BHV-1 vaccine induces cross-protection against BHV-5 disease in cattle.

Protection against BHV-5 disease induced by inactivated BHV-1 or BHV-5 based vaccines was analysed. Two groups of calves were subcutaneously immunized with an inactivated BHV-1 or BHV-5 based vaccine. A third group was not vaccinated and used as control. In the post-vaccination period, we studied the humoral and cellular immune response resulting similar to both groups. The efficacy of the vaccines was tested after intranasal challenge of the calves with a virulent Argentinean BHV-5 isolate (A-663). All control animals developed neurological signs associated with BHV-5 infection and high levels of virus shedding. Calves immunized with the BHV-1 and BHV-5 inactivated vaccines were protected against BHV-5 disease. Our study provides evidence that strongly support the existence of cross-protection between BHV-1 and BHV-5 in calves. Even though this has already been suggested by previous works, this is the first time an exhaustive study of the immune response is performed and typical clinical BHV-5 meningoencephalitis signs are reproduced in an experimental BHV-5 challenge trial.

Bovine herpes virus gD protein produced in plants using a recombinant tobacco mosaic virus (TMV) vector possesses authentic antigenicity.

A tobacco mosaic virus (TMV)-based vector was utilized for expression of a cytosolic form of the bovine herpesvirus type 1 (BHV-1) protein glycoprotein D (gDc). Nicotiana benthamiana plants were harvested 7 days after inoculation with RNA transcripts derived from the TMV-gDc recombinant virus. Recombinant gDc protein of expected electrophoretic mobility accumulated in inoculated leaves to a concentration of about 20 micrograms/g of fresh leaf tissue. Oil-based vaccines were formulated with crude foliar extracts to immunize mice parentally. After a single injection, animals developed a sustained and specific response to both the isolated gD and native virus particles. Cattle vaccinated with the same gDc containing extracts developed specific humoral and cellular immune responses directed against both the viral gD and BHV-1 particles. Most importantly, animals vaccinated with the plant-produced gDc showed good levels of protection after challenge with the virulent BHV-1. Virus excretion was drastically reduced in these animals, reaching levels comparable to animals vaccinated with a commercial BHV-1 vaccine. The positive immunological characterization obtained for the gDc, indicated that an important part of the natural conformation was retained in the plant recombinant protein.

Induction of a virus-specific antibody response to foot and mouth disease virus using the structural protein VP1 expressed in transgenic potato plants.

We have recently communicated the oral and parental immunogenicity of the structural protein VP1 of foot and mouth disease virus (FMDV) expressed in different transgenic plants. Those results clearly indicated the necessity of increasing the expression of the foreign genes in the transgenic plant to avoid additional steps toward the purification and/or concentration of the antigen of interest. Here, we report the production of transgenic potatoes plants containing the VP1 gene cloned under the regulatory activity of either a single (pRok2) or a double (pRok3) copy of the S35 cauliflower mosaic virus (CaMV 35S) promoter, as a strategy for increasing the level of VP1 gene expression. The presence of the VP1 gene in the plants was confirmed by polymerase chain reaction (PCR) and its specific transcription activity was demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR). The results showed that, although the immunized animals presented a FMDV VP1 specific antibody response and protection against the experimental challenge, no significant differences were demonstrated in the immunizing activity of plant extracts obtained from the pRok2 or pRok3 transformed plants. These results confirm those previously obtained using other plant species allowing the possibility of using plants as antigen expression vectors, and demonstrated that at least in the potato system, the use of double CaMV 35S promoter does not cause a significant increase in the level of the VP1 expressed.

Adjuvant effects of sulfolipo-cyclodextrin in a squalane-in-water and water-in-mineral oil emulsions for BHV-1 vaccines in cattle.

The antibody and cell mediated immune responses induced by BHV-1 were analysed in cattle after vaccination and challenge exposure to the virulent strain LA of BHV-1. Animals were vaccinated intramuscularly (IM) with inactivated virus vaccines against BHV-1 containing either a water in mineral oil adjuvant (W/O), a water in mineral oil adjuvant plus Avridine (W/O+Avridine) or sulfolipo-cyclodextrin in squalane in-water emulsion (SL-CD/S/W). No significant differences were registered in the antibody response induced by the three evaluated vaccines. However, the BHV-1 specific cell-mediated immunite response was stronger and appeared earlier when SL-CD/S/W was included in the formulation. The efficacy of the vaccines was also evaluated after intranasal challenge of the calves with a virulent BHV-1 LA strain. Animals vaccinated with SL-CD/S/W had reduced virus excretion and clinical symptoms compared with the mock-vaccinated animals. Comparison of levels of BHV-1 specific IgG2 and IgG1 with virus shedding revealed that, regardless of the adjuvant administered, animals showing BHV-1 specific IgG2/IgG1 ratios higher than 1 were those with a significant lower number of individuals shedding virus. Additionally, animals vaccinated with SL-CD/S/W presented no post-vaccinal reactions. These factors, combined with the higher efficacy and the ease of manipulation of the biodegradable oil, makes the vaccine formulated with this new adjuvant an important contribution for the veterinary vaccines industry.

