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Immunogenicity of a single intranasal dose of IBD vaccine based on Poly-Lactide-Co-Glycolide (PLG) microparticles in broiler chickens Dandapat SS.*, Rajan , Banerjee D., Dash B.B.1, Kataria J.M., Yadav M.P. Director's Laboratory Indian Veterinary Research Institute, Izatnagar-243 122 (U.P.) *Corresponding author
1Division of Avian Diseases
Abstract In this study, immunogenicity of the microencapsulated IBD virus (intermediate strain) was investigated after intranasal delivery of the viral antigens incorporated in PLG microparticles. Although the size of virus entrapped microparticles ranged from 1–10 µm, majority were of less than 2 µm and they contained viral antigens to the extent of 1.4 to 1.7% by weight of microspheres. A single intranasal dose (75 µg) of the experimental microsphere vaccine was able to induce both mucosal and systemic immunity in poultry. The immunized chicks showed significantly higher levels of virus specific IgG with the peak titre of (log10) 3.77 at the 3rd wk post-immunization (PI) and also protective levels of neutralizing antibodies (log10 NI 2) up to 6-week PI, which were comparable to that of the conventionally vaccinated chicks. In the tracheal washings, considerably higher levels of virus specific IgA were detected with the peak titre 40 at the 3rd wk PI, which gradually decreased to 24 at 6th wk PI. Intranasal delivery of microspheres induced lymphocyte blastogenic response with peak stimulation index of (1.360±0.031) at the 3rd wk PI, which was significantly (p<0.05) higher than that of the unvaccinated control group. The immunized chicks also afforded resistance against challenge infection with virulent IBD virus, which was comparable to the conventional vaccine. Top | Mucosal immunization strategies are being considered to be more appropriate than parenteral injections of vaccine antigens for better protective immunity because most of the infectious agents enter the body and exert their pathophysiological effects through invasion of mucosal surfaces. Although parenteral immunization is effective against systemic infections, it cannot induce local IgA antibody response, which is essential to protect the mucosal surfaces against foreign agents (Mestecky and McGhee, 1987). Mucosal associated lymphoid tissues (MALT) have been considered to be the largest immunological organ of the body and antigenic stimulation of MALT can induce humoral and cell mediated immunity at both local and systemic sites (McGhee et al., 1992). The main function of MALT is to protect the mucous membranes against colonization and invasion by the potentially dangerous microbes encountered as the mucosal tissues act as first line of defence against invasion by such microorganisms (Tomasi and Bienenstock, 1968). Nasopharyngeal associated lymphoid tissues (NALT) have a lymphoepithelium like that in intestinal mucosae and contain M cells for selective antigen uptake (Spit et al., 1989). |
In recent years, microencapsulation of vaccine antigens has received considerable attention due to its potential for protecting the antigens against adverse microenvironment in the mucosal milieu and for controlled release of antigens (O'Hagan, et al., 1993). Biodegradable microspheres composed of poly (lactide-co-glycolide) (PLG) have been shown to be an effective vehicle for the delivery of antigen and adjuvant for potentiating response to antigen when administered via mucosal or systemic route (Morris et al., 1994; Jones et al., 1996; O'Hagan, 1997). Unlike soluble antigens, mucosally administered antigens entrapped in PLG microparticles can induce good local and systemic antibody response (Eldridge et al. GG, 1991) as well as cell mediated immunity (Partidos et al., 1996; O'Hagan, 1997). |
Infectious bursal disease (IBD) is an acute, highly contagious, immunosuppressive viral disease of young chicken caused by an Avibirnavirus (Dobos et al., 1979). Infected birds develop immunodeficiency as the virus has affinity to replicate in the bursa of Fabricius. Immuno-suppression increases the susceptibility to other pathogens and is also responsible for vaccination failure particularly against Ranikhet disease (Allan et al., 1972). The economic importance of both clinical and sub clinical diseases warrants the search for and the use of efficient vaccines. Besides its economic importance, IBD serves as a model of mucosal infection. In order to assess the efficacy of mucosal immunization, in the present study the virus specific immune responses have been evaluated after intranasal immunization of the broiler chicks with microencapsulated IBD virus incorporated in PLG microspheres. |
