Poly(lactide-co-glycolide) microspheres: A potent oral delivery system to elicit systemic immune response against inactivated rabies virus

Vaccine 27 (2009) 2138–2143

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Poly(lactide-co-glycolide) microspheres: A potent oral delivery system to elicit systemic immune response against inactivated rabies virus R. Ramya a,∗ , P.C. Verma a , V.K. Chaturvedi a , P.K. Gupta b , K.D. Pandey a , M. Madhanmohan c , T.R. Kannaki a , R. Sridevi a , B. Anukumar a a b c

Biological Products Division, Indian Veterinary Research Institute, Izatnagar 243122, UP, India Animal Biotechnology Division, Indian Veterinary Research Institute, Izatnagar 243122, UP, India Research and Development centre, Indian Immunologicals Ltd., Gachibowli, Hyderabad 500032, India

a r t i c l e

i n f o

Article history: Received 1 October 2008 Received in revised form 23 January 2009 Accepted 29 January 2009 Available online 6 February 2009 Keywords: Poly(lactide-co-glycolide (PLG) Rabies Oral immunization

a b s t r a c t Rabies is an endemic, fatal zoonotic disease in the developing countries. Oral vaccination strategies are suitable for rabies control in developing countries. Studies were performed to investigate the suitability of poly(lactide-co-glycolide) (PLG) microspheres as an oral delivery system for ␤-propiolactone inactivated concentrated rabies virus (CRV). Immune responses induced by encapsulated (PLG + CRV) and un-encapsulated inactivated rabies virus after oral and intraperitoneal route administrations were compared. The anti-rabies virus IgG antibody titer, virus neutralizing antibody (VNA) titers obtained by mouse neutralization test (MNT) and IgG2a and IgG1 titers of mice group immunized orally with PLG + CRV showed significantly (p < 0.001) higher response than the group immunized orally with un-encapsulated CRV. There was no significant difference (p > 0.05) between groups inoculated by intraperitoneal route. The stimulation index (SI) obtained by lymphoproliferation assay of PLG + CRV oral group also showed significantly (p < 0.001) higher response than the group immunized orally with un-encapsulated CRV, suggesting that oral immunization activates Th1-mediated cellular immunity. Immunized mice of all experimental groups were challenged intracerebrally with a lethal dose of virulent rabies virus Challenge Virus Standard (CVS). The survival rates of mice immunized orally with PLG + CRV and CRV alone were 75% and 50%, respectively, whereas intraperitoneally immunized groups showed 100% protection. The overall results of humoral, cellular immune response and survival rates of mice immunized orally with PLG + CRV were significantly (p < 0.001) higher than those of mice immunized orally with CRV alone. These data suggest that the PLG encapsulated inactivated rabies virus can be used for oral immunization against rabies. © 2009 Elsevier Ltd. All rights reserved.

1. Introduction Rabies, a fatal neuroencephalomyelitis is transmitted through the bite of rabid animals. Human mortality from endemic canine rabies was estimated to be 55,000 deaths per year in Asia and Africa [1]. Although vaccination of domestic and wild animals and removal of stray dogs have been effective in rabies control in developed countries, similar approaches have been difficult to implement in developing countries, primarily because vaccination of animals and humans exposed at risk of rabies is expensive. In addition wild animals which constitute reservoirs for the propagation of the virus are not readily accessible for vaccination and hence an alternative approach to limit the spread of the disease is warranted [2]. Oral immunization which has been successful in control of rabies in