A comparison of methods for measuring the antibody response in mice and cattle following vaccination against foot and mouth disease.

We present a comparison of methods for evaluating the potency of foot and mouth disease vaccine in the laboratory. The anti-FMDV antibodies (Ab) in vaccinated mice were tested by liquid phase (lp) ELISA, solid phase (sp) ELISA and virus neutralization (VN), and were compared with the Ab titres detected by lpELISA, which is the official test in Argentina for testing the potency of FMD vaccines and protection against a virulent challenge in cattle. The results demonstrated that it is possible to relate the Ab levels induced in vaccinated mice with both the Ab and protective responses elicited in cattle. Furthermore, it was found that the anti-FMDV Ab titres in mice detected by lpELISA 14 days after vaccination should be an accurate parameter for predicting the results of the challenge test in cattle. Thus, this test in mice appears to be an inexpensive and rapid alternative for testing FMD vaccines in cattle.

Detection and characterization of functional T-cell epitopes on the structural proteins VP2, VP3, and VP4 of foot and mouth disease virus O1 campos.

Foot and mouth disease virus (FMDV) is the cause of a widespread infectious disease affecting cloven-hoofed animals. It is controlled by vaccination with immune-inactivated virus grown in tissue culture. However, peptide vaccines represent a safer alternative to the current virus-inactivated immunogens. Their design requires the identification and evaluation of the sequences recognized by T- and B-lymphocytes. Four structural proteins, VP1, VP2, VP3, and VP4, comprise the viral capsid of the FMDV, but only VP1 has been extensively studied regarding the existence of relevant T-cell epitopes. Here, we utilize a murine model to present a functional T-cell epitope mapping on the complete sequences of VP2, VP3, and VP4 of FMDV O1 Campos. We used two in vitro assays to describe 13 amino acid sequences, each one of them including at least one T-cell epitope. The in vivo T-cell helper function of these sequences was studied in an adoptive cell-transfer assay in mice. Immunization experiments with a fusion peptide containing one of the sequences characterized were also done comparing the helper activity of this sequence with other T-cell epitopes included in the major immunogenic region of VP1.

Protection of mice against challenge with foot and mouth disease virus (FMDV) by immunization with foliar extracts from plants infected with recombinant tobacco mosaic virus expressing the FMDV structural protein VP1.

A tobacco mosaic virus (TMV)-based vector has been used to express in plants the complete open reading frame coding for VP1, the major immunogenic protein of foot and mouth disease virus (FMDV). In vitro RNA transcripts were inoculated into Nicotiana benthamiana plants and detectable amounts of recombinant VP1 were identified by Western blot as soon as 4 days postinfection. Foliar extracts prepared from infected leaves were injected intraperitoneally into mice and all of the immunized animals developed a specific antibody response to both the complete virus particle and the major immunogenic region as determined by ELISA and Western blot analysis. Most importantly, all immunized mice developed a protective immune response against experimental challenge with virulent FMDV. To our knowledge, this is the first report showing the expression of a complete open reading frame of an antigenic foreign protein in plants, using a recombinant plant virus, in sufficient quantity to permit use of the crude plant extract as an experimental immunogen to protect animals against virus challenge.

Improvement of the immune response to foot and mouth disease virus vaccine in calves by using Avridine as adjuvant.