Top Material and methods Embryonated chicken eggs Seven-day-old embryonated chicken eggs (ECE) procured from the Hatchery Unit of Central Avian Research Institute (CARI), Izatnagar were further incubated at 37°C for use in various experiments. Experimental chicks Apparently healthy day-old broiler chicks were procured from CARI, Izatnagar and reared under standard managemental practices with proper hygienic condition. The chicks were provided with balanced ration (obtained from CARI) and clean drinking water ad libitum during the entire period of experiment, after obtaining approval of the animal ethics committee. Preparation of viral antigens The cell culture adapted IBD vaccine virus (Georgia strain) at the 3rd passage level, obtained from the Virology Laboratory of the Division of Avian Diseases, IVRI, Izatnagar was propagated further in primary chicken embryo fibroblast (CEF) culture for bulk preparation. The virus infected cell culture fluid was harvested by three successive cycles of freezing and thawing and clarified by centrifugation at 10,000 rpm for 30 min at 4°C. The virus was concentrated by direct pelleting through high speed centrifugation at 1,00,000 g for 2 h using Sorval Pro-80 ultracentrifuge machine. The pelleted virus was finally resuspended in 2ml of Tris-NaCl-EDTA (TNE) buffer (pH 7.2) and the total protein concentration was determined as per method of Lowry et al. (1951). Titration of virus Serial ten-fold dilutions (10−1 to 10−6) of IBD virus were prepared and added into the 96-well tissue culture plate (100 µl/well) taking 5 wells for each dilution. Then, 100 µl of CEF cell suspension having a concentration of 2 x 106 cells/ml in growth medium was added to each well. The plate was incubated at 37°C in 5% CO2 incubator for 3–5 days, which was observed daily for the development of CPE. Fifty percent tissue culture infectivity dose (TCID50) was calculated as per the method described by Reed and Muench (1938). Preparation of virus entrapped PLG microspheres Microspheres containing IBD virus was prepared by an emulsion based solvent evaporation technique as described by Greenway et al. (1998) with some modifications. Briefly, the pelleted IBD virus suspension was emulsified with PLG solution at 10,000 rpm for 6 min. using homogenizer (PRO-260, HAVARD Apparatus, UK) to produce a water-in-oil emulsion. Then, 2% (w/v) aqueous solution of polyvinyl alcohol (PVA) was added and further homogenized to produce water-in-oil-in-water (w/o/w) double emulsion. Subsequently, the emulsion was stirred overnight at 4°C after adding sufficient distilled water to allow solvent evaporation and microparticle formation. Finally, the microspheres were pelleted by centrifugation at 8,000 rpm for 10 min and washed once with 0.1% PVA and then twice with distilled water. The microspheres with entrapped IBD virus were lyophilized and stored at 4°C for further use. Similarly, empty microspheres (without any viral antigen) were also prepared for use as control. Characterization of microspheres The size and morphology of the microspheres were determined by scanning electron microscopy (SEM) as per O'Hagan et al. (1994). Briefly, a small pinch of the lyophilized microspheres were coated on the metallic slub by ion sputtering or gold coating and then observed under different magnifications using Jeol JSM 840 x Scanning electron microscope. Viral antigen load in the microspheres was determined by measuring the protein content in the hydrolysate of a known quantity of microspheres by using bicinchoninic acid (BCA) protein assay kit (Sigma chemicals, USA) as per the method described by Guiterro et al. (2002). Experimental vaccination in chicks A total of one hundred and fifty day-old chicks (synthetic strain of broilers) were randomly divided into 3 groups of 50 chicks in each and immunized at 10th day of age. The chicks of group-I (MS) were vaccinated with microencapsulated IBD vaccine formulation. The appropriate quantity of microspheres was suspended in PBS, so that in 0.1 ml inoculum around 75 µg viral antigens could be inoculated per chick. The chicks of group-II (CV) were vaccinated with commercial IBD and RDF vaccines through occulonasal route with one booster dose of IBD vaccine after 3 weeks. The group-III (C) chicks were kept as unvaccinated control, which were sham-immunized intranasally with the empty microspheres. Evaluation of immune responses induced by the microencapsulated IBD virus Assessment of humoral immune responses
Serum samples were collected randomly from five chicks of each group at weekly intervals up to 6 weeks post immunization (PI). The virus-specific antibody levels in the pooled sera of immunized chicks were measured by indirect ELISA and serum neutralization test (SNT). The IgA levels in the tracheal washings were measured by indirect ELISA.