∗ Corresponding author. Tel.: +91 40 23000211; fax: +91 40 23005958. E-mail address: [email protected] (R. Ramya). 0264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2009.01.129

wild animals uses live attenuated vectors or attenuated vaccines [3,4]. These technologies may not be readily available for vaccine manufactures in developing countries. However inactivated virus vaccines are of wide use in developing countries. Hence vaccine formulations using inactivated virus are attractive options for alternative (oral/mucosal) immunization strategies. To be viable, oral vaccination may be improved by using adjuvants or gastrointestinal protectants to maintain antigen integrity [5]. A suitable delivery system that minimizes the antigen dose, boosters and offers protection from the stomach acids is needed to control rabies using oral vaccination. Among a variety of inert and biodegradable polymers for controlled release studied so far, poly(lactide-co-glycolide) (PLG) particles made from lactic and glycolic acids holds promise for delivery of variety of antigens to the immune system [6]. PLG microspheres have been used in the delivery of a variety of antigens because of their stability, non-toxicity [7] and ability to induce strong immune response in various animal models [8–12]. This study was under taken to investigate the protective efficacy of orally delivered ␤-propiolactone (BPL) inactivated rabies

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virus (RV) encapsulated in PLG microspheres in mice. The immune responses induced by oral vaccination were compared with intraperitoneal route of immunization. 2. Materials and methods 2.1. Cells and viruses BHK-21 cell line (baby hamster kidney cell line, ATCC, USA) was cultured at 37 ◦ C and 5% CO2 in a humidified incubator using Dulbecco’s modified Minimum Essential Medium (DMEM, GIBCO, USA) supplemented with 10% (v/v) heat inactivated fetal bovine serum (FBS, GIBCO, USA) and antibiotics (100 IU of penicillin/ml and 100 ␮g of streptomycin/ml). Rabies virus (PV-11 strain, Institut Pasteur, France) was propagated using BHK-21 C13 cells grown in 150 cm2 tissue culture flasks [13]. Challenge Virus Standard (CVS) mouse brain strain of rabies virus (Institut Pasteur, France) was used for mouse protection test to determine the vaccine potency and mouse neutralization test (MNT) to determine the virus neutralizing antibody (VNA) titers. 2.2. Preparation of concentrated BPL-inactivated rabies virus (CRV) The rabies virus harvests obtained from infected BHK-21 cells grown in tissue culture flasks was inactivated with BPL (1:4000) and the degree of inactivation was determined by mouse inoculating test [14]. The concentrated, inactivated rabies virus was prepared as described elsewhere [15,16]. The pellet was resuspended in small volume of STE buffer (0.15 M NaCl, 0.01 M Tris and 0.001 M EDTA, pH 7.5) and protein was estimated using bicinchoninic acid (BCA) protein assay kit (Bangalore Genei, India). The purified rabies viral proteins were visualized on 10% SDS-PAGE after staining with Coomassie brilliant blue stain. For the detection of rabies viral proteins the concentrated rabies virus was resolved over10% SDS-PAGE and transferred onto PVDF membrane (Millipore, USA). The blot was probed with rabbit anti-rabies hyperimmune serum (1:500). Binding of specific antibodies to CRV was detected using anti-rabbit IgG-HRPO conjugate (1:5000, Bangalore Genei, India). 2.3. Preparation and characterization of PLG microspheres PLG microsphere (Sigma, USA) containing purified CRV was prepared as described by Rosas et al. [17]. Briefly 6% (W/V) of PLG (50:50) was dissolved in 5 ml of dichloromethane (Merck, India) and emulsified with 5 mg of purified rabies virus antigen by highspeed homogenization for 2–4 min. The primary emulsion (W/O) was mixed with 25 ml of 1 mM HEPES buffer, pH 7.5 containing 8% polyvinyl alcohol (MW 30,000–70,000; Sigma, USA) and emulsified by high-speed homogenization for 5 min in order to form double emulsion (W/O/W). Finally, 50 ml of 2% isopropanol solution was added and stirred for 1 h. The microspheres were collected by centrifugation at 10,000 × g for 15 min and washed thrice with distilled water and stored at −20 ◦ C after freeze drying. PLG microspheres treated similarly but without antigen were used as control in animal immunization experiments. The size and surface morphology of prepared microspheres were visualized by scanning electron microscopy (SEM). 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 in a scanning electron microscope (JEOL JSM 840X, Japan) and the images captured. Viral antigen load in the microspheres was determined by measuring the protein content using BCA method after known quantity (20 mg) of microspheres were hydrolyzed by using 3 ml of 5% (W/V) SDS in 0.1 M NaOH solution and stirring overnight at room temperature. The mixture was

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Table 1 Experimental groups of mice used in this study. The mice were inoculated with appropriate vaccine as indicated. Sl. no.

