empty capsids have been produced in insect cells and larvae using the baculovirus expression system, although protein yield and efficiency of capsid assembly have been highly variable. In this work, two strategies were compared for the expression of FMDV A/Arg/01 empty capsids: infection with a dual-promoter baculovirus vector coding for the capsid precursor (P12A) and the protease 3C under the control of the polyhedrin and p10 promoters, respectively (BacP12A-3C), or a single-promoter vector coding the P12A3C cassette (BacP12A3C). Expression levels and assembly into empty capsids were analyzed in insect cells and larvae. We observed that the use of the single-promoter vector allowed higher levels of expression both in insect cells and larvae. Recombinant capsid proteins produced by both vectors were recognized by monoclonal antibodies (mAbs) directed against conformational epitopes of FMDV A/Arg/01 and proved to self-assemble into empty capsids (75S) and pentamers (12S) when analyzed by sucrose gradient centrifugation.

Comparative inhibitory activity of the stilbenes resveratrol and oxyresveratrol on African swine fever virus replication.

Stilbenols are polyphenolic phytoalexins produced by plants in response to biotic or abiotic stress. These compounds have received much attention because of their significant biological effects. One of these is their antiviral action, which has previously been documented for two members of this class, namely resveratrol and oxyresveratrol. Here we tested the antiviral effect of these two compounds on African swine fever virus, the only member of the newly created family Asfarviridae and a serious limitation to porcine production worldwide. Our results show a potent, dose-dependent antiviral effect of resveratrol and oxyresveratrol in vitro. Interestingly, this antiviral activity was found for these synthetic compounds and also for oxyresveratrol extracted from new natural sources (mulberry twigs). The antiviral effect of these two drugs was demonstrated at concentrations that do not induce cytotoxicity in cultured cells. Moreover, these antivirals achieved a 98-100% reduction in viral titers. Both compounds allowed early protein synthesis but inhibited viral DNA replication, late viral protein synthesis and viral factory formation.

DIVA diagnostic of Aujeszky's disease using an insect-derived virus glycoprotein E.

Commercial vaccines against Aujeszky's disease are mainly formulated using deleted versions of attenuated or inactivated Pseudorabies virus (PRV) particles lacking of the structural glycoprotein E (gE). Complementary diagnostic assays used to differentiate infected from vaccinated animals (DIVAs), are based on the detection of serum antibodies against gE. A recombinant version of the PRV gE protein was expressed in a baculovirus vector system in Trichoplusia ni insect larvae in order to obtain this diagnostic reagent for large scale diagnosis at reduced costs. A recombinant gE gene (gEr), lacking of signal peptide and transmembrane domains, was cloned into a modified baculovirus vector to allow glycosylation of the protein and its subsequent exportation to the extracellular space. Analysis by SDS-PAGE, Western-blotting and glycoprotein staining revealed that a glycosylated protein of the expected electrophoretic mobility was obtained in infected larvae. Time course experiments revealed that maximum expression levels were reached 72h post-infection using 10(4)pfu of the recombinant baculovirus (BACgEr) per inoculated larva. An indirect PRV gE-ELISA was developed using gEr as a coating antigen. A comparison between larvae-derived PRV gE-ELISA and two commercially available PRV diagnostic kits showed good correlation between assays and better sensitivity when testing certain sera pig samples using the gEr ELISA. More than 30,000 ELISA determinations could be performed from crude extracts obtained from a single larva infected with the recombinant baculovirus, indicating the feasibility of this strategy for inexpensive production of glycosylated antigens for PRV diagnosis.

Development of a low-cost, insect larvae-derived recombinant subunit vaccine against RHDV.

