Muhamuda K, Madhusudana SN , Apte M and  Ravi V, Department of Neurovirology, Nimhans, Bangalore

Abstract

Introduction

Rabies is a fatal zoonotic viral infection of the central nervous system that is transmitted by bite of rabid animal and is capable of infecting all mammal species. It is one of the oldest disease known to mankind that continues to kill thousands of people every year in spite of the availability of effective vaccines and sera to prevent it. It continues to be a major public health problem in India and other developing countries. As per the latest national survey conducted in 2003 about 20,000 people die of rabies e very year in India and in more than 95 per cent cases the transmitting vectors are dogs1. Rabies is also emerging as an important disease in North America as several cases of human rabies have been reported due to exposure to bats2. Australia, which was hitherto considered rabies free, has also reported human rabies deaths due to bites from bats3,4. Rabies is present uniformly in all states of India except in Andaman, Nicobar and Lakshadweep islands.

The infection occurs by inoculation of the virus into the bite wound through the saliva of the infected animals. After a prolonged and variable incubation period ranging from few days to few years, rabies presents with two types of clinical manifestations: 80% encephalitic form and 20% paralytic form. Laboratory diagnosis may not be required in the first form but paralytic rabies need laboratory confirmation.

Though several sensitive and specific tests are available for diagnosis of rabies, most laboratories in India still use Negri body demonstration for diagnosing rabies, with which both false positive and false negative results are sometimes reported. The main reason for this is the limited supply of diagnostic reagents that are often imported. There is an urgent need to develop these reagents indigenously and ensure their adequate supply. Reagents based on monoclonal antibodies will be ideal as they will be highly specific and false positive results sometimes seen with polyclonal antibodies can be avoided.

Though rabies is 100% fatal in humans, it is also 100% preventable if the state of the art modern prophylactic measures are instituted soon after the exposure. One of the important parameter of post exposure treatment is passive immunization in the form of local infiltration of rabies immunoglobulin (RIG). Presently 2 types of RIGs are used viz: equine rabies immunoglobulin (ERIG) and human rabies immunoglobulin (HRIG). While the former causes some serious side effects, the latter is very expensive for use in developing countries like India. Moreover, with imposing restrictions on the use of horses for production of biologicals, the production and supply of ERIGs may be affected. Keeping this possibility the WHO has initiated efforts to replace ERIGS with murine monoclonal antibodies (Mabs). Therefore, we thought it is worthwhile to produce murine Mabs in our laboratory, which may have potential for both passive Immunotherapy as well as in rabies diagnostics. The present paper describes the production, characterization and initial evaluation of these Mabs.

Materials and Methods

Production of hybridomas: Hybridomas that secreted monoclonal antibodies specific to rabies virus proteins were produced by the fusion of SP2/0 cells with splenocytes of BALB/C mice immunized with rabies vaccine (Verorab, PV strain) by using established procedures5. Initial screening of Mabs was performed by ELISA using strips coated with purified rabies virus, purified G and N protein. These were produced by previously described and established techniques 6. The clones producing G Mabs were subcloned by limiting dilution to get individual clones, which were then expanded.

Screening of clones producing G Mabs:  Initial screening was done by ELISA using strips coated with G protein. The supernatants from positive clones were tested for their neutralizing capacity against CVS strain of rabies virus by performing rapid fluorescent focus inhibition test (RFFIT) as advocated by WHO with some modifications7 . Instead of tissue culture chambers we used flat bottomed 96 well tissue culture plates (Sigma) and the cell line used was BHK-21 (CL-13) obtained from National Institute of Cell Sciences (NCCS). Clones that secreted high titered neutralizing antibodies were used for production of ascitic fluid in mice.

Production of Ascitic fluid: Ascitic fluids were obtained from BALB/C mice primed with 0.3ml Pristane (Sigma) injected intraperitoneally and then inoculated 2 weeks later with 5 X 106 hybridoma cells. Tumors and ascitic fluid developed in approximately 2 weeks. The ascitic fluid was collected, separated from cells, and purified by precipitation using the salting-out method with 40% saturated ammonium sulfate. The concentration of the protein was calculated by Folin-phenol method8 .

Isotyping and sub typing: Isotypes and Subtypes were determined with a mouse Mab Isotyping kit (Sigma) as per the procedure described by the manufacturer. This was done with both cell culture supernatants and purified ascitic fluid.

Confirmation of specificity: SDS- PAGE and Native electrophoresis was carried out with purified virus. Proteins were then electrotransfered to nitrocellulose membranes. Nonspecific binding of the membrane were blocked by incubating the membranes in 5% defatted milk in Tris-buffered saline (TBS)-Tween. Blocked membranes were incubated overnight with either pure hybridomas supernatant or ascitic fluid diluted 1:10,000. Membranes were washed with TBS-Tween. Blots were processed for colorimetric detection of the primary Mab by using a goat anti-mouse horseradish peroxidase (HRP) conjugate (Bangalore, Genei) with diaminobenzidine and H2O2.