Groups

No. of mice

Route

Antigen dose/mice (␮g)

1 2 3 4 5 6 7

CRV PLG + CRV PLGa CRV PLG + CRV PLGa Naive

15 15 15 15 15 15 15

Oral Oral Oral IP IP IP –

50 50 – 50 50 – –

IP: intraperitoneal; PLG: poly(lactide-co-glycolide); ␮g: microgram. a Control groups inoculated with equivalent quantity of empty microspheres.

centrifuged and the protein content of supernatant (hydrolysate) was determined. Antigenicity of the CRV released from microspheres was determined by Dot-ELISA using rabies virus specific rabbit polyclonal serum and compared with results obtained using pre-encapsulated CRV [18]. 2.4. Immunization and sample collection Swiss albino mice (3–4 weeks old; n = 105) were used in this study. The animals were maintained and used according to the guidelines of Council for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). The experiment groups are shown in Table 1. Mice were randomly assigned to each experimental group. Mice were inoculated with respective antigen in adequate quantity of microspheres suspended in PBS. Blood samples were obtained on day 0, 7, 14, 21, 28, 35 and 42 post-immunization. Splenocytes from vaccinated and control Swiss albino mice were isolated after lysis of erythrocytes using RBC lysis buffer (Sigma, USA). The viability of splenocytes was >95% as determined by trypan blue dye exclusion method. 2.5. ELISA and determination of rabies virus specific Ig titers Serum IgG, IgG1 and IgG2a antibody responses were determined by ELISA. Ninety-six-well Maxisorp immunoplates (Nunc, Denmark) were coated with appropriately diluted CRV (100 ␮l) in carbonate/bicarbonate buffer (pH 9.6) and incubated overnight at 4 ◦ C. After removal of unbound antigens, the un-reacted sites were blocked using 200 ␮l of 3% BSA by incubation at 37 ◦ C for 1 h. The test sera (100 ␮l) and control sera were subjected to serial two-fold dilutions with phosphate-buffered saline–Tween 20 (PBS containing 0.05% Tween 20, V/V; PBST) as a diluent and incubated at 37 ◦ C for 1 h. After washing thrice with PBST, 100 ␮l/well of appropriately diluted HRP-conjugated IgG raised against mouse IgG, IgG1and IgG2a (Bangalore Genei, India) were added to the plates and incubated at 37 ◦ C for 1 h. After removal of unbound conjugate, positive binding was detected by adding substrate (OPD/H2 O2 ) and incubation for 15 min at 37 ◦ C in dark. The enzyme–substrate reaction was stopped by adding 100 ␮l/well of 1.25 M sulphuric acid and absorbance read at 492 nm. Rabies virus specific antibody titers were expressed as the reciprocal of the highest serum dilution that showed an OD492 value above the cut-off value, which was defined as the average OD492 value of seven non-immunized sera ± 3 standard deviations. 2.6. Mouse neutralization test MNT was carried out essentially as described [19]. Briefly, the heat inactivated sera samples were serially diluted and incubated with 50 LD50 of CVS at 37 ◦ C for 90 min. The presence of unneutralized virus in the mixture was checked by intracerebral inoculation of mice (n = 10) for each serum dilution. Control groups

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Fig. 1. (A) SDS-PAGE analysis of concentrated BPL-inactivated rabies (CRV). Lane 1: molecular weight marker. Lanes 2 and 3: CRV (different concentration). Bands corresponding to the major viral protein G, N, P and M are marked. (B) Western blot analysis of CRV showing immunoreactivity with hyperimmune serum raised against rabies. Lane 1: molecular weight marker. Lane 2: CRV.

were inoculated with 100 LD50 , 50 LD50 or 10 LD50 of CVS. The inoculated mice were observed daily for 21 days and presence of any symptom typical of rabies such as hind limb paralysis were noted. The VNA titer of test sera samples was calculated based on the percentage of survivors in each group. A standard rabies immunoglobulin (SRIG) was included as a positive control for comparison.