The epidemiological analysis of the cattle population during the eradication plan of foot and mouth disease (FMD) in Argentina clearly indicated a higher incidence of the disease in animals within their first year of age. It is important to improve the efficacy of the vaccination in those animals. In a previous report, we have shown the effect of an immunomodulator, Avridine (Avr), in the enhancement of the immune response elicited by FMD virus (FMDV) vaccines in experimental hosts [Berinstein, A., Pérez Filgueira, M., Schudel, A., Zamorano, P., Borca, M., Sadir, A.M., 1993. Avridine and LPS from Brucella ovis: effect on the memory induced by foot-and-mouth disease virus vaccination in mice. Vaccine 11, 1295-1301]. In this report, we analyze the effect of Avr in the improvement of the anti-FMDV immune response elicited in young animals immunized with a tetravalent vaccine. The anti-FMDV antibody response was evaluated using a liquid-phase blocking sandwich ELISA (LPBE) [Smitsaart, E.N., Zanelli, M., Rivera, I., Fondevila, N., Compaired, D., Maradei, E., Bianchi, T., O'Donnell, V., Schudel, A.A., 1998. Assessment using ELISA of the herd immunity levels induced in cattle by foot and mouth disease oil vaccines. Prev. Vet. Med 33, 283-296] while the cellular response was detected using an antigen specific lymphoproliferative test [Zamorano, P., Wigdorovitz, A., Chaher, M., Fernández, F., Sadir, A., Borca, M., 1994. Localization of B and T cell epitopes on a synthetic peptide containing the major immunogenic site of FMDV O1 Campos. Virology 201, 383-387]. The results show that, while no differences were detected in the cellular response, the anti-FMDV antibody reaction was significantly (<0.05) higher in animals immunized with the immunogen containing Avr. At 90 days post vaccination, 89-100% of the animals immunized with Avr presented predicted protection (PP) higher than 82% while just 50-61% of the animals immunized with vaccine without immunomodulator presented that characteristic. Also, it is shown that the increase in the anti-FMDV antibody titre in animals immunized with the vaccine containing Avr was mediated by an increase in the levels of both IgG1 and IgG2 which presented a significative correlation with LPELISA antibodies titres. It is concluded that the addition of Avr in the FMDV vaccines improve the immune status of the calves, the cattle population that suffers the highest epidemiological risk.

Induction of a protective antibody response to foot and mouth disease virus in mice following oral or parenteral immunization with alfalfa transgenic plants expressing the viral structural protein VP1.

The utilization of transgenic plants expressing recombinant antigens to be used in the formulation of experimental immunogens has been recently communicated. We report here the development of transgenic plants of alfalfa expressing the structural protein VP1 of foot and mouth disease virus (FMDV). The presence of the transgenes in the plants was confirmed by PCR and their specific transcription was demonstrated by RT-PCR. Mice parenterally immunized using leaf extracts or receiving in their diet freshly harvested leaves from the transgenic plants developed a virus-specific immune response. Animals immunized by either method elicited a specific antibody response to a synthetic peptide representing amino acid residues 135-160 of VP1, to the structural protein VP1, and to intact FMDV particles. Additionally, the immunized mice were protected against experimental challenge with the virus. We believe this is the first report demonstrating the induction of a protective systemic antibody response in animals fed transgenic plants expressing a viral antigen. These results support the feasibility of producing edible vaccines in transgenic forage plants, such as alfalfa, commonly used in the diet of domestic animals even for those antigens for which a systemic immune response is required.

Effect of Mycobacterium sp. wall and Avridine on the antibody response, IgG isotype profile and proliferative response induced by foot and mouth disease virus (FMDV) vaccination in cattle.

Different immunomodulators have been previously tested in our laboratory as enhancers of the specific immune response to FMDV vaccines in a murine model [2-4]. Here, we present results of two of these immunomodulators, a water-soluble fraction of the cell wall of Mycobacterium sp. (WSF) and a synthetic lipoamide, Avridine (AV), which were tested in bovines included in FMDV oil vaccines. Two different concentrations of inactivated viral antigen were employed and the effect of different concentrations of the adjuvants were studied when added to the lower viral dose. It is shown that the inclusion of these adjuvants in the higher concentration in vaccines formulated with low antigen concentration induced the same antibody levels as those induced by vaccines containing twice the concentration of virus, and no adjuvants, and as a commercial formulation which performed with 100% of protection in the potency test. The IgG isotypes profiles induced in these experimental vaccines differed from those elicited by the commercial and control vaccines. Both IgG1 and IgG2 were augmented by the experimental formulations. These adjuvants, specially the WSF, also enhanced the cellular immune response against the FMDV in antigen driven proliferation assays, thus acting on a broad range of immune mechanisms.

Modulation of the antigen presentation activity in foot and mouth disease virus (FMDV) vaccines by two adjuvants: avridine and a water soluble fraction of Mycobacterium sp.

