Analía Berinstein

Protective immunity against foot-and-mouth disease virus induced by a recombinant vaccinia virus.

We report the construction of a recombinant vaccinia virus expressing the precursor for the four structural proteins of FMD virus (FMDV) (P1) strain C3Arg85 using a procedure for isolation of recombinant vaccinia viruses based solely on plaque formation. Adult mice vaccinated with this recombinant vaccinia virus elicited high titers of neutralizing antibodies against both the homologous FMDV and vaccinia virus, measured by neutralization assays. Liquid phase blocking sandwich enzyme-linked immunosorbent assays (ELISAs) using whole virus as antigen showed high total antibody titers against homologous FMDV, similar to those induced by the conventional inactivated vaccine. When ELISAs were carried out with heterologous strains A79 or O1Caseros as antigens, sera from animals vaccinated with the recombinant virus cross-reacted. Mice boosted once with the recombinant vaccinia virus were protected against challenge with infectious homologous virus. These results indicate that recombinant vaccinia viruses are efficient immunogens against FMDV when used as a live vaccine in a mouse model.

Enhancement of the immune response elicited with foot-and-mouth disease virus vaccines by an extract of the Mycobacterium sp. wall

The immunomodulating effect of an extract of the cell wall of Mycobacterium sp. (WSF, Vetrepharm Inc., London, Canada) in foot-and-mouth disease virus inactivated vaccines was tested in a murine model. The duration of immunity, protection, stimulation of immunocompetent cells acting on the long-lasting secondary response and possible tissue damage were examined. The incorporation of 10 micrograms WSF into aqueous and oil vaccines induced a high and long-lasting specific antibody response. The neutralizing titres of these antibodies were significantly higher than those observed in animals immunized with vaccines lacking WSF and conferred protection for at least 7 months. The data collected in repopulation assays indicate that WSF participates in the activation of immune cell populations involved in long-lasting memory. This results in an efficient B-cell secondary response even in the absence of T cells, lasting at least 5 months. No adverse reactions were detected. The enhancement effect of WSF on the immune response to foot and mouth disease vaccines observed in the murine model indicates the possibility of its inclusion in aqueous vaccines to be tested in cattle.

Expression of Hemagglutinin-Neuraminidase glycoprotein of Newcastle Disease Virus in agroinfiltrated Nicotiana benthamiana plants

The worldwide need for producing safer and less expensive vaccines with minor manufacture and processing requirements, together with the advances made through biotechnology, has promoted the development of efficient alternative tools to conventional vaccines. One of these is the use of plants or plant cell culture as production platforms of vaccine antigens with potential use as immunogens. We have already described the use of transgenic potato plants as immunogens against Newcastle Disease Virus (NDV), although the amount of the recombinant antigen recovered was low. The main objective of the work presented here was to enhance the expression of the HN glycoprotein of NDV through a protein targeting strategy and a promoter change. We have cloned the HN coding region under the regulation of the rubisco small subunit promoter in 5 different versions in a subcellular localization strategy, and we have established that the construct harboring the complete HN gene with its own signal peptide, fused to KDEL retention peptide, rendered the best expressed/accumulated HN protein level whether a transient or a stable transformation assay was performed. We conclude that agroinfiltration results in a simple and useful tool for selecting suitable genetic constructions to be used in stable plant transformation and, moreover, it could be used as a method to produce immunogens for vaccine developments.

Transient expression of VP2 in Nicotiana benthamiana and its use as a plant-based vaccine against Infectious Bursal Disease Virus

Infectious Bursal Disease Virus (IBDV) is the etiological agent of an immunosuppressive and highly contagious disease that affects young birds. This disease causes important economic losses in the poultry industry worldwide. The VP2 protein has been used for the development of subunit vaccines in a variety of heterologous platforms. In this context, the aim of this study was to investigate VP2 expression and immunogenicity using an experimental plant-based vaccine against IBDV. We determined that the agroinfiltration of N. benthamiana leaves allowed the production of VP2 with no apparent change on its conformational epitopes. Chickens intramuscularly immunized in a dose/boost scheme with crude concentrated extracts developed a specific humoral response with viral neutralizing ability. Given these results, it seems plausible for a plant-based vaccine to have a niche in the veterinary field. Thus, plants can be an adequate system of choice to produce immunogens against IBDV.

Activation of the immune response against Infectious Bursal Disease Virus after intramuscular inoculation of an intermediate strain.

Infectious bursal disesase is a highly contagious, wide spread immunosuppressive chicken disease caused by the Infectious Bursal Disease Virus (IBDV). IBDV is a two segmented double-strand RNA virus, member of the Birnaviridae family. In order to study the interaction between IBDV and the immune system, chickens were exposed to an intermediate IBDV strain by intramuscular route, and using Real Time PCR the expression of a panel of avian cytokines and chemokines in duodenum, spleen and bursa of Fabricius was analyzed. Also, splenic nitrite (NO₂) production and the frequencies of different mononuclear cell populations were evaluated by Griess reaction and flow cytometry, respectively. Intramuscular (i.m.) IBDV inoculation promoted an over expression of proinflammatory cytokines IL-6, IL-15 and gIFN in spleen, which correlated with an increase of gIFN plasma concentration measured by ELISA, together with an increment of NO₂ concentration in splenocyte supernatants at 1dpi. Results obtained in the present work showed that IBDV of intermediate virulence, given i.m., induced similar effects to those previously described for highly virulent IBDV in early innate immune responses. Considering that the i.m. route is the route of choice for the delivery of new generation vaccines, and that the use of recombinant antigens also requires the addition of adjuvants for proper immune stimulation, results presented here could contribute to identify suitable cytokines to be used or to be stimulated when utilizing subunit vaccines, for the improvement of prevention tools for avian health.