2.7. Challenge study The protective efficacy of the different groups of immunized mice were assessed by challenging eight mice randomly selected from each group with 0.03 ml of 20 LD50 of mouse adapted rabies virus CVS strain. After inoculation by intracerebral route the mice were observed for 14 days for symptoms of rabies. Protection index was calculated based on the percentage of survivors in each group.

2.8. Lymphoproliferation assay Stimulation index was measured by lymphoproliferation assay using MTT [3-[4,5-dimethylthiazole-2-yl]2,5-diphenyl tetrazolium bromide] dye [20]. Briefly CRV immunized mouse splenocytes (1 × 106 splenocytes/ml) were seeded in 96-well flat-bottom tissue culture plate (NunclonTM , Denmark). The splenocytes were stimulated with CRV (10 ␮g/ml) or Concanavalin A (Con A; 20 ␮g/ml) and incubated at 37 ◦ C for 72 h in a CO2 incubator. Twenty microliters of MTT (5 mg/ml in PBS) was added to all the wells and the plates were incubated for 4 h at 37 ◦ C in a CO2 incubator. One hundred and 50 ␮l of dimethyl sulfoxide (DMSO) was added to dissolve the formazon crystals formed by metabolization of MTT and the plates were incubated for 15 min at 37 ◦ C. The optical density (OD) was measured at 520 nm with reference background color reduction at 650 nm in a microplate reader.

3. Results 3.1. Characterization of CRV The analysis of CRV by reducing SDS-PAGE revealed the presence of major structural proteins such as G protein (65 kDa), N protein (57 kDa), P protein (38 kDa) and M protein (25 kDa) (Fig. 1A). Western blot analysis of the CRV produced expected polypeptide profile (Fig. 1B).

3.2. Characterization of PLG microspheres SEM observation of the PLG prepared by double emulsion method exhibited varying size (range of 0.5–2.5 ␮m) of microspheres and majority of them were less than 1 ␮m in diameter (Fig. 2). All the microspheres had spherical, intact and smooth surface regardless of their size. There was no difference in surface morphology of empty and antigen loaded microspheres. The loading efficiency of the rabies antigen in microspheres varied from 50 to 55% as determined by BCA assay. To check for in vitro toxicity, ten mice were injected subcutaneously with a 10× vaccination dose. No abnormal clinical signs or death were observed in these animals during the observation period of 14 days (data not shown).

2.9. Statistic analysis Unpaired Student’s t-test was used to analyze differences of means between groups. Statistical analyses were performed using OriginPro 7.5 SR4 analysis software (Origin Lab Corporation, USA).

Fig. 2. Scanning electron micrograph showing PLG microspheres prepared with CRV (2500×).

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Fig. 3. Detection of rabies virus specific IgG antibody titer by indirect ELISA. Rabies virus specific IgG antibody titer of each immunized groups was determined by indirect ELISA. The serum samples from each group (n = 10) were collected at 0, 7, 14, 21, 28, 35 and 42 days post-immunization. The ELISA antibody titers were calculated as the log10 of the reciprocal antibody dilution that showed an OD492 value above the cut-off value, which was defined as the average OD492 value of seven non-immunized sera ± 3 standard deviation. Each symbol represents mean level of serum antibody, expressed as geometric mean ± standard deviation (GMT ± S.D.). Each curve represents kinetics of antibody production induced by various immunized groups. ***p < 0.001.

