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Veterinary Immunology and Immunopathology 140 (2011) 291296

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Veterinary Immunology and Immunopathology

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Short communication

Immune responses to hemagglutinin-neuraminidase protein of peste des petits ruminants virus expressed in transgenic peanut plants in sheep
Abha Khandelwal a,1 , Gourapura. J. Renukaradhya a,b,2 , Malleshappa Rajasekhar b , G. Lakshmi Sita a , Melkote S. Shaila a,
a b

Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India Project Directorate on Animal Disease Monitoring and Surveillance, Indian Council of Agricultural Research, Hebbal, Bangalore 560024, India

a r t i c l e

i n f o

a b s t r a c t
Peste des petits ruminants (PPR) is an acute, highly contagious disease of small ruminants caused by a morbillivirus, Peste des petits ruminants virus (PPRV). The disease is prevalent in equatorial Africa, the Middle East, and the Indian subcontinent. A live attenuated vaccine is in use in some of the countries and has been shown to provide protection for at least three years against PPR. However, the live attenuated vaccine is not robust in terms of thermotolerance. As a step towards development of a heat stable subunit vaccine, we have expressed a hemagglutinin-neuraminidase (HN) protein of PPRV in peanut plants (Arachis hypogea) in a biologically active form, possessing neuraminidase activity. Importantly, HN protein expressed in peanut plants retained its immunodominant epitopes in their natural conformation. The immunogenicity of the plant derived HN protein was analyzed in sheep upon oral immunization. Virus neutralizing antibody responses were elicited upon oral immunization of sheep in the absence of any mucosal adjuvant. In addition, anti-PPRVHN specic cell-mediated immune responses were also detected in mucosally immunized sheep. 2010 Elsevier B.V. All rights reserved.

Article history: Received 18 July 2010 Received in revised form 28 November 2010 Accepted 8 December 2010 Key words: Peste des petits ruminants virus Hemagglutinin-neuraminidase Transgenic peanut Oral immunogenicity

1. Introduction Peste des petits ruminants (PPR) is a severe, rapidly spreading disease of small ruminants. It is characterized by sudden onset of depression, fever, serous discharges from eyes and nose, sores in mouth, disturbed breathing and cough, foul smelling diarrhea and death. PPR is endemic in parts of sub-Saharan Africa, the Middle East, and Asia, and it was rampant in sub-Saharan Africa, the Arabian Penin-

Corresponding author. Tel.: +91 80 22932702; fax: +91 80 23602697. E-mail address: shaila@mcbl.iisc.ernet.in (M.S. Shaila). 1 Present address: Monsanto Company, 700 Chestereld Parkway West, St. Louis, MO 63017, USA. 2 Present address: Food Animal Health Research Program, Ohio Agricultural Research and Development Center, Department of Veterinary Preventive Medicine, The Ohio State University, Wooster, OH 44691, USA. 0165-2427/$ see front matter 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2010.12.007

sula and the entire Indian subcontinent (Roeder, 1999). An outbreak of PPR has also been reported in the Ngari region of western Tibet, Peoples Republic of China (Wang et al., 2009). PPRV is an important member of the genus Morbillivirus in the Paramyxoviridae family. PPRV is antigenically closely related to rinderpest virus (RPV) (Anderson and McKay, 1994). Although the existing live attenuated vaccine for rinderpest disease has been shown to protect against PPR, the use of rinderpest vaccine to control PPR is now contradicted since antibodies to rinderpest virus compromise serosurveillance for rinderpest, and thereby the Global Rinderpest Eradication programme (Anderson and McKay, 1994). A traditional vaccine based on attenuated live virus (Nigerian strain) has been shown to provide protection at least for three years (Diallo et al., 1989). However, the available vaccine requires maintenance of cold chain from production to nal immunization. Further, the