We have previously demonstrated that the presence of the antigen presenting cells (APC) is critical in the induction and maintenance of the immune response in animals infected or immunized with inactivated FMDV. The use of immunological adjuvants has been repeatedly shown to be essential for the improvement of the immunogenicity in FMDV vaccines. Specifically, we have previously shown that the addition of the synthetic lipoamide Avridine (AVR) or a water soluble fraction of Mycobacterium sp. (WSF) significantly increased the immune response and protection against FMDV challenge. Here, we study the effect of these adjuvants on the induction of APC activity in mice immunized with inactivated FMDV. Both adjuvants were able to induce a long lasting antibody response which correlates with an efficient APC activity. Experiments using sequential cell transfers showed that the presence of the APC activity is not related with the efficiency of keeping free antigen in the vaccinated host. Interestingly, APC from animals immunized with AVR as adjuvant elicited virus neutralizing antibodies, while those APC obtained from donors vaccinated using WSF as adjuvant (or just an oil emulsion) induced anti-FMDV detectable only by ELISA. The analysis of the antibody response to a well studied synthetic peptide raised evidences that indicate that this difference could be explained by a differential presentation of viral B epitopes when different adjuvants were used. These results suggest that the induction of APC should be considered as one of the critical factors in the process of improving the immunogenicity of experimental FMDV vaccines.

Induction of anti foot and mouth disease virus T and B cell responses in cattle immunized with a peptide representing ten amino acids of VP1.

We previously demonstrated that the immunization of cattle with a synthetic peptide representing the amino acid sequence of foot and mouth disease virus (FMDV) type O1 Campos VP1 residues 135-160 (p135-160), containing immunodominant T and B epitopes, was able to induce a strong neutralizing antibody (NA) response. The epitope mapping of p135-160 identified T and B epitopes in the area restricted to amino acid residues 135-144 (Zamorano et al. 1994, Virology 201; 1995, Virology 212). We are now reporting that, although immunization with a synthetic peptide covering amino acids 135-144 (p135-144) failed to elicit an anti-FMDV response, a synthetic peptide representing a tandem duplication of the VP1 epitope 135-144 (p135-144 x 2) was very efficient in inducing a strong NA response in cattle. Both the antibody and T cell responses elicited by p135-144 x 2 were highly specific for the VP1 135-144 sequence since no reactivity was detected against synthetic peptides representing the 140-160 sequence of VP1. Additionally, both responses to B and T epitopes were long lasting in the immunized cattle. These results constitute a good example of the improvement of the immune response by rational handling of precisely identified B and T epitopes. To our knowledge, this is the shortest native amino acid sequence to induce a significant NA response to FMDV in cattle.

Protective immune response to foot-and-mouth disease virus with VP1 expressed in transgenic plants.

It has been reported recently that genes encoding antigens of bacterial and viral pathogens can be expressed in plants in a form in which they retain native immunogenic properties. The structural protein VP1 of foot-and-mouth disease virus (FMDV), which has frequently been shown to contain critical epitopes, has been expressed in different vectors and shown to induce virus-neutralizing antibodies and protection in experimental and natural hosts. Here we report the production of transformed plants (Arabidopsis thaliana) expressing VP1. Mice immunized with leaf plant extracts elicited specific antibody responses to synthetic peptides representing amino acid residues 135 to 160 of VP1, to VP1 itself, and to intact FMDV particles. Additionally, all of the immunized mice were protected against challenge with virulent FMDV. To our knowledge, this is the first study showing protection against a viral disease by immunization with an antigen expressed in a transgenic plant.

Duration of the foot-and-mouth disease virus antibody response in mice is closely related to the presence of antigen-specific presenting cells.

Natural and experimental hosts infected with foot-and-mouth disease virus (FMDV) develop a long-lasting immune response that is closely related to the presence of anti-FMDV antibodies (Ab). We show here that spleen cells from animals which had been infected 3 or more months previously induced an anti-FMDV-Ab response in untreated animals which lasted more than 210 days after cell transfer. Persistence of infectious virus was excluded since virus isolation or detection of the viral genome by PCR in donor splenocytes were consistently negative. The role of antigen presentation (AP) in this phenomenon was studied in vivo by using irradiated splenocytes from virus-sensitized donor mice. Although these irradiated cells were unable to induce anti-FMDV-Ab in normal or irradiated recipient mice, they elicited a strong secondary reaction in FMDV-pre-sensitized recipients. The presence of AP cells (APC) presenting FMDV epitopes (FMDV/APC) was also analysed in mice sensitized to FMDV in different ways. A close correlation between FMDV/APC and the presence of anti-FMDV-Ab was found in infected mice as well as in mice immunized with different doses of inactivated virus, with or without adjuvants. Experiments in vivo and in vitro showed that the APC activity can be specifically blocked with either anti-MHC class II monoclonal antibody or anti-FMDV antiserum, and is dependent on the presence of T cell function. These results strongly suggest that persistent FMDV/APC are responsible for the existence and maintenance of an anti-virus immune response regardless of the immunization method used.