Determining neutralization titer:  This was done at both cell culture stage and after obtaining ascitic fluid of inoculated mice. The neutralizing titer was determined by doing a RFFIT (see above) using 100 FFD50 (Fluorescent focus dose) of CVS strain of rabies virus. The titers were expressed as the reciprocal of the highest dilution of the neutralizing Mab showing 50 % reduction of fluorescent foci of infected BHK 21 cells when compared to virus control.

Results

Results of Fusion Experiments: Splenocytes were fused with myeloma cells in the ratio of 1:10 and plated into three 96 well plates. Amongst the three 96 wells plated, 100 wells (35%) showed the presence of hybridomas. Amongst the 100 clones 50 (50%) were positive in the screening ELISA, 20/50 were reactive to the Glycoprotein of rabies virus, 20/50 were reactive to the Nucleoprotein of the rabies virus whilst the remaining 10/50 were reactive to whole rabies virus antigen but negative in G and N ELISA.

Amongst the 20 clones reactive to G protein, it was noted that five clones were positive at high titers by ELISA. These five clones were chosen for subcloning. Amongst the 20 clones reactive to N protein, it was noted that five clones were positive at high titers by ELISA as well as the intracytoplasmic fluorescence on acetone fixed infected cell smears by indirect Immunofluorescence assay. These five clones were chosen for subcloning and further characterization.

Sub-cloning: After subcloning 20 G single clones were obtained, out of which 11 were highly neutralizing G clones, and nine were non neutralizing G clones. Fourteen stable N sub-clones were obtained with high ELISA OD values and also reacted with BHK-21 cells infected with CVS strain of rabies virus and showed intracytoplasmic fluorescence on acetone fixed smears by IFA suggesting that they recognize the rabies virus nucleoprotein.

Production of ascitic fluid : Eight neutralizing G clones were selected for the production of ascites in BALB/C mice based on the high titers in RFFIT. With each of these clones 15ml ascitic fluid was obtained by intraperitoneal inoculation of 1X106-7.7 hybridoma cells. Five N clones were selected for the production of ascites in BALB/C mice based on the high titers in the Immunofluorescence assay.

Characterization of Mabs (Ascitic fluid):  Eight Neutralizing G Mabs obtained as ascitic fluid were titrated by RFFIT to estimate the neutralizing titers and were also characterized in terms of Isotypes and Subtypes. The neutralizing G Mab titer in ascitic fluid varied between 1:10,000 and 1:100,00,000; and belong to IgG 2a subtype (Table 1), while the N Mab IFA titer varied between 1:1000 and 1:5000 and belonged to IgG 2b subtype (Table 2).

Confirmation of the reactivity of G and N Mabs: Immunoblotting using purified CVS strain of rabies virus confirmed the specificity of G and N Mabs. Initially when the neutralizing G Mabs were evaluated in SDS-PAGE followed by Immunoblotting, none of the neutralizing G Mabs showed a positive reaction with G protein (65 kDa). Therefore the experiment was repeated with native PAGE, followed by the Immunoblot, all clones recognized the glycoprotein bands thereby suggesting that all neutralizing G Mabs were directed against a conformational epitope rather than a linear epitope. Nine nonneutralizing G Mabs showed a positive reaction on SDS-PAGE followed by the Immunoblotting suggesting that all non-neutralizing G Mabs were directed against a linear epitope. On the other hand, all the five N Mabs reacted with 57kDa protein (N protein); both in SDS-PAGE as well as in native PAGE confirming that they are specific for N protein and suggesting that they recognize both conformational and linear epitope.

Table-5
Cinical feature of rabies at admission

82.44% had taken no treatment. Only 5.85 percent of Rabies Patient washed the wounds with soap and water and 17.02 percent of the Rabies patients received post-exposure anti-rabies vaccination  and many of them did not take full course of vaccination either due to ignorance or due to non- availability of vaccine.

Discussion

Monoclonal antibodies (Mabs) have played an important role in medical and allied fields, ever since their discovery in 1975 by Kohler and Milstein9. They have been used in diagnosis of infectious diseases, immunological disorders, as well as in Immunotherapeutics of infectious diseases and cancer. The usefulness of monoclonal antibodies is as a result of three important characteristics they possess: (i) their specificity of binding, (ii) their homogeneity and (iii) their ability to be produced in unlimited quantities.  Utility of Mabs in rapid diagnosis of rabies is another possibility. Neutralizing Mabs to rabies virus have a potential for use in passive immunotherapy and may serve to replace currently used equine polyclonal antisera. However, to the best of our knowledge Mabs to rabies virus have not been produced and characterized in India to date. This work was therefore undertaken with the objective of producing Mabs to rabies proteins and to study their utility in the diagnosis and immunotherapy of the disease.