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efcacy of vaccine can only be tested if vaccination provides a way to differentiate between vaccinated and infected or recovered animals and this cannot be achieved by a conventional vaccine, since one cannot distinguish vaccine induced antibodies from those produced in response to viral infection. For coordinated eradication of PPR, a recombinant subunit vaccine, which is heat stable and cost effective, is needed. Cost-effective bioproduction of recombinant proteins in large scale can be achieved through development of transgenic plants. If the protective antigen is expressed in edible parts of the plant, it can be consumed orally to provide immunization (Tacket et al., 1998, 2000; Wigdorovitz et al., 1999). This food based oral immunization has several advantages over parenteral immunization, which include the induction of both mucosal and systemic immune response (McGhee et al., 1992). Specic humoral defense is provided by both serum and secretory antibodies, predominantly of the IgA isotype, which are selectively transported by a receptor-mediated mechanism into external secretions providing the rst line of defense against the pathogens that invade or colonize the mucosal surfaces. Mucosal immunity has been gaining increased attention for vaccine development and immune therapeutics. Mucosal surfaces cover the largest surface area in the body and almost 80% of total immune cells are present in the mucosaassociated lymphoid tissues and at mucosal sites, guarding against the entry of pathogens into the body (Singh et al., 2001). Therefore, induction of mucosal immunity is likely to make an important contribution towards protection. The plant expressing candidate antigen for oral immunization can be used as a fodder. Based on this criterion, Arachis hypogea (Beardsley et al., 1983) (peanut) plant was selected as a plant of choice. The HN protein of PPRV has earlier been shown to possess hemagglutination activity (Seth and Shaila, 2001), and the same protein exhibits neuraminidase activity also (Devireddy et al., 1998). Puried HN protein has been shown to confer protection against challenge with virulent lapinized RPV in a rabbit model (Devireddy et al., 1998). Puried recombinant extracellular baculovirus carrying HN protein of PPRV has also been shown to elicit both humoral and cell-mediated immune responses in goats (Sinnathamby et al., 2001). We had earlier expressed the HN protein in transgenic pigeon pea which was shown to possess neuraminidase activity (Prasad et al., 2004). In the present work, we show that transgenic peanut (Arachis hypogea L.) plants express the HN protein, which is enzymatically active and antigenic. We have demonstrated induction of both neutralizing antibody response as well as cell-mediated immune responses in orally immunized sheep. 2. Materials and methods 2.1. Construction of binary vector A pGemT recombinant vector containing the full length HN gene from an Indian isolate of the virus PPRV/Ind/AP94/1 was earlier constructed in the laboratory (Shyam et al., unpublished). The HN gene released from

pGemHN DNA as PstI/NcoI fragment was end lled and subcloned into the binary vector pBI121 replacing the reporter gene uidA. Thus, in the recombinant binary vector pBI HN, the 1.9 kb HN gene is under the control of constitutively expressed CaMV 35S promoter. pBI HN was mobilized into Agrobacterium tumefaciens (GV3 101) using the procedure described previously (Holsters et al., 1978). 2.2. Genetic transformation of peanut Transgenic peanut plants generated using pBI121 served as the control and termed as vector-transformed peanut plants. Transgenic peanut plants expressing hemagglutinin-neuraminidase protein were generated via Agrobacterium-mediated transformation (Venkatachalam et al., 2000) using shoot apices with one cotyledon intact as the explants, and direct regeneration of the explants was obtained following the method described previously (McKently et al., 1995). 2.3. PCR analysis Presence of the transgene HN was conrmed by PCR amplication using gene specic primer sequences. Plant DNA for PCR analysis was isolated as described previously (Wang et al., 1993). The HN specic primers correspond to: nucleotides 124 (5 -ACAATGGCTGCACAAAGGGAAAGG3 ), and nucleotides 957977 (5 -GTTCCAAAGAG AGAGCCTCTT-3 ), which amplify a length of 957 bp DNA specic to HN gene. 2.4. Recombinant proteins Recombinant hemagglutinin (H) protein of Rinderpest virus (RPV) expressed in insect cells secreted into the medium (SecH) (Naik and Shaila, 1997) was used as capture antigen for the detection of HN specic antibodies. Recombinant nucleocapsid protein of RPV expressed in E. coli was puried by ultra centrifugation on a CsCl gradient as described earlier (Mitra-Kaushik et al., 2001). The full length M gene of RPV (RBOK) in pBluesript KS+ vector (Kindly provided by Dr. M. Baron, Institute for Animal Health, Pirbright, UK) was subcloned into pRSET expression vector and expressed in E. coli BL21 (DE3) (Shaji and Shaila, unpublished). 2.5. Western blot analysis The expression of PPRV-HN protein in transgenic leaves was evaluated by Western Blot analysis using polyclonal PPRV-HN antibody (Devireddy et al., 1998). Five hundred mg of leaf material was homogenized in the presence of liquid nitrogen in 3 ml of cold PBS containing 0.1% Tween20 (PBST) and 1 mM Phenylmethylsulfonyl uoride (PMSF). The leaf debris were removed by centrifugation at 2000 g for 15 min at 4 C and the supernatant was further centrifuged at 2000 g for 30 min at 4 C. The supernatant was concentrated by lyophilization and boiled for 10 min in SDS sample buffer (Sambrook et al., 1989) prior to analysis by 10% SDS-PAGE gel. Protein bands were transferred to nitrocellulose membrane by electroblotting. After blocking with