In Dot-ELISA the hydrolysate of the CRV loaded microspheres showed immunoreactivity with RV specific hyperimmune sera and positive dots were formed similar to that of the pre-encapsulated CRV, indicating the retention of antigenicity after microencapsulation (data not shown). 3.3. In vivo antibody response in mice The ability of PLG microspheres for oral and intraperitoneal (I/P) delivery of CRV to induce specific immune responses was evaluated. One week post-immunization, the mice (n = 10) were test bled and their sera tested in ELISA to determine anti-RV antibody titer. The anti-RV antibody titers were determined for all experimental groups up to 42 days post-immunization (dpi). The highest mean ELISA titer of 2.893 ± 0.018 was observed in PLG + CRV oral group which was significantly (p < 0.001) higher than CRV (1.991 ± 0.024) at 42 dpi. In the case of intraperitoneal immunized group there were no significant differences between PLG + CRV (2.870 ± 0.033) and CRV alone (2.893 ± 0.011) at 42 dpi (Fig. 3). 3.4. Mouse neutralization test MNT was done to determine VNA titers in the immune mice sera (n = 10). A VNA titer of 4 IU/ml was observed in PLG + CRV oral group which was significantly (p < 0.001) higher than the group immunized orally with CRV alone (2 IU/ml) on 35 dpi. The highest mean VNA titer of 8 IU/ml was observed in the following intraperitoneal immunized groups CRV, PLG + CRV. There was no significant difference (p > 0.05) between encapsulated and un-encapsulated groups immunized by intraperitoneal route on 35 dpi (Fig. 4). These results suggest that the PLG encapsulated CRV can induce a virus neutralizing antibody response by oral route. However, intraperitoneal administration may induce a higher antibody response in mice. 3.5. Protection against challenge In order to compare the protective efficacy, different groups of immunized mice were challenged intracerebrally with 20 LD50 of CVS strain on 42 dpi. In the group immunized orally with PLG + CRV 75% protection was observed whereas mice immunized with CRV orally exhibited 50% protection. The mice groups immunized with CRV alone (I/P), PLG + CRV (I/P) resisted the challenge and all of them (100%) were protected. The unvaccinated control mice failed to

Fig. 4. Detection of rabies neutralizing antibody titer by MNT. VNA titer of 35 dpi sera was determined by mouse neutralization test. Briefly the heat inactivated sera samples were serially diluted and incubated with 50 LD50 of CVS at 37 ◦ C for 90 min. The presence of un-neutralized virus in the mixture was checked by inoculating intracerebrally in mice (n = 10) for each serum dilution. Virus neutralizing antibody titer of the test sera samples was calculated based on the percentage of mice survived in each group. The VNA titer was expressed as IU/ml. ***p < 0.001.

resist the challenge and died between 7 and 10 days post-challenge (Table 2). 3.6. Cell-mediated immune response The induction of cellular immunity in immunized groups was assessed in vitro by lymphoproliferation assay. As shown in Fig. 5, the level of proliferative responses in the mice immunized orally with PLG + CRV were significantly higher (1.855 ± 0.03, p < 0.001) Table 2 Protection levels of different groups of mice challenged with 20 LD50 CVS strain (mouse adapted).

of

Sl. no.

Groups

No. of mice challenged

No. of mice died

Protection (%)

1 2 3 4 5 6 7

CRV oral PLG + CRV oral PLG oral CRV IP PLG + CRV IP PLG IP Naive

8 8 8 8 8 8 8

4 2 8 0 0 8 8

50 75 0 100 100 0 0

rabies virus

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Fig. 5. Stimulation index (SI) of splenocytes from immunized and control mice stimulated with Concanavalin A and BPL inactivated rabies virus (CRV). The end point was determined by colorimetric MTT dye reduction test. The stimulation index was calculated by OD of stimulated–OD of un-stimulated/OD of unstimulated. Data are expressed as the mean ± S.D. (n = 6 mice per group). ***p < 0.001.