Although rabies virus has five proteins, this study was restricted to the characterization and utility of Mabs to G and N protein of rabies virus. Rabies G protein plays an essential role in induction of protective immunity in animals and man by producing virus neutralizing antibodies. All the presently available rabies vaccines protect against infection mainly by eliciting neutralizing antibodies. Furthermore, protection can also be induced by passively transferring antibodies to G protein to the exposed individual

Fusion of SP2/O myeloma cells with splenocytes obtained from mice immunized with PV strain rabies virus resulted in production of large numbers of antibody-producing hybrid cells. Although the fusion experiments resulted in the generation of 50 secretory hybridomas, for practical reasons, only 10 of these were further studied for characterization and utility. Based on high ELISA OD values, five of G hybridomas and five of N hybridomas were sub cloned to obtain individual secreting clones. Single clones obtained after sub cloning were characterized using various tests such as ELISA, RFFIT, IFA, and Immunoblotting.  Among the 20 anti-G Mabs obtained after sub cloning, 11 G Mabs had neutralizing ability and the virus neutralizing titers of these Mabs varied between 1:1,000 and 1:5,000 by RFFIT ( data not shown). Other nine G Mabs were non-neutralizing and 14 clones were reactive to N protein by IFA. The isotyping and subtyping of the Mabs revealed that they belonged to IgG1, IgG2a, IgG2b, and IgM. Among these 8 G clones and 5 N clones were chosen to produce ascitic fluid in mice. The titers of these Mabs obtained as ascitic fluid is shown in Table 1 and Table 2. Thus we obtained G Mabs with a very high neutralizing titer against CVS strain of rabies virus.

The rabies virus G protein induces conformational and linear epitope-specific virus neutralizing antibodies. The G protein has 8 antigenic sites (I-VI, “a” and G1). Sites VI and G1 are defined as linear or nonconformational, whereas the others are conformation dependent epitope and readily destroyed on denaturation. In this study, we have not determined the epitope specificity of neutralizing G Mabs. However, we know that all the 8 neutralizing Mabs, recognized conformational epitopes as these failed to react with denatured glycoprotein.  All the 8 Mabs, neutralized CVS strain of rabies virus in very high dilutions. The N Mabs produced in this study recognized both linear and conformational epitopes and reacted strongly in the immunofluorescence using CVS infected mouse brains. We are now in the process of evaluating these Mabs against different street virus strains isolated from dog brains submitted to our laboratory for diagnosis of rabies. The G Mabs are being processed for their neutralization capacity against these street strains of rabies virus.

Thus our study resulted in production of neutralizing G Mab and immunofluorescence reactive to N Mab with very high titers. Further studies are underway to see the feasibility of using these Mabs in Immunodiagnosis of rabies using N Mabs and passive immunotherapy using G Mabs.

Reference:

1. Association for Prevention and Control of Rabies in India (APCRI)   Report of the national multicentric rabies survey 2003. Bangalore, APCRI, 2004

2. Noah DL , Drenzek CL, Smith JS et al. The epidemiology of human rabies in the United States.1980 to 1996. Ann Intern Med 1998;11:922-930.

3. Hooper PT, Lunt RA, Gould R et al. A new Lyssavirus: The first endemic          rabies related virus recognized in Australia. Bull Inst Past.1997;95:209-218

4. Mackenzie JS. Emerging viral diseases: An Australian perspective. Emerg. Infect. Dis. 1999;5: 1-8

5. Wiktor TJ, Koprowski H. Monoclonal antibodies against rabies virus produced by somatic cell hybridization: detection of antigenic variants. Proc Natl Acad Sci USA. 1978;75 (8):3938-42.

6. Perrin P, Vermisse P, Delagneau JF, Lucas G, Rollin P, Sureau P. The influence of Immunosorbent on rabies antibody EIA: advantages of purified glycoprotein.  J Biol Stand , 1986;14:95-102

7. Smith JS, Yager PA, Baer GM. A rapid reproducible test for determining rabies Neutralizing antibodies. In Meslin FX, Kaplan MM, Koprowsky H. eds. Laboratory techniques in rabies. 4th ed. , Geneva, WHO, 1996 pp 181-192

8. Lowry OH, Rosebrough AL, Farr and Randall RJ.  Protein measurement with the Folin phenol reagent. J. Biol. Chem, 1951; 193: 265-275.

9. Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975 , 7; 256(5517):495-7.

Table 1
Characterization of neutralizing G Mabs (ascitic fluid)

Table 2
Characterization of N Mabs (Ascitic Fluid)

                                                                   IFA= Indirect immunofluorescence.