Virus specific IgG levels by indirect ELISA
The virus specific IgG antibody levels were measured by indirect ELISA as per Collins et al. (1993). The ELISA plates (TPP, France) were first coated with the purified IBD virus (2 µg/ well in 50 µl) and then 50 µl of serial two-fold dilutions of sera samples in PBS were added in triplicate wells. The rabbit anti-chicken IgG-HRPO conjugate (50 µl/ well) at the dilution of 1:20000 was used for detection of IgG in the serum samples. Absorbance was read at 492 nm using Multiskan-Ex microplate reader (Thermo Labsystem). The positive cut off O.D. value was taken as 0.1 above the mean O.D. of negative serum. Assay for virus specific neutralizing antibody
Virus specific neutralizing antibody levels in the serum of immunized chicks were determined by SNT in CEF culture as per the standard method using constant serum and decreasing concentration of virus. The 50% end point in each case was determined by the method of Reed and Muench (1938). The difference between 50% end point titers of virus (control) and virus+serum mixture was taken as the Neutralization Index (NI). Assessment of IgA antibody levels in tracheal washings
Tracheal secretions were collected aseptically as per the method described by Hawkes et al. (1983) from three randomly selected chicks of each group. The IgA antibody levels in the tracheal secretions of the immunized chicks were assessed by indirect ELISA as per the method described by Mukiibi-Muka and Jones (1999). Briefly, the ELISA plates were coated with purified IBD viral antigen (2 µg/ well in 50 µl) and serial two-fold dilutions of tracheal washings were added (50 µl/well) and then incubated at 37°C for 2 h. Then, goat anti-chicken IgA-HRPO conjugate (Serotec, U.K.) at the dilution of 1:20,000 was used for detection of IgA antibody. The positive cut off O.D. value was taken as 0.1 above the mean O.D. of negative serum.
Assessment of cell mediated immune response
The cell mediated immunity was evaluated by in vitro lymphoproliferation assay using MTT colorimetric method as described by Mosmann (1983). Briefly, the peripheral blood mononuclear cells (PBMC) were separated by using Histopaque-1077 (Sigma, USA). The cell suspension (2 × 106 cells/ml) was added (100 µl/well) to 96-well flat bottom tissue culture plate (TPP, France). Then, 100 µl of RPMI-1640 growth medium containing either Con A @ 20 µg/ ml (positive control) or antigen @ 20 µg/ml was added in triplicate wells. After 96 h of incubation, 20 µl of MTT solution (5 mg/ ml in PBS) was added to each well and further incubated for 4 h. Finally, dimethyl sulfoxide (DMSO) was added (150 µl/well) in order to dissolve formazan crystals. The intensity of colour development was measured by taking absorbance (OD) at 550 nm and the mean stimulation indices for each group were calculated.
Resistance to challenge infection with virulent IBD virus At the end of 6th wk PI, the immunized chicks were given challenge infection with virulent IBD virus (obtained from the Division of Avian diseases, IVRI, Izatnagar) at the dose rate of 104.5 EID50/chick in 0.2 ml inoculum through oral route. The protection was assessed on the basis of presence of IBD antigen in the bursa of chicks of different experimental groups, checked by agar gel precipitation test (AGPT) at 3rd and 7th day PC by sacrificing 4 chicks from each group. The bursa and other visceral organs were also examined for gross and histopathological lesions. To assess the immunosuppressive effect due to invasion of bursa by the virulent IBD virus, the chicks were inoculated with RDF vaccine one week after the IBD virus challenge and antibody titres against RDF were assessed up to 2 weeks period. Top Results Viral antigen The IBD virus obtained at 3rd passage level having titre of 103.5 TCID50/ ml was further propagated in chicken embryo fibroblast culture and at the end of 8th passage the titre was found to be 105.5 TCID50/ ml. The protein concentration of the pelleted virus was 4.5 mg/ml. Plg microspheres A heterogenous population of microspheres having spherical shape with rough and porous surface was seen under Scanning electron microscope. Although the size of the microspheres ranged from 1–10 µm, majority of them were less than 2 µm in diameter (Fig. 1). The protein content in the hydrolysate of different batches of microsphere preparations was found to be 420–510 µg/ml as determined by using Bicinchoninic acid (BCA) protein assay kit. Antigen load was calculated and expressed as the percentage (w/w) of dry weight of microspheres, which was found to be 1.4–1.7%. However, no protein was detected in the hydrolysate of empty microspheres.