than in the group immunized with CRV alone (1.057 ± 0.03). Stimulation index of intraperitoneal group also showed significant difference (p < 0.001) between encapsulated (2.017 ± 0.02) and unencapsulated CRV (1.256 ± 0.01) suggesting that PLG can help to induce strong cell-mediated immune responses. 3.7. Th1 (IgG2a) and Th2 (IgG1) response Anti-RV IgG1 and IgG2a titers were measured in mice sera from different immunized groups by ELISA. There was significant (p < 0.001) difference of IgG2a/IgG1 response between encapsulated and un-encapsulated inactivated rabies virus administered by oral route. In the case of intraperitoneal route there was no significant difference (p > 0.05) in the IgG2a/IgG1 response (Fig. 6). Interestingly, a group of mice immunized with CRV alone (oral) showed low levels of IgG1 and IgG2a responses. Taken together, these observations indicate that mice immunized orally with PLG + CRV are able to respond to the inactivated rabies virus and produce strong Th1 response. 4. Discussion Rabies is endemic in Asia and Africa and rabid dogs are considered responsible for more than 90% of human rabies deaths. Various types of human and animal vaccines are used worldwide for the prevention of rabies. This includes inactivated cell culture,

Fig. 6. Detection of rabies virus specific IgG1 and IgG2a antibody titer by indirect ELISA. Rabies virus specific IgG1 and IgG2a antibody titer of each immunized groups was determined by indirect ELISA at 35 days post-immunization. The ELISA antibody titers were calculated as the log10 of the reciprocal antibody dilution that showed an OD492 value above the cut-off value, which was defined as the average OD492 value of seven non-immunized sera ± 3 standard deviations. Each bar represents mean level of serum antibody, expressed as geometric mean ± standard deviation (GMT ± S.D.). ***p < 0.001.

modified live, recombinant and subunit vaccines. Among these inactivated cell culture vaccine is the most commonly used. Vaccination of stray dogs has been a major obstacle for the control of rabies in developing countries. Oral vaccination of dogs against rabies is a promising supplementary method for dog rabies control [21]. There are reports describing a bait delivery system for delivering oral rabies vaccine to stray dogs in India [22]. Baer et al. [23] demonstrated that oral vaccination of foxes by inactivated vaccine resulted in protection against live virus challenge and induction of serum-neutralizing antibodies. Rupprecht et al. [5] suggested that adjuvants or gastrointestinal protectants are necessary to deliver inactivated rabies vaccines orally and induce a protective immune response. Among the several antigen delivery systems available, PLG 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 [24]. Unlike soluble antigens, mucosally administered antigens entrapped in PLG microspheres can induce good local and systemic antibody response as well as cell-mediated immunity [25]. Oral delivery of many bacterial and viral antigens based on PLG microspheres have been reported [8–12]. PLG encapsulated antigen preparations administered by mucosal route can induce local and systemic antibody response [9] as well as cell-mediated immunity [25]. In the present study oral vaccination of mice using PLG microspheres encapsulated CRV was attempted and immune responses were compared with intraperitoneal administered CRV. Encapsulation of CRV by PLG microspheres did not result in degradation of the CRV. Dot-ELISA and Western blot of CRV hydrolyzed from the PLG microspheres indicated that the antigenic polypeptides were present in the vaccine formulations. The PLG encapsulated CRV was able to induce a humoral immune response in mice as shown by the indirect ELISA using CRV as antigen (Fig. 3). The antibody responses induced by oral inoculation of PLG encapsulated CRV were significantly higher than responses induced by CRV alone. Protection of antigen in the gastric environment and presentation of antigen to lymphoid cells in the gut by PLG microspheres may have resulted in a stronger humoral immune response. Degradation of the un-encapsulated CRV may have resulted in a weaker antibody response. The glycoprotein of rabies is responsible for inducing strong humoral immune response including induction of neutralizing antibody response. It is possible that PLG encapsulation helps in maintaining the antigen (glycoprotein) integrity. Further, the MNT results indicate that PLG encapsulated CRV is better than CRV alone in inducing virus neutralizing antibodies and also antiviral resistance mechanisms resulting in protection of mice. In this respect, the intraperitoneal administered CRV and PLG encapsulated CRV were superior. Highest neutralization titers were observed in these groups. Intraperitoneal administrations may present CRV to a larger surface area for efficient uptake by peritoneal macrophages. Cell-mediated immunity plays an important role in recovery from attenuated rabies virus infection and in the post-exposure prophylaxis of rabies [26–28]. Dietzschold et al. [29] demonstrated that T-cell activation is a major protective component in the immune response against RV infection, considering that antibody levels are low in the brain because of the blood brain barrier [30], and lymphocytes infiltrate relatively easily into the brain after virus infection. Lymphoproliferative responses as indicated by the MTT assay were higher in the PLG encapsulated CRV immunized by the oral route. PLG microspheres have been reported to induce cellmediated response against M. tuberculosis 38 kDa protein [31] and recombinant HIV protein [32]. The lymphoproliferative responses may involve induction of antigen specific T cells after engulfment and processing of PLG encapsulated CRV by antigen presenting cells