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Fig. 1. Functional PPRV-HN protein was expressed on transformed peanut plants. (A) The expression of HN protein in transformed peanut plants was analyzed by Western blot analysis using PPRV-HN specic polyclonal antibody. Lanes 17 protein extracts from individual PPRV-HN transformed plant designated as DGHN2/4, DGHN2/5, DGHN2/9, DGHN2/11, DGHN2/18, DGHN2/24, DGHN2/33, lane 8: protein extract from non-transformed peanut plant. (B and C) Neuraminidase activity in peanut plant-derived HN protein in T0 (B) and T1 (C) generations was analyzed by a standard neuraminidase assay. GM represents the leaf extract from vector-transformed peanut plant and NGn represents the leaf extract from non-transformed peanut plant. Specic activity is dened as nmoles of sialic acid released per hour per mg of protein at 37 C.

3% gelatin in PBST, the blot was incubated with pre-cleared serum which was obtained by incubating non-transformed protein extract with rabbit anti-HN (1:5000) in PBS at 4 C overnight. After washing, the blot was incubated with anti rabbit-ALP conjugate at RT and developed with BCIP/NBT substrate. 2.6. Neuraminidase assay Total protein from leaves of transformed and vectortransformed peanut plants was isolated using the method previously described (McGarvey et al., 1995). The release of product, sialic acid was estimated using a colorimetric method as described previously (Aymard-Henry et al., 1973). 2.7. Animals and oral immunization Sheep (local breed) aged between 6 months to 2 years maintained at a farm, Veterinary College, Bangalore were used for the oral immunization. Animals were maintained and used in experiments in accordance with the standards of the Institutional Laboratory Animal Care and Use Committee, Indian Institute of Science. Five animals were fed with 5 g of fresh leaves per animal from non-transformed plants for ve-weeks at weekly intervals, and 5 animals were fed with 5 g of fresh peanut leaves expressing HN protein. Animals were given with ad libitum of feed and water at all times. 2.8. Cells and viruses Vero cells were obtained from National center for cell Science, Pune, India, and were maintained in MEM supplemented with 5% fetal calf serum (Gibco-BRL, USA). A tissue

