Marisela Suárez

The bovine IFN-ω1 is biologically active and secreted at high levels in the yeast Pichia pastoris

Abstract The gene coding for bIFN- ω 1 was isolated from bovine genomic DNA by polymerase chain reaction (PCR). Recombinant bIFN- ω 1 was expressed in the yeast Pichia pastoris and high levels of the recombinant protein (0.4 mg ml −1 ) were secreted to the culture media. The obtained bIFN- ω 1 showed a cross-species antiviral activity on four mammalian cell lines of calf, pig, hamster and human origin, but this activity was absent on Madin–Darby canine kidney (MDCK) cells. A delivery carrier was developed to permit a better release of bIFN- ω 1. When compared with a control group, an increase in 6 days in the corpus luteum lifespan was obtained in cyclic ewes following three interferon (IFN) intrauterine administrations on days 9, 10 and 11 post-estrus. In summary, these results demonstrated for the first time that biologically active recombinant bIFN- ω 1 was highly secreted by P. pastoris showing antiviral activity in different cell lines and an antiluteolytic effect in cyclic ewes, with no detrimental effects on the animals.

A single dose of the novel chimeric subunit vaccine E2-CD154 confers early full protection against classical swine fever virus

Classical swine fever is an economically important, highly contagious disease of swine worldwide. Subunit vaccines are a suitable alternative for the control of classical swine fever. However, such vaccines have as the main drawback the relatively long period of time required to induce a protective response, which hampers their use under outbreak conditions. In this work, a lentivirus-based gene delivery system is used to obtain a stable recombinant HEK 293 cell line for the expression of E2-CSFV antigen fused to porcine CD154 as immunostimulant molecule. The E2-CD154 chimeric protein was secreted into the medium by HEK293 cells in a concentration around 50mg/L in suspension culture conditions using spinner bottles. The E2-CD154 immunized animals were able to overcome the challenge with a high virulent CSF virus strain performed 7days after a unique dose of the vaccine without clinical manifestations of the disease. Specific anti-CSFV neutralizing antibodies and IFN-γ were induced 8days after challenge equivalent to 14days post-vaccination. The present work constitutes the first report of a subunit vaccine able to confer complete protection by the end of the first week after a single vaccination. These results suggest that the E2-CD154 antigen could be potentially used under outbreak conditions to stop CSFV spread and for eradication programs in CSF enzootic areas.

Humoral and cellular immune response in mice induced by the classical swine fever virus E2 protein fused to the porcine CD154 antigen

The development of subunit vaccines against classical swine fever is a desirable goal, because it allows discrimination between vaccinated and infected animals. In this study, humoral and cellular immune response elicited in inbred BALB/c mice by immunization with a recombinant classical swine fever virus (CSFV) E2 protein fused to porcine CD154 antigen (E2CD154) was assessed. This model was used as a predictor of immune response in swine. Mice were immunized with E2CD154 emulsified in Montanide ISA50V2 or dissolved in saline on days 1 and 21. Another group received E2His antigen, without CD154, in the same adjuvant. Montanide ISA50V2 or saline served as negative controls for each experimental group. Animals immunized with 12.5 and 2.5 μg/dose of E2CD154 developed the highest titers (>1:2000) of CSFV neutralizing antibodies. Moreover, CSFV specific splenocyte gamma-interferon production, measured after seven and twenty-eight days of immunization, was significantly higher in mice immunized with 12.5 μg of E2CD154. As a conclusion, E2CD154 emulsified in Montanide ISA50 V2 was able to induce a potent humoral and an early cellular immune response in inbred BALB/c mice. Therefore, this immunogen might be an appropriate candidate to elicit immune response in swine, control CSF disease and to eliminate CSFV in swine.

Corrigendum to “Efficacy of E2 glycoprotein fused to porcine CD154 as a novel chimeric subunit vaccine to prevent classical swine fever virus vertical transmission in pregnant sows”

Here we evaluated the effect of double vaccination with a novel subunit marker vaccine candidate based in the CSFV E2 glycoprotein fused to the porcine CD154 to prevent CSFV vertical transmission. A lentivirus-based gene delivery system was used to obtain a stable recombinant HEK 293 cell line for the expression of E2 fused to porcine CD154 molecule. Six pregnant sows were distributed in two groups and at 64days of gestation animals numbered 1-4 (group 1) were vaccinated via intramuscular inoculation with 50μg of E2-CD154 subunit vaccine. Animals from group 2 (numbered 5 and 6, control animals) were injected with PBS. Seventeen days later sows from group 1 were boosted with the same vaccine dose. Twenty-seven days after the first immunization, the sows were challenged with a virulent CSFV Margarita strain and clinical signs were registered. Samples were collected during the experiment and at necropsy to evaluate immune response and virological protection. Between 14 and 18days after challenge, the sows were euthanized, the foetuses were obtained and samples of sera and tissues were collected. E2-CD154 vaccinated animals remained clinically healthy until the end of the study; also, no adverse reaction was shown after vaccination. An effective boost effect in the neutralizing antibody response after the second immunization and viral challenge was observed and support the virological protection detected in these animals after vaccination. Protection against CSFV vertical transmission was found in the 100% of serums samples from foetus of vaccinated sows. Only two out of 208 samples (0.96%) were positive with Ct value about 36 corresponding to one tonsil and one thymus, which may be non-infective viral particles. Besides, its DIVA potential and protection from vertical transmission, the novel CSFV E2 bound to CD154 subunit vaccine, is a promising alternative to the live-attenuated vaccine for developing countries.