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[33], these responses may involve expression of cytokines [31,32]. In this study, the T helper cell immune responses were demonstrated to be the Th1 type as shown by the higher IgG2a responses in mice orally immunized with PLG encapsulated CRV. This type of responses has been described for PLG encapsulated antigens earlier [31,32]. The Th1 type response will help in protection against live rabies virus challenge [26–28]. Earlier studies [33] showed that PLG encapsulated antigens are efficiently phagocytosed by macrophages (APC) and presenting the antigen by cytotoxic MHC class I pathway resulting in CMI response. Compared with un-encapsulated CRV immunized groups, SI values of T-cell proliferation assay were much higher in mice immunized with PLG encapsulated antigens given by oral and intraperitoneal route (Fig. 6) and all the PLG encapsulated groups showed significantly higher or comparable immune response with un-encapsulated CRV group immunized by oral and I/P route, respectively, suggesting that PLG delivery system activates both Th1 and Th2 pathways. Survival from lethal challenge could be predicted based on the VNA titers obtained by MNT. Since the VNA titers in this study were determined without complement, these neutralizing antibodies would have been able to inhibit the binding of the virus to the rabies virus specific receptors or the fusion process of the virus as described by Dietzschold et al. [34]. Hence, it is reasonable to assume that VNA inhibits virus growth in the brain and VNA titers predictive of challenge study results. The 50% protection observed in CRV (oral) without PLG may largely be attributed due to the whole viral protein remaining after degradation in the stomach. These proteins may have stimulated a protective immune response. In summary, the anti-rabies virus IgG antibody response, VNA response obtained by MNT and IgG2a and IgG1 antibody response of mice group immunized orally with PLG + CRV showed significantly higher response than the group immunized orally with un-encapsulated CRV. Results presented here show that PLG encapsulated rabies vaccine formulations can induce protective systemic immune response by oral route. Significantly higher immune response of the PLG encapsulated groups to that of the CRV alone oral group suggesting, that the PLG microspheres were efficiently phagocytosed by macrophages and presented to the immune system without altering the immunogenicity of the antigen. PLG microspheres can be developed in to a suitable oral delivery system for inactivated rabies virus. The microencapsulation technique using PLG polymer may be useful in the mass oral immunization programme to control rabies in stray dogs and wild animals. Future studies will determine the effect of the booster immunization; dose–response and potency studies in dogs to determine the usefulness of the PLG encapsulated CRV for rabies control in developing countries. Acknowledgements We gratefully acknowledge Director, Indian Veterinary Research Institute, Izatnagar, India, for providing necessary facilities to carry out this research. We are thankful to Dr. T. Nagarajan, Dr. D. Thiagarajan and Dr. V.A. Srinivasan for their critical comments. References [1] WHO Expert Consultation on Rabies. World Health Organization. Technical Report Series 2005;931:1–88. [2] LeBlois H, Tuffereau C, Blancou J, Artois M, Aubert A, Flamand A. Oral immunization of foxes with avirulent rabies virus mutants. Vet Microbiol 1990;23:259–66. [3] Brochier B, Kieny MP, Costy F, Coppens P, Bauduin B, Lecocq JP, et al. Largescale eradication of rabies using recombinant vaccinia-rabies vaccine. Nature 1991;354:520–2. [4] Schneider LG. Rabies virus vaccines. Dev Biol Stand 1995;84:49–54.

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