A 10-amino-acid linear sequence of VP1 of foot and mouth disease virus containing B- and T-cell epitopes induces protection in mice.

The area of foot and mouth disease virus (FMDV) comprising residues 140 and 160 of capsid protein VP1 has been used extensively as an immunogen in natural and experimental hosts. A detailed epitope mapping of this region, however, has not been reported. For this purpose a synthetic peptide containing the residues 135 to 160 (p135-160) of VP1 of FMDV O1 Campos was analyzed for its T- and B-cell epitopes. The p135-160 is highly immunogenic, either by itself or coupled to a carrier protein (BSA), elicits a long-lasting neutralizing antibody response in mice, and provides solid protection against virulent challenge. By using a set of synthetic 10mer overlapping peptides, which cover the entire sequence 135-160 of VP1, we have shown that at least four discrete B epitopes are regularly distributed along the peptide. Although immunization with each of the 10mers coupled with BSA as a carrier protein induced peptide-specific antibody responses, individually none of the 10mers was able to induce neutralizing antibodies. However, anti-135-160 antibodies sorted by immunoaffinity chromatography using each of the 10mers revealed the existence of at least four discrete neutralizing sites: one spanning residues 135-144, at least two more between residues 140 and 154, and another in the region 150-160. Moreover, T-cell epitopes were identified, both by antigen-dependent proliferation assays and by adoptive cell transfer. By both methods, a T-cell epitope was located in the area comprising residues 135-144; the cell transfer experiment, which seems to be more sensitive, also identified a second T-cell epitope between residues 150 and 160. Interestingly, when the region 135-144, which contains both B- and T-cell epitopes, was in a tandem repeat configuration it induced a strong neutralizing antibody response in mice and solid protection against the challenge.

Isotype profiles induced in Balb/c mice during foot and mouth disease (FMD) virus infection or immunization with different FMD vaccine formulations.

The IgG isotype response in Balb/c mice infected with FMDV or immunized with different vaccine formulations using inactivated virus particles as antigen was analyzed at various times post-inoculation. For this purpose an ELISA based on polyclonal antibodies for detection and quantification of mouse IgG isotypes with FMD virus (FMDV) specificity was developed. Three immunomodulators, which have been shown to be very effective in inducing strong and long-lasting antibody responses (Bahnemann, Arch. Virol. 1975, 47, 47-56; Polatnik and Bachrach, Appl. Microbiol. 1964, 12, 368-376), were employed to formulate different vaccines using aqueous and oil vehicles: a water-soluble fraction of the cell wall of Mycobacterium sp., a purified extract of lipopolysacharide from Brucella ovis and a synthetic lipoamide, Avridine. Infected animals between 14 and 60 days post-inoculation (d.p.i.) showed responses dominated by IgG2b, followed by IgG1, IgG2a and IgG3, respectively. The IgG3 isotype was the first, together with IgG1, to be elicited during the first 7 days after infection, whereas no IgG3 activity was detected in vaccinated animals at any time. With formulations including immunomodulators, persisting high levels of IgG2b (similar to those of infected animals) were detected until 180 d.p.i., while with conventional vaccines IgG2b responses were detected up to 60 d.p.i. Animals vaccinated with formulations including these immunomodulators presented an augmented resistance to viral challenge at 210 d.p.i. in relation with those immunized with conventional vaccines. The possible relationship of these differences in the isotype response and protection is discussed.

Recognition of B and T cell epitopes by cattle immunized with a synthetic peptide containing the major immunogenic site of VP1 FMDV 01 Campos.

The precise location of B and T cell epitopes have been established in a peptide containing the major immunogenic site (residues 135-160) of FMDV strain 01 Campos (01C) VP1. The peptide (p135-160), administered free or conjugated to bovine serum albumin, induced complete protection in guinea pigs and a strong neutralizing antibody (NAb) response in cattle. Using a set of partially overlapping peptides it was shown that although several B cell epitopes were distributed along the p135-160, the residues responsible for the induction of NAb were restricted to the amino acids 135-144. In addition, bovines immunized with the p135-160 showed a strong proliferative response to this peptide but a very poor response against purified virus. Two T-epitopes were located in the 135-160 sequence: one on the region 135-144 and the other in the region 150-160. The immunodominance of these two T cell epitopes was confirmed in cattle immunized with inactivated virus vaccines.