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.

BacMam immunization partially protects pigs against sublethal challenge with African swine fever virus.

Lack of vaccines and efficient control measures complicate the control and eradication of African swine fever (ASF). Limitations of conventional inactivated and attenuated virus-based vaccines against African swine fever virus (ASFV) highlight the need to use new technologies to develop efficient and safe vaccines against this virus. With this aim in mind, in this study we have constructed BacMam-sHAPQ, a baculovirus based vector for gene transfer into mammalian cells, expressing a fusion protein comprising three in tandem ASFV antigens: p54, p30 and the extracellular domain of the viral hemagglutinin (secretory hemagglutinin, sHA), under the control of the human cytomegalovirus immediate early promoter (CMVie). Confirming its correct in vitro expression, BacMam-sHAPQ induced specific T-cell responses directly after in vivo immunization. Conversely, no specific antibody responses were detectable prior to ASFV challenge. The protective potential of this recombinant vaccine candidate was tested by a homologous sublethal challenge with ASFV following immunization. Four out of six immunized pigs remained viremia-free after ASFV infection, while the other two pigs showed similar viremic titres to control animals. The protection afforded correlated with the presence of a large number of virus-specific IFNγ-secreting T-cells in blood at 17 days post-infection. In contrast, the specific antibody levels observed after ASFV challenge in sera from BacMam-sHAPQ immunized pigs were indistinguishable from those found in control pigs. These results highlight the importance of the cellular responses in protection against ASFV and point towards BacMam vectors as potential tools for future vaccine development.

Enhancing DNA immunization by targeting ASFV antigens to SLA-II bearing cells.

One of the main criticisms to DNA vaccines is the poor immunogenicity that they confer on occasions, at least in large animals. Confirming this theory, immunization with plasmid DNA encoding two African swine fever virus genes in frame (pCMV-PQ), failed in inducing detectable immune responses in pigs, while it was successful in mice. Aiming to improve the immune responses induced in swine, a new plasmid was constructed, encoding the viral genes fused in frame with a single chain variable fragment of an antibody specific for a swine leukocyte antigen II (pCMV-APCH1PQ). Our results clearly demonstrate that targeting antigens to antigen professional cells exponentially enhanced the immune response induced in pigs, albeit that the DNA vaccine was not able to confer protection against lethal viral challenge. Indeed, a viremia exacerbation was observed in each of the pigs that received the pCMV-APCH1PQ plasmid, this correlating with the presence of non-neutralizing antibodies and antigen-specific SLA II-restricted T-cells. The implications of our discoveries for the development of future vaccines against African swine fever virus and other swine pathogens are discussed.

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.

Insect larvae biofactories as a platform for influenza vaccine production.

Increased production capacity is one of the most important priorities for seasonal and pandemic influenza vaccines. In the present study, we used a baculovirus-insect larvae system (considered small, living biofactories) to improve the production of recombinant influenza virus H1N1 hemagglutinin (HA). Insect larvae produced four-fold more HA protein than insect cells per biomass unit (1 g of fresh larvae weight). A single infected Trichoplusia ni larva produced up to 113 µg of soluble and easily purified recombinant HA, an amount similar to that produced by 1.2×10(8) Sf21 insect cells infected by the same baculovirus. The use of the KDEL endoplasmic reticulum retention signal fused to the HA protein further increased recombinant protein production. Larvae-derived HA was immunogenically functional in vaccinated mice, inducing the generation of hemagglutination inhibition antibodies and a protective immune response against a lethal challenge with a highly virulent virus. The productivity, scalability and cost efficiency of small, living biofactories based on insect larvae suggest a broad-based strategy for the production of recombinant subunit vaccines against seasonal or pandemic influenza as an alternative to fermentation technologies.

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.

Optimization and validation of recombinant serological tests for African Swine Fever diagnosis based on detection of the p30 protein produced in Trichoplusia ni larvae.

We describe the validation of an enzyme-linked immunosorbent assay (ELISA) and confirmatory immunoblotting assays based on a recombinant p30 protein (p30r) produced in insect larvae using a baculovirus vector. Such validation included the following: (i) the scaling up and standardization of p30r production and the associated immunoassays, (ii) a broad immunological analysis using a large number of samples (a total of 672) from Spain and different African locations, and (iii) the detection of the ASF virus (ASFV)-antibody responses at different times after experimental infection. Yields of p30r reached up to 15% of the total protein recovered from the infected larvae at 3 days postinfection. Serological analysis of samples collected in Spain revealed that the p30r-based ELISA presented similar sensitivity to and higher specificity than the conventional Office International des Epizooties-approved ASFV ELISA. Moreover, the p30r ELISA was more sensitive than the conventional ELISA test in detecting ASFV-specific antibodies in experimentally infected animals at early times postinfection. Both the recombinant and conventional ELISAs presented variable rates of sensitivity and specificity with African samples, apparently related to their geographical origin. Comparative analyses performed on the sequences, predicted structures, and antigenicities of p30 proteins from different Spanish and African isolates suggested that variability among isolates might correlate with changes in antigenicity, thus affecting detection by the p30r ELISA. Our estimations indicate that more than 40,000 ELISA determinations and 2,000 confirmatory immunoblotting tests can be performed with the p30r protein obtained from a single infected larva, making this a feasible and inexpensive strategy for production of serological tests with application in developing countries.

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.

Placa móvil carotídea / Mobile carotid plaque

Introducción. La literatura sólo observa la publicación de siete casos de placa móvil sobre carótida cuya descripción imprecisa dificulta que se diferencie de las lesiones móviles de tipo trombo. Por otra parte, la historia natural de este tipo de lesiones no es conocida y su tratamiento resulta controvertido por cuanto existe la tendencia de elegir el tipo de tratamiento a seguir de acuerdo con el `potencial embolígeno' de la afección. Casos clínicos. Se describen dos casos de placa móvil sobre carótida. El diagnóstico se ha practicado en ambos casos mediante ecografía Doppler. El primer caso es un paciente neurológicamente sintomático, cuya placa móvil está asociada a una estenosis superior al 70 por ciento. Se le se indica tratamiento quirúrgico. El segundo caso es neurológicamente asintomático. En el estudio mediante ecografía Doppler se observa el fragmento de placa móvil sobre la carótida común, en la zona del seno carotídeo, sobre una estenosis del 30-50 por ciento. En la zona distal de la misma, sobre el bulbo de la carótida interna, se encuentra una estenosis >70 por ciento. En este caso se indica tratamiento médico debido al deteriorado estado general del paciente. Ambos pacientes han evolucionado favorablemente. Conclusiones. Sumados al segundo caso aquí descrito, son tres los pacientes con placa móvil carotídea (neurológicamente asintomáticos) que han evolucionado favorablemente con tratamiento médico. Mientras no se disponga de más datos acerca de la historia natural de estas lesiones no deberían considerarse, por sí mismas, motivo suficiente para indicar la endarterectomía carotídea. La posible indicación de tratamiento quirúrgico estará en función del grado de estenosis carotídea (AU)

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.