Evaluation of immune responses to the microencapsulated IBD virus IBD virus specific IgG levels
The IBD virus specific IgG levels in the pooled sera of immunized chicks at different intervals of PI and up to 2-wk PC are presented in Fig. 2. In case of the chicks of group-I, which were immunized with the microencapsulated IBD virus, the peak antibody titre was (log10) 3.77 at the 3rd wk PI, which gradually declined to (log10) 3.6 by 6th wk PI. The peak antibody titre of the chicks receiving conventional vaccine (group-II) was (log10) 3.69 at the 2nd wk PI. The control chicks of group-III did not show any marked antibody response up to the 6th wk PI. After challenge infection, there was a gradual increase in antibody titres in all the experimental groups and it was found that the IgG titres in the serum of group-I, II and III were (log10) 3.84, 3.77 and 3.0, respectively at 2nd wk PC.
IBD virus specific neutralizing antibody response
The neutralizing antibody responses against IBD virus (log10 NI) in the pooled sera of chicks of various groups at different period post immunization are presented in Fig. 3. The microencapsulated IBD virus as well as the conventional IBD vaccine showed significant levels of neutralizing antibodies up to the 6th wk PI. The peak neutralizing antibody levels of group-I was recorded at 3rd wk PI with the log10 NI values of 3.0 followed by a gradual decline to 2.2 at 6th wk PI. The peak neutralizing index of group-II (conventional) was 2.8 at the 2nd wk PI, which went down to 1.9 at 6th wk PI. The log10 NI values of control group remained low (<1.0) throughout the observation period.
IBD virus specific IgA levels
The IBD virus specific IgA titres in the pooled tracheal washings of chicks of various experimental groups at different intervals of post immunization are depicted in Fig. 4. It was found that in case of group-I, there was a considerable IgA antibody level with the peak titre of 40 at the 3rd wk PI, which gradually decreased to 24 at the 6th wk PI. The peak antibody titre of the conventional group (II) was 40 at 4th wk PI, which went down to 20 at the 6th wk PI. However, no IgA antibody against IBD virus was detected in the chicks of control group up to 6th wk PI.
IBD virus specific lymphocyte blastogenic response
IBD virus specific blastogenic response of the peripheral blood lymphocytes of chicks of various experimental groups as assessed by MTT colorimetric assay at different period post immunization are presented in Fig. 5. In group-I and II, the mean stimulation index (SI) values showed initial increase followed by gradual decline towards 5th to 6th wk PI. The peak stimulation index in group-I was (1.336±0.031) at 3rd wk PI, which was significantly (p<0.05) higher than that of control group that showed the mean SI of (1.014±0.08). The peak stimulation index value of group-II (conventional) was (1.317±0.06) at 4th wk PI that went down to (1.242±0.05) at 6th wk PI. The mean S.I. values of both the vaccinated groups were significantly higher (p<0.05) as compared to the control group up to 6th wk PI.
Resistance to challenge infection with virulent IBD virus In the vaccinated chicks, the resistance to virulent IBD virus challenge was assessed on the basis of detection of viral antigen in the bursa of Fabricius (BF), histopathological changes in the BF and the immunosuppressive effect on humoral immune system after challenge infection.
Top Discussion Evidences on importance of mucosal immunization and the potentialof MALT have provided an opportunity for vaccinologists to design vaccines and immunization strategies, which enhance mucosal immune responses against a variety of infectious agents. IBD virus has portal of entry via exposed mucosal surfaces, which calls for the protection at mucosal sites. Hence, in our study efforts were made to evaluate the mucosal as well as systemic immune responses induced by the microencapsulated IBD virus, delivered through nasal route in poultry with an aim to explore the possibility of using PLG microspheres fordevelopment of a nasal vaccine for IBD. |
Intranasal administration of microsphere vaccine formulation containing IBD virus was able to induce mucosal immune response at localsites as revealed by a considerable IgA level in the tracheal washings of the immunized chicks. The IgA response at mucosal sites is one of the essential features of protective immunity as the IgA antibody plays major role in prevention of adherence of microorganisms to mucosal surfaces (Walker et al., 1972; Williams and Gibbons, 1972) and also causes neutralization of viruses (Waldmann et al., 1986). |
