FIELD OF INVENTION

The present invention relates to the field of immunology in particular to the monoclonal antibodies against Rabies.

BACKGROUND OF THE INVENTION

Rabies is a zoonotic viral disease which infects wild as well as domestic animals. The virus is transmitted in mammalians through close contact with saliva from the infected animals. The disease has been widely distributed all over the world in tropical areas. The annual number of human deaths worldwide caused by rabies is estimated to be between 40,000 and 70,000 for densely populated countries in Africa and Asia where rabies is endemic.

The rabies virus is a bullet-shaped, enveloped, single-stranded RNA virus classified in the Rhabdovirus family and Lyssavirus genus. The genome of rabies virus codes for five viral proteins: RNA-dependent RNA polymerase (L); a nucleoprotein (N); a phosphorylated protein (P); a matrix protein (M) located on the inner side of the viral protein envelope; and an external surface glycoprotein (G). The G protein (62-67 KDa) is a type-I glycoprotein composed of 505 amino acids that has two to four potential N-glycosylation sites, of which only one or two are glycosylated depending on the virus strains. The G protein forms the protrusions that cover the outer surface of the virion envelope and is known to induce virus-neutralizing antibodies.

Rabies can be treated or prevented by both passive and active immunizations. Rabies post-exposure prophylaxis includes prompt local wound care and administration of both passive (anti-rabies immunoglobulins) and active (vaccines) immunizations. Currently, the anti-rabies immunoglobulins (RIG) are prepared from the serum samples of either rabies virus-immune humans (HRIG) or rabies virus-immune horses (ERIG). According to WHO, combined immunoglobulin treatment is the best specific treatment available for the post exposure prophylaxis of rabies in humans. Human (HRIG) and equine (ERIG) immunoglobulin are used for post-exposure prophylaxis.

Usually more than 10 million people are reported to receive PEP (post Exposure Prophylaxix) every year (Dietzschold, B., W.H. Wunner, T.J. Wiktor, A.D. Lopes, M. Lafon, C.L., Smith, and H. Koprowski. 1983. Characterization of an antigenic determinant of the glycoprotein that correlates with pathogenicity of rabies virus. Proc. Natl. Acad. Sci. USA 80:70-74 .

However, a comparatively high incidence of adverse reaction including serum sickness caused by heteroantigens and infectious disease caused by undetected infectious agents are reported. In addition an insufficient supply of therapeutical immunoglobulins has made post exposure treatment more difficult.

Over the last decade, a variety of recombinant techniques have been developed that have revolutionized the generation of antibodies and their engineering. Particularly, the development of antibody libraries and display technologies, such as phage display have greatly influenced antibody preparation. In general, the established generation of antibody libraries in phages includes the cloning of repertoires of immunoglobulin genes or parts thereof for display on the surface of the phages. The starting material for preparing antibody libraries has been RNA isolated from the total population of peripheral blood lymphocytes or B cells from immunized or non-immunized donors. A problem associated with the use of the total population of peripheral blood lymphocytes or B cells for preparing antibody libraries is that functionally relevant and therapeutically effective antibodies against pathogenic organisms such as bacteria are underrepresented in these libraries.

This problem has now been solved by using RNA from a subset of antibody-producing B cells, i.e., IgM memory B cells, for the production of antibody libraries. Pathogenic organisms are known to have evolved many evasive techniques to avoid detection or attack from the immune system. For example, many bacteria display huge variation in their surface antigens or at least the antigenic sites on which the immune system focuses. Therefore, antibodies designed to protect against these bacteria should be capable of recognizing many antigens to provide the maximum coverage of the
most common infections; however, because of extensive antigen variation, coverage of all strains of a type of bacterium by an antibody is difficult to accomplish. Furthermore, although antibodies that are cross-reactive between strains are required, antibodies that are additionally cross-reactive between species of bacteria are preferred as these would be more attractive to develop and use clinically.

SUMMARY OF THE INVENTION

In the present invention, the recombinant human antibody Fab fragments was selected from naive antibody library. Biopanning was performed for selection of anti rabies virus specific antibody fragments. The eluted phages obtained after third round of panning was used to infect Escherichia coli TGI and 96 individual clones were tested for binding to target antigen. As indicated by the phage ELISA, specific clones showed positive binding to antigen. Furthermore, PCR and enzymatic analysis showed the presence of Fab antigen binding fragments. Selected Fab plasmids were transformed into Escherichia