culture adapted vaccine strain of RPV (RBOK) (Plowright and Ferris, 1957) was obtained from the Institute of Animal Health and Veterinary Biologicals, Bangalore, India, and vaccine strain of PPRV (Nig 75/1) was provided by Dr. A. Diallo, CIRAD-EMVT, France. To prepare infected cell lysate, RPV or PPRV infected Vero cells showing 70% CPE (approximately 2448 h post-infection) were resuspended in PBS at 1 107 cells per ml, and then cells were lysed and stored at 20 C. 2.9. Virus neutralization test Serum samples collected at various time points postimmunization were tested for the presence of both homologous and cross-neutralizing antibodies in atbottom 96-well plates as described previously (Barrett et al., 1989). Vaccine strain of PPRV (Nig 75/1) and vaccine strain of RPV (RBOK strain) were grown on Vero cells and titrated employing TCID50 method (Reed and Muench, 1938). 2.10. Proliferation of PBMC from immunized sheep Sheep were bled through jugular vein puncture at specied times and collected in EDTA. The blood was diluted 1:2 in sterile PBS and subjected to Ficoll-Hypaque density gradient centrifugation at 3000 rpm for 30 min. The buffy coat was diluted with PBS and centrifuged. After a wash, cells were resuspended in RPMI 1640 supplemented with 10% FBS. In vitro proliferation assay was setup with 2 105 lymphocytes per well in the presence of difference concentrations of PPRV antigens in a nal volume of 200 l and incubated at 37 C for 5 days. The 3 Hthymidine incorporation was measured after pulsing the cells with one microCurie {3H}-thymidine (specic activ-


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Table 1 In vitro analysis of neutralization of PPRV infectivity by serum collected from sheep after oral immunization with PPRV-HN transgenic or control leaves. Neutralization titer Source of antigen Transgenic HN leaves Sheep no. 2.1 2.2 2.3 2.4 2.5 1.1 1.2 1.3 1.4 1.5 0 DPI 0 0 0 0 0 0 0 10 0 0 7 DPI 1280 640 80 640 160 0 10 20 0 0 14 DPI 1280 1280 80 640 320 0 20 0 0 0 21 DPI 1280 320 160 1280 1280 0 0 0 0 0 28 DPI 1280 80 320 320 320 0 0 0 40 0 35 DPI 1280 80 80 320 160 0 0 0 0 0

Control leaves

ity 6500 mCi/mmol; Amersham, UK) for 16 h and harvested onto glass ber lters, using a cell harvester. The radioactivity in the lters after thorough washing was measured in a Rachbeta liquid scintillation spectrometer. 3. Results and discussion To achieve genetic transformation of peanut plants, Agrobacterium tumefaciens (GV3 101) containing recombinant plasmid pBI HN was used in our study. Primarily transformed plants were analyzed for the presence of transgene by gene specic PCR and the integration of the HN gene into the plant genomic DNA by Southern hybridization (data not shown). The lines having single integration were further analyzed for the expression of HN protein by SDS-PAGE separation of leaf extracts and immunoblotting with anti-HN antibodies. The protein extracts from the transformed plants showed the expression of approximately 72 kDa PPRV-HN specic protein, and a similar extract from the vector control plant did not show any specic signal (Fig. 1A). The neuraminidase activity associated with HN protein was tested with extracts from six plant lines expressing HN protein. The presence of enzymatically active PPRV-HN was detected in primary transformed lines (T0 generation) (Fig. 1B) and in the progeny (T1 generation) (Fig. 1C), suggesting a stable integration and inheritance of the transgene in at least 50% of the transformed plants. In one line (DGHN2/11), neuraminidase activity was detected until T2 generation (data not shown). Further, the antigenic authenticity of the peanut-derived HN protein was tested for its reactivity using different PRRV-HN conformation specic mAb (Renukaradhya et al., 2002). The protein extract from transgenic lines reacted specically with all the four mAb generated against different antigenic epitopes of HN protein, implying that the HN protein expressed in the peanut plant is conformationally similar to PPRV-HN made in the virus-infected cells or HN protein on the virus envelope (Fig. 2). Sheep fed with transgenic leaves expressing PPRV-HN elicited HN-specic antibodies in serum, and the specicity of those antibodies was tested positive by immunohistochemical staining of the virus-infected cells (data not shown). Also the sera of experimental animals reacted specically with the Vero cells infected PPRV antigens and not with the control uninfected cell antigens, such reactiv-