It is also evident from our results that microencapsulated IBD virus could induce a satisfactory level of circulating IgG, detected in the serum of the immunized chicks, which was maintained up to 6-wkPI and even after the challenge infection. There was also a significant level of neutralizing antibody in the serum with log10 NI ≥ 2.0, which is also considered to be very important feature of protective immune response. These findings indicated that intranasal delivery of the virus with PLG microspheres is also efficient in eliciting good humoral responses at systemic sites. Induction of good local and systemic antibody responses by the mucosally administered antigen entrapped in PLG microparticles has also been reported earlier (Eldridge et al., 1991; Moldoveanu et al., 1993; Greenway et al., 1998; Baras et al., 2000; Guiterro et al., 2002) for various bacterial, viral and parasitic antigens. The trends of lymphocyte blastogenic response indicated that intranasally administered microsphere-IBD vaccine formulation was able to induce cell mediated immune response. Our findings correlate with that of other workers (Partidos et al.,1996; O'Hagan, 1997; Mutiwiri et al.,2002) who have reported that CMI could be elicited by mucosal immunization of antigen encapsulated in the PLG microparticles. |
Intranasal immunization with the microencapsulated IBD virus could afford resistance to the challenge infection as the invasion of bursa by the virulent virus was prevented and IBD viral antigen could not be detected in the bursa of 6 out of 8 chicks tested, which was also comparable to that of the conventional IBD vaccine. The restricted invasiveness of the virulent IBD virus in case of immunized chicks was also revealed from the milder histopathological lesions in the bursa as compared to that of the control chicks. The antibody response to RDF vaccination in the immunized chicks during post challenge period was not affected indicating that immunized chicks could afford resistance to challenge infection and there was no significant immunosuppressive effect due to invasion of bursa by the virulent IBD virus. |
From this study, it can be concluded that mucosal delivery of IBD virus through PLG microspheres was effective as it could elicit both mucosal and systemic immune responses and afforded moderate protection against IBD in poultry almost up to the marketing age of the broiler (6-8 wk). This has again proved the immunogenic potential of biodegradable microspheres as novel antigen delivery system. However, optimization of dose of the microencapsulated vaccine formulation is needed so as to further increase the magnitude and duration of protective immunity. |
Top Figures | Fig. 1: Scanning Electron Micrograph showing morphology of the PLG microspheres (2500X).
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| | Fig. 2: IgG levels in the pooled sera samples of different groups of immunized chicks as measured by indirect ELISA. MS : Microencapsulated IBD virus; CV : Conventional vaccine; C : unvaccinated control
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| | Fig. 3: Levels of virus specific neutralizing antibody (log10 NI) in different experimental groups as determined by SNT.
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| | Fig. 4: IgA response in the tracheal washings of different groups of experimental chicks, as measured by indirect ELISA.
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| | Fig. 5: Blastogenic response of peripheral blood lymphocytes in different groups of chicks as measured by MTT colorimetric method.
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| | Fig. 6: Section of bursa of Fabricius of IBD infected chicks : (A) showing mild depletion of lymphocytes in case of vaccinated chicks and (B) significant depletion and degeneration of lymphocytes in the unvaccinated control (H&E, 16 x 6.3).
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Tables | Table 1: Detection of IBD antigen and histopathological lesions in the bursa of Fabricius (BF) of the immunized chicks after challenge infection.
| | Experimental groups | No. of chicks | 3DPC** | IBD Ag in BF* 7 DPC** | Total | Histopathological lesions in BF |
| | Group-I (MS) | 8 | 0/4 | 2/4 | 2/8 | Mild oedema and depletion of lymphocytes | | Group-II (CV) | 8 | 0/4 | 2/4 | 2/8 | Mild oedema and depletion of lymphocytes | | Group-III (C) | 8 | 4/4 | 4/4 | 8/8 | Marked depletion and degeneration of lympho-cytes and infiltration of mononuclear cells |
| *IBD Ag detected by AGPT in the bursa of Fabricius; | **4 chickssacrificed from each group at 3rd and 7th day PC. |
| | | Table 2: HI antibody response against RDF vaccine in the immunized chicks challenged with virulent IBD virus.
| | Experimental groups | Mean HI titre before RDF inoculation | Mean HI titre* after RDF inoculation |
| | 1 wk | 2 wk |
| | Group I (MS) | < 1.0 | 2.6 | 4.8 | | Group II (CV) | 3.8** | 4.6 | 5.2 | | Group III (C) | < 1.0 | 1.6 | 2.6 |
| *Each value is average of HI titres (log2) determined in sera samples of 4 chicks from each group against 4 HA units of RD virus. | **This anti-RDV HI titre is due to conventional vaccination with RDF. | | Group-I (MS) : chicks were immunized with microspheres containing IBD virus. | | Group-II (CV) : chicks had received RD and IBD conventional vaccines as per normal schedule. | | Group-III (C) : Unvaccinated control chicks. |
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| Acknowledgements The authors are thankful to the Director, IVRI, Izatnagar for the valuable suggestions and providing infrastructural facilities. This research was financially supported by the ICAR under National Agricultural Technology Programme (NATP) with the project code number 28 (1)/2000-NATP/CGP-II/220 dated 25.5.2001. Top References | | AllanW.H., FaragherJ.T., CullenG.A.