Vaccine antigens against rabbit hemorrhagic disease virus (RHDV) are currently derived from inactivated RHDV obtained from livers of experimentally infected rabbits. Several RHDV-derived recombinant immunogens have been reported. However, their application in vaccines has been restricted due to their high production costs. In this paper, we describe the development of an inexpensive, safe, stable vaccine antigen for RHDV. A baculovirus expressing a recombinant RHDV capsid protein (VP60r) was used to infect Trichoplusia ni insect larvae. It reached an expression efficiency of 12.5% of total soluble protein, i.e. approximately 2 mg of VP60r per larva. Preservation of the antigenicity and immunogenicity of the VP60r was confirmed by immunological and immunization experiments. Lyophilized crude larvae extracts, containing VP60r, were stable, at room temperature, for at least 800 days. In all cases, rabbits immunized with a single dose of VP60r by the intramuscular route were protected against RHDV challenge. Doses used were as low as 2 microg of VP60r in the presence of adjuvant or 100 microg without one. Orally administered VP60r in the absence of an adjuvant gave no protection. The potential costs of an RHDV vaccine made using this technology would be reduced considerably compared with producing the same protein in insect cells maintained by fermentation. In conclusion, the larva expression system may provide a broad-based strategy for production of recombinant subunit antigens (insectigens) for human or animal medicines, especially when production costs restrain their use.

Haemodynamic strategy for treatment of diastolic anterograde giacomini varicose veins.

OBJECTIVES: To assess the diagnosis and outcome of a haemodynamic strategy for the treatment of primary varicose veins associated with anterograde diastolic flow (ADF) in the Giacomini vein (GV). METHODS: ADF in the GV, with the escape point located at the saphenopopliteal junction, was demonstrated in 15 patients (15 limbs) by duplex ultrasound. No other escape points were seen in this group. ADF was defined as the flow present in the relaxing phase after isometric contraction of the lower limb, measured in the standing position. Duplex and clinical follow-up was performed prospectively at 1 week, at 1, 3, 6, and 12 months and once per year thereafter, between 1998 and 2001. Surgery consisted of flush division of the GV from the small saphenous vein (SSV) and division of the incompetent collateral veins from the GV. RESULTS: GV diameter showed an average reduction from 6 to 4 mm 33 months after surgery. Fourteen patients (93%) showed no symptoms or varicose veins. GV reconnection and recurrent ADF was demonstrated in two patients (13%). CONCLUSIONS: ADF is a rare condition associated with primary varicose veins. ADF occurs when there is a closed venovenous shunt with recirculation in the muscular diastole. This implies that, although a part of the circuit is ascendant, the re-entry point must be located downstream to the escape point. Accurate duplex assessment is required to distinguish this atypical haemodynamic condition from an abnormal systolic circuit bypassing a deep vein obstruction. Interruption of the GV above its junction with the SSV abolished ADF with an acceptable rate of recurrences.

Expression of porcine CD163 on monocytes/macrophages correlates with permissiveness to African swine fever infection.

Monocytes-macrophages, the target cells of African swine fever virus (ASFV) are highly heterogeneous in phenotype and function. In this study, we have investigated the correlation between the phenotype of specific populations of porcine macrophages and their permissiveness to ASFV infection. Bone marrow cells and fresh blood monocytes were less susceptible to in vitro infection by ASFV than more mature cells, such as alveolar macrophages. FACS analyses of monocytes using a panel of mAbs specific for porcine monocyte/macrophages showed that infected cells had a more mature phenotype, expressing higher levels of several macrophage specific markers and SLA II antigens. Maturation of monocytes led to an increase in the percentage of infected cells, which correlated with an enhanced expression of CD163. Separation of CD163+ and CD163- monocytes demonstrated the specific sensitivity of the CD163+ subset to ASFV infection. In vivo experiments also showed a close correlation between CD163 expression and virus infection. Finally, mAb 2A10 and, in a lower extent, mAb 4E9 were able to inhibit, in a dose-dependent manner, both ASFV infection and viral particle binding to alveolar macrophages. Altogether, these results strongly suggest a role of CD163 in the process of infection of porcine monocytes/macrophages by ASFV.

Durability of reflux-elimination by a minimal invasive CHIVA procedure on patients with varicose veins. A 3-year prospective case study.