ity was also absent in preimmune serum (data not shown). In addition, high levels of HN specic neutralizing antibodies were detected in HN transgenic plant fed sheep, which were absent in respective control sera (Table 1). This was observed just one-week after feeding the transgenic plant, even in the absence of any mucosal adjuvant. Interestingly, the generated HN-specic antibodies in serum also crossneutralized the in vitro infectivity of RPV to Vero cells, albeit to a lesser extent. All these data suggests the anti-PPRV-HN specic immune response in sheep orally fed with peanutderived HN protein. It is known that Langerhan cells of the human and rat buccal mucosa process and present antigens. Trans-epithelial delivery of replicating as well as non-replicating measles virus antigens through buccal mucosa elicited satisfactory CTL response (Etchart et al., 1997, 2001). The specic CTL response induced by buccal immunization to measles virus N protein protected mice against a lethal intracerebral challenge to a related canine distemper virus, which shares its unique CTL epitopes. Therefore, it is conceivable that plant expressed antigens when used orally elicit cell-mediated as well as humoral immune responses at the buccal mucosa. At the end of ve-oral feedings, PBMC isolated from peripheral blood of experimental and control sheep were restimulated in vitro using different concentrations of PPRV

Fig. 2. Peanut-derived PPRV-HN protein retained its antigenic epitopes. Hemagglutinin-neuraminidase protein present in the peanut plantextract was analyzed for its antigenic authenticity by using PPRV-HN specic monoclonal antibodies by ELISA. Empty bars represent protein extract from HN transgenic peanut plant and lled bars represent vectortransformed peanut plant.

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antigens. The results indicated that lymphocytes present in blood from sheep that received transgenic peanut plants proliferated substantially upon restimulation with PPRV antigens (Fig. 3A, C and E). This specic proliferation was absent in PBMC of sheep that received control nontransformed leaves (Fig. 3B, D and F). As a control, the PBMC were also stimulated with uninfected Vero cell extract and Matrix protein of RPV, as expected non-specic proliferation of lymphocytes was not detected. This suggested that PPRV-HN specic prolonged cell-mediated immune responses in sheep orally immunized with HN transgenic peanut plant leaves was present. To determine the PPRV-HN specic memory immune responses in orally immunized sheep, 10-weeks after the last oral booster, three of the experimental sheep were injected with a sub-immunogenic dose of PPRV vaccine (PPRV Nig75/1) parenterally. PBMC isolated from peripheral blood was collected on 0, 2, and 4 weeks postvaccination, and were restimulated in vitro with PPRV

antigens. Interestingly, we detected enhanced lymphocytes proliferation in two of the three vaccine received sheep, at both 2 and 4 weeks post-vaccination (data not shown). These results suggested the presence of HN specic memory immune response in sheep to oral immunization with transgenic plants. Peanut plant expressed HN protein of PPRV possessed neuraminidase activity when tested up to T2 generation. In recent times, the efcacies of plant derived antigens have been tested in target species, such as in humans. In two independent studies, human volunteers fed either with transgenic potato expressing heat labile enterotoxin (LT-B) of E. coli (Tacket et al., 1998), and potato tubers expressing recombinant Norwalk virus capsid protein (Tacket et al., 2000) elicited specic immune responses. Consistent with that, our study demonstrated the efcacy of transgenic peanut plant by oral immunization to a morbillivirus protein in sheep in the absence of any mucosal adjuvant.

Fig. 3. PPRV-HN specic lymphoproliferative response in sheep orally immunized with HN transformed peanut plants. PBMC isolated from individual sheep fed orally with HN (A, C, and E) or vector (B, D, and F) transformed peanut plants post-ve weeks of oral immunization were restimulated using PPRV-HN antigens at different concentrations and then subjected to a standard lymphocytes proliferation assay. The lled square represents antigens from PPRV infected cells as in vitro re-stimulating antigens and empty squares represent antigens from uninfected cells.