(1972).
Vet. Rec.,
90:
511. TopBack | | BarasB., BenoitM-A., Poulain-GoldefroyO., SchachtA.M., CapronA., GillardJ., RiveauG.
(2000).
Vaccine,
18:
1495. TopBack | | CollinsM.A., GoughR.E., AlenanderD.J.
(1993).
Avian Pathol.,
22:
469. TopBack | | DobosP., HillB.J., HalletR., KellsD.T.C., BechtH., TeningesD.
(1979).
J. Virol.,
32:
593. TopBack | | EldridgeJ.H., StaasJ.K., MeulbrockJ.A., McGheeJ.R., TiceT.R., GilleyR.M.
(1991).
Mol. Immunol.,
28:
287. TopBack | | EylesJ.E., SharpG.J.E., WilliamsonE.D., SpiersI.D., AlparH.O.
(1998).
Vaccine,
16:
698. TopBack | | GreenwayT.E., EldridgeJ.H., LudwigG., StaasJ.K., SmithJ.F., GilleyR.M., SuzanneM.M.
(1998).
Vaccine,
15:
1314. TopBack | | GutierroI., HernandezR.M., IgartuaM., GasconA.R., PedrazJ.L.
(2002).
Vaccine,
20:
2181. TopBack | | HawkesR.A., DarbyshireJ.H., PetersR.W.
(1983).
Avian Pathol.,
12:
331. TopBack | | JonesD.H., McBrideB.W., FarrarG.H.
(1996).
J. Biotechnol.,
44:
29. TopBack | | LowryO.H., RosenbroughN.J., FarrA.L., RandallR.J.
(1951).
J. Biol.Chem.,
193:
265. TopBack | | McGheeJ.R., MesteckyJ., DertzbaughM.T., EldridgeJ.H., HirasawaM., KiyonoH.
(1992).
Vaccine,
10:
75. TopBack | | MesteckyJ., McGheeJ.R.
(1987).
Adv. Immun.,
40:
153. TopBack | | MoldoveanuZ., NovakM., HuangW.Q., GilleyR.M., StaasJ.K., SchaferD., CompansR.W., MesteckyJ.
(1993).
J. Infect. Dis.,
167:
84. TopBack | | MorrisW., SteinhoffM.C., RussellP.K.
(1994).
Vaccine,
12:
5. TopBack | | MosmannT.
(1983).
J. Immunol. Meth.,
65.55. TopBack | | Mukibi-MukaG., JonesR.C.
(1999).
Avian Path.,
28:
54. TopBack | | MutwiriG., BowersockT., KidaneA., SanchezM., GerdtsV., BabiukL.A., GriebelP.
(2002).
Vet. Immunol. Immunopath.,
87:
269. TopBack | | O'HaganD.T.
(1997).
Prospects for the development of new and improved vaccines through the use of microencapsulaton technology. In: New Generation vaccines eds.
LevineM.M., WoodrowG.C., KaperJ.B., CobonG.S., MarcelDekker, Inc.,
New York, NY, p.
215-228. TopBack | | O'HaganD.T., JefferyH., DavisS.S.
(1994).
Int. J. Pharm.
103:
37. TopBack | | O'HaganD.T., McGheeJ.P., HolmgrenJ., HolmgrenJ., MowatA.Mcl., DonachieA.M., MillsK.H.G., GaisfordW., RahmanD., ChallacombeS.J.
(1993).
Vaccine,
11:
149. TopBack | | PartidosC.D., VohraP., JonesD.H., FarrarG.H., StewardM.W.
(1996).
J. Immunol. Meth.,
195:
135. TopBack | | ReedL.J., MuenchH.
(1938).
Am. J. Hyg.,
27:
493. TopBack | | SpitB.J., HendriksenE.G.J., BruijntjesJ.P., KuperC.F.
(1989).
Cell and Cancer Research.
255:
193. TopBack | | TomasiJr. T.B., BienenstockJ.
(1968).
Adv. Immunol.,
8:
1. TopBack | | WaldmannR.H., StoneJ., BergmannK.C. et al.
(1986).
Ann. J. Med. Sci,
292:
367. TopBack | | WalkerW.A., IsselbacherK.J., BlochK.J.
(1972).
Science,
177:
608. TopBack | | WilliamsR.C., GibbonsR.J.
(1972).
Science,
177:
697. TopBack |
| |
|
|
|