OBJECTIVES: to assess the outcome of a conservative and haemodynamic method for insufficient veins on an ambulatory basis (French acronym, "CHIVA") with preservation of the greater saphenous vein (GSV) for treatment of primary varicose veins. METHODS: duplex incompetence of the sapheno-femoral junction (SFJ) and the GSV trunk, with the re-entry perforating point located on a GSV tributary was demonstrated in 58 patients with varices (58 limbs). The re-entry point was defined as the perforator, whose compression of the superficial vein above its opening eliminates reflux in the GSV. Duplex scanning was performed preoperatively and at 7 days, and patients were followed prospectively at 1, 3, 6, 12, 24, and 36 months after CHIVA. Operation consisted in flush ligation and division from the GSV of the tributary containing the re-entry perforating vein (no additional high ligation is included). If reflux returned, SFJ interruption was performed in a second surgical procedure. RESULTS: the GSV diameter showed an average reduction from 6.6 to 3.9 mm 36 months after surgery. Reflux in the GSV system was demonstrated in all but five (8%) patients. Of the 53 patients with recurrent reflux, 46 underwent SFJ interruption. CONCLUSIONS: elimination of reflux in the GSV after the interruption of insufficient collaterals is only temporary.

Antigenic and immunogenic properties of a chimera of two immunodominant African swine fever virus proteins.

A chimera of the two immunodominant African swine fever (ASF) virus proteins p54 and p30 was constructed by insertion of the gene CP204L into a Not I restriction site of E183L gene. The resulting chimeric protein p54/30, expressed by a recombinant baculovirus in insect cells and in Trichoplusia ni larvae, retained antigenic determinants present in both proteins and reacted in Western blot with a collection of sera from inapparent ASF virus carrier pigs. Remarkably, pigs immunized with the chimeric protein developed neutralizing antibodies and survived the challenge with a virulent African swine fever virus, presenting a reduction of about two logs in maximum viremia titers with respect to control pigs. In conclusion, this study revealed that the constructed chimeric protein may have utility as a serological diagnostic reagent and for further immunological studies that may provide new insights on mechanisms of protective immunity to ASFV.

African swine fever virus protein p54 interacts with the microtubular motor complex through direct binding to light-chain dynein.

Dynein is a minus-end-directed microtubule-associated motor protein involved in cargo transport in the cytoplasm. African swine fever virus (ASFV), a large DNA virus, hijacks the microtubule motor complex cellular transport machinery during virus infection of the cell through direct binding of virus protein p54 to the light chain of cytoplasmic dynein (LC8). Interaction of p54 and LC8 occurs both in vitro and in cells, and the two proteins colocalize at the microtubular organizing center during viral infection. p50/dynamitin, a dominant-negative inhibitor of dynein-dynactin function, impeded ASFV infection, suggesting an essential role for dynein during virus infection. A 13-amino-acid domain of p54 was sufficient for binding to LC8, an SQT motif within this domain being critical for this binding. Direct binding of a viral structural protein to LC8, a small molecule of the dynein motor complex, could constitute a molecular mechanism for microtubule-mediated virus transport.

High-yield expression of a viral peptide vaccine in transgenic plants.

A high-yield production of a peptide vaccine in transgenic plants is described here. A 21-mer peptide, which confers protection to dogs against challenge with virulent canine parvovirus, has been expressed in transgenic plants as an amino-terminal translational fusion with the GUS gene. Transformants were selected on the basis of their GUS activities, showing expression levels of the recombinant protein up to 3% of the total leaf soluble protein, a production yield comparable to that obtained with the same epitope expressed by chimeric plant viruses. The immunogenicity of the plant-derived peptide was demonstrated in mice immunized either intraperitoneally or orally with transgenic plant extracts, providing the suitability of the GUS fusions approach for low-cost production of peptide vaccines.

[Indications for carotid endarterectomy without arteriography. A validation study using eco-Doppler]. / Indicaciones de endarterectomía carotídea sin arteriografía. Estudio de validación mediante eco-Doppler.