A. Khandelwal et al. / Veterinary Immunology and Immunopathology 140 (2011) 291296 McKently, A.H., Moore, G.A., Doostdar, H., Niedz, R.P., 1995. Agrobacterium-mediated transformation of peanut (Arachis hypogaea L.) embryo axes and the development of transgenic plants. Plant Cell Rep. 14, 699703. Mitra-Kaushik, S., Nayak, R., Shaila, M.S., 2001. Identication of a cytotoxic T-cell epitope on the recombinant nucleocapsid proteins of Rinderpest and Peste des petits ruminants viruses presented as assembled nucleocapsids. Virology 279, 210220. Naik, S., Shaila, M.S., 1997. Characterization of membrane-bound and membrane anchor-less forms of hemagglutinin glycoprotein of Rinderpest virus expressed by baculovirus recombinants. Virus Genes 14, 95104. Plowright, W., Ferris, R.D., 1957. Cytopathogenicity of rinderpest virus in tissue culture. Nature 179, 316. Prasad, V., Satyavathi, V.V., Sanjaya, Valli, K.M., Khandelwal, A., Shaila, S.M., Lakshmi Sita, G., 2004. Expression of biologically active hemagglitininneuraminidase protein of Peste des petits ruminants virus in transgenic pigeon pea (Cajanus cajan (L) Mill sp.). Plant Sci. 166, 199205. Reed, L.J., Muench, L., 1938. A simple method of estimating fty per cent endpoints. Am. J. Hyg. 27 (3), 493497. Renukaradhya, G.J., Sinnathamby, G., Seth, S., Rajasekhar, M., Shaila, M.S., 2002. Mapping of B-cell epitopic sites and delineation of functional domains on the hemagglutinin-neuraminidase protein of peste des petits ruminants virus. Virus Res. 90, 171185. Roeder, P.L., 1999. Recognizing Peste des petits ruminants: a eld manual. FAO Animal Health Manual 5, 28. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual, second edition. Cold Spring Harbor Laboratory Press. Seth, S., Shaila, M.S., 2001. The hemagglutinin-neuraminidase protein of peste des petits ruminants virus is biologically active when transiently expressed in mammalian cells. Virus Res. 75, 169177. Singh, M., Briones, M., OHagan, D.T., 2001. A novel bioadhesive intranasal delivery system for inactivated inuenza vaccines. J. Control. Release 70, 267276. Sinnathamby, G., Renukaradhya, G.J., Rajasekhar, M., Nayak, R., Shaila, M.S., 2001. Immune responses in goats to recombinant hemagglutinin-neuraminidase glycoprotein of Peste des petits ruminants virus: identication of a T cell determinant. Vaccine 19, 48164823. Tacket, C.O., Mason, H.S., Losonsky, G., Clements, J.D., Levine, M.M., Arntzen, C.J., 1998. Immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato. Nat. Med. 4, 607609. Tacket, C.O., Mason, H.S., Losonsky, G., Estes, M.K., Levine, M.M., Arntzen, C.J., 2000. Human immune responses to a novel norwalk virus vaccine delivered in transgenic potatoes. J. Infect. Dis. 182, 302305. Venkatachalam, P., Geetha, N., Khandelwal, A., Shaila, M.S., Lakshmi Sita, G., 2000. Agrobacterium-mediated genetic transformation and regeneration of transgenic plants from Cotyledon explants of groundnut (Arachis hypogaea L.) via somatic embryogenesis. Curr. Sci. 78, 11301136. Wang, H., Qi, M., Cutler, A.J., 1993. A simple method of preparing plant samples for PCR. Nucleic Acids Res. 21, 41534154. Wang, Z., Bao, J., Wu, X., Liu, Y., Li, L., Liu, C., Suo, L., Xie, Z., Zhao, W., Zhang, W., Yang, N., Li, J., Wang, S., Wang, J., 2009. Peste des petits ruminants virus in Tibet, China. Emerg. Infect Dis. 15, 299301. Wigdorovitz, A., Carrillo, C., Dus Santos, M.J., Trono, K., Peralta, A., Gomez, M.C., Rios, R.D., Franzone, P.M., Sadir, A.M., Escribano, J.M., Borca, M.V., 1999. 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. Virology 255, 347353.

In summary, our data has demonstrated the feasibility of plant-derived PPRV-HN protein to be used as a source of edible vaccine. Notably, our results demonstrated the enhanced cell-mediated secondary immune responses in sheep orally immunized with transgenic peanut leaves carrying HN protein. Thus, oral mucosal immunization using HN protein of PPRV could prove to be advantageous in developing countries where conventional vaccine may be unstable due to difculties in maintaining proper coldchain. Further, experiments in target animals are necessary to test protection in edible PPRV-HN orally immunized animals against PPRV challenge. Conict of interest The authors declare no nancial or commercial conict of interest. References
Anderson, J., McKay, J.A., 1994. The detection of antibodies against peste des petits ruminants virus in cattle, sheep and goats and the possible implications to rinderpest control programmes. Epidemiol. Infect. 112, 225231. Aymard-Henry, M., Coleman, M.T., Dowdle, W.R., Laver, W.G., Schild, G.C., Webster, R.G., 1973. Inuenzavirus neuraminidase and neuraminidase-inhibition test procedures. Bull. World Health Organ. 48, 199202. Barrett, T., Belsham, G.J., Subbarao, S.M., Evans, S.A., 1989. Immunization with a vaccinia recombinant expressing the F protein protects rabbits from challenge with a lethal dose of rinderpest virus. Virology 170, 1118. Beardsley, T.R., Pierschbacher, M., Wetzel, G.D., Hays, E.F., 1983. Induction of T-cell maturation by a cloned line of thymic epithelium (TEPI). Proc. Natl. Acad. Sci. U.S.A. 80, 60056009. Devireddy, L.R., Raghavan, R., Ramachandran, S., Subbarao, S.M., 1998. Protection of rabbits against lapinized rinderpest virus with puried envelope glycoproteins of peste-des-petits-ruminants and rinderpest viruses. Acta Virol. 42, 299306. Diallo, A., Taylor, W.P., Lefevre, P.C., Provost, A., 1989. Attenuation of a strain of rinderpest virus: potential homologous live vaccine. Rev. Elev. Med. Vet. Pays Trop. 42, 311319. Etchart, N., Buckland, R., Liu, M.A., Wild, T.F., Kaiserlian, D., 1997. Class I-restricted CTL induction by mucosal immunization with naked DNA encoding measles virus haemagglutinin. J. Gen. Virol. 78 (Pt 7), 15771580. Etchart, N., Desmoulins, P.O., Chemin, K., Maliszewski, C., Dubois, B., Wild, F., Kaiserlian, D., 2001. Dendritic cells recruitment and in vivo priming of CD8+ CTL induced by a single topical or transepithelial immunization via the buccal mucosa with measles virus nucleoprotein. J. Immunol. 167, 384391. Holsters, M., de Waele, D., Depicker, A., Messens, E., van Montagu, M., Schell, J., 1978. Transfection and transformation of Agrobacterium tumefaciens. Mol. Gen. Genet. 163, 181187. McGarvey, P.B., Hammond, J., Dienelt, M.M., Hooper, D.C., Fu, Z.F., Dietzschold, B., Koprowski, H., Michaels, F.H., 1995. Expression of the rabies virus glycoprotein in transgenic tomatoes. Biotechnology (NY) 13, 14841487. McGhee, J.R., Mestecky, J., Dertzbaugh, M.T., Eldridge, J.H., Hirasawa, M., Kiyono, H., 1992. The mucosal immune system: from fundamental concepts to vaccine development. Vaccine 10, 7588.