Baculovirus treatment fully protects mice against a lethal challenge of FMDV

Foot-and-mouth disease virus (FMDV) causes a highly contagious and economically devastating disease that affects cattle, swine, goat and sheep among others. FMDV is able to overcome the initial host innate immune response by inhibiting the induction of antiviral molecules at both the transcriptional and the translational levels. It has been demonstrated that FMDV A/Arg/2001 causes the death of adult C57Bl/6 mice within 72h. We evaluated the capacity of Autographa californica nuclear polyhedrosis virus (AcNPV), an insect virus with potent innate immunostimulating effects, to promote early protection against FMDV A/Arg/2001 challenge in C57Bl/6 mice. Groups of 8-9 weeks old female mice were injected intravenously with AcNPV and challenged with a lethal dose of FMDV at different times post-administration. Our results showed that pretreatment of mice with a single injection of AcNPV 3h or 3 days before FMDV challenge resulted in complete abrogation of mortality and complete or partial suppression of viremia, respectively. Furthermore, no signs of disease were observed. AcNPV could be a valuable tool to improve the design of a novel vaccine that protects as an adjuvant at early times post-vaccination.

Mucosal immunity induced by orally administered transgenic plants

Oral immunization is an efficient means to induce protection at the portal entrance for many pathogens. Therefore, the design of efficient edible vaccines through transgenic plants represents a challenging alternative to the traditional injectable ones. We have previously reported the construction of transgenic potato plants expressing the genes coding for the immunogenic proteins of Newcastle Disease Virus (NDV) and their immunogenicity in mice. All mice receiving transgenic plant extracts in incomplete Freunds adjuvant produced specific antibodies. Animals fed with transgenic leaves also showed a specific response against NDV. The aim of the present study was to continue the evaluation of the mucosal immune response. Adult Balb/c mice were fed with potato leaves for a month and on day 36 mucosal samples were collected. ELISAs performed on intestinal washes showed that transformed plants elicited the synthesis of NDV-specific IgG and IgA antibodies. In addition, anti-NDV IgA antibodies were detected in supernatants of cultured small intestine fragments of mice fed with the recombinant immunogens, suggesting the presence of NDV-specific IgA secreting plasma cells in the intestinal tissue. Moreover, we detected specific anti-NDV antibodies in intestinal fluids after oral immunization with F and HN transgenic plants. Also, indirect immunofluorescence on intestinal tissue was performed. The present results suggest that these immunogens, F and HN glycoproteins of NDV, when orally administered, would enhance the number of IgA(+) B cells, and the cytotoxic cellular immune response via CD8(+) T cells, found in the gut lamina propria that is in accordance with our first findings.

Differences in the virulence of two strains of Foot-and-Mouth Disease Virus Serotype A with the same spatiotemporal distribution.

During the 2000-2001 epidemic of Foot-and-Mouth Disease Virus (FMDV) in Argentina, two FMDV serotype A viruses were identified among others. Since different pathogenic properties between these virus strains were noticed in cattle, we evaluated several biological properties and features of FMDV A/Arg/00 and FMDV A/Arg/01 in order to compare these viruses in terms of virulence and pathogenicity. Our results indicate that FMDV A/Arg/00 grows less efficiently than FMDV A/Arg/01, exemplified by smaller sized plaques, retarded one-step growth curves and overall low viral yields. Also, FMDV A/Arg/00 displayed the lowest specific infectivity in suckling mice requiring 50-fold more infectious particles than FMDV A/Arg/01 to generate a LD50 in suckling mice. Finally, FMDV A/Arg/00 did not cause death in adult C57Bl/6 mice even at high doses (10(7)-10(6)PFU) whereas FMDV A/Arg/01 resulted lethal in doses as low as 10(2)PFU. Overall, we were able to demonstrate that these virus strains differ from each other in terms of virulence and pathogenicity.

Avridine and LPS from Brucella ovis: Effect on the memory induced by foot-and-mouth disease virus vaccination in mice

Foot-and-mouth disease is one of the more economically important diseases among meat-producing biungulate species. In contrast to natural infection, current foot-and-mouth disease virus (FMDV) vaccines, prepared with inactivated virus and adjuvants, elicit short-lived protection. The immunomodulating effect on FMDV vaccines of avridine and lipopolysaccharide of Brucella ovis (LPS) was tested in a murine model. The duration of immunity, protection, stimulation of immunocompetent cells producing a long-lasting secondary response and immunoglobulin (Ig) isotypes were examined. The incorporation of either immunomodulator into aqueous and oil vaccines induced a long-lasting specific antibody response. The neutralizing titres and protection were significantly higher than those observed in animals immunized with control vaccines. Data collected from repopulation assays indicated that the immunomodulators used participate in the activation of immune cell populations involved in long-lasting memory. This resulted in an efficient B-cell secondary response even in the absence of T cells, which were necessary for the stimulatory effect of the immunomodulators in donor mice. Avridine and LPS stimulated IgG1, IgG2a and IgG2b production, which was correlated with the improvement of the protection induced by these vaccines.

Infectious Bursal Disease Virus non-structural protein VP5 is not a transmembrane protein

Infectious Bursal Disease Virus (IBDV) causes a highly relevant poultry disease that affects young chickens causing, among other effects, immunosuppression. IBDV is a bi-segmented double stranded RNA virus. The smaller ORF of larger RNA segment encodes VP5, a 17-kDa non-structural protein. Although it is an important protein for viral replication cycle, the definition of its specific role and subcellular localization remains unclear. In the present work we demonstrate, using imaging techniques, that VP5 is not a type II transmembrane protein but an intracellular membrane-associated protein. This finding might provide evidences of VP5 interaction with cellular proteins and its functions.