INTRODUCTION: The possibility of diagnosing carotid stenosis and carrying out surgery without arteriography has increased with the use of different diagnostic methods. The eco-Doppler has been shown to be a useful method, although it requires previous individualised validation. OBJECTIVE: We aim to validate our Vascular Diagnostic Laboratory in the diagnosis of stenosis of the carotid bifurcation using eco-Doppler as compared with angiography, and the therapeutic indication thus obtained. PATIENTS AND METHODS: We made a prospective study for one year of 62 consecutive patients diagnosed on eco-Doppler as having carotid stenosis of over 70% and subsequent carotid arteriography. The treatment indicated is given, without waiting for the result of the angiography, on clinical evaluation, computerized tomography or cranial magnetic resonance and eco-Doppler, and subsequently on angiography. The degree of correlation of both methods, both for the therapeutic indication and for the degree of stenosis was determined. RESULTS: In five cases there was discrepancy and the therapeutic indication was different. Three of these were related to the diagnosis of carotid occlusion and in the other two cases there were bilateral lesions of over 50% on eco-Doppler, which were classified as minor on angiographic study. In the group with stenosis of over 70% with < 50% contralateral stenosis, eco-Doppler showed sensitivity and specificity of 100%, with a kappa correlation index = 1. CONCLUSION: It is possible to indicate carotid endarterectomy in patients with unilateral stenosis greater than 70% and contralateral stenosis < 50%, based on the eco-Doppler studies done in our Vascular Diagnostic Laboratory.

Oral immunogenicity of the plant derived spike protein from swine-transmissible gastroenteritis coronavirus.

Transgenic plants represent an inexpensive alternative to classical fermentation systems for production of recombinant subunit vaccines. Transgenic potato plants were created that express the N-terminal domain of the glycoprotein S (N-gS) from Transmissible gastroenteritis coronavirus (TGEV), containing the major antigenic sites of the protein. Extracts from potato tubers expressing N-gS were inoculated intraperitoneally to mice, and the vaccinated mice developed serum IgG specific for TGEV. Furthermore, when potato tubers expressing N-gS were fed directly to mice, they developed serum antibodies specific for gS protein, demonstrating the oral immunogenicity of the plant derived spike protein from TGEV.

In vitro inhibition of the replication of haemorrhagic septicaemia virus (VHSV) and African swine fever virus (ASFV) by extracts from marine microalgae.

We have screened for in vitro inhibition of viral replication with extracts from the following marine microalgae: Porphyridium cruentum, Phaeodactylum tricornutum, Tetraselmis suecica, Chlorella autotrophica, Dunaliella tertiolecta, Dunaliella bardawil, Isochrysis galbana, Isochrysis galbana var Tiso, Ellipsoidon sp. and Tetraselmis tetrathele. We have used as viral models two enveloped viruses of significant economic importance, the viral hemorrhagic septicemia virus (VHSV) of salmonid fish and the African swine fever virus (ASFV). The aqueous extracts from P. cruentum, C. autotrophica and Ellipsoidon sp., produced a significant inhibition of the in vitro replication of both viruses in a dose-dependent manner. That this inhibition could be due to sulfated polysaccharides was suggested because the same pattern of viral inhibition was obtained by using exocellular extracts from microalgae enriched in these compounds and/or dextran sulfate of high molecular weight. However, the inhibition of viral replication did not correlate with the percentage of sulfatation of the exocellular polysaccharides. Extracts from marine microalgae may have prophylactic utility against fish and mammalian viral diseases.

Functionality and cell anchorage dependence of the African swine fever virus gene A179L, a viral bcl-2 homolog, in insect cells.

The African swine fever virus gene A179L has been shown to be a functional member of the ced9/bcl-2 family of apoptosis inhibitors in mammalian cell lines. In this work we have expressed the A179L gene product (p21) under the control of the baculovirus polyhedrin promoter using a baculovirus system. Expression of the A179L gene neither altered the baculovirus replication phenotype nor delayed the shutoff of cellular protein synthesis, but it extended the survival of the infected insect cells to very late times postinfection. The increase in cell survival rates correlated with a marked apoptosis reduction after baculovirus infection. Interestingly, prevention of apoptosis was observed when recombinant baculovirus infections were carried out in monolayer cell cultures but not when cells were infected in suspension, suggesting a cell anchorage dependence for p21 function in insect cells. Cell survival was enhanced under optimal conditions of cell attachment and cell-to-cell contact as provided by extracellular matrix components or poly-D-lysine. Since it was observed that cytoskeleton organization varied depending on culture conditions of insect cells (grown in monolayer versus grown in suspension), these results suggested that A179L might regulate apoptosis in insect cells only when the cytoskeletal support of intracellular signaling is maintained upon cell adhesion. Thus, cell shape and cytoskeleton status might allow variations in intracellular transduction of signals related to cell survival in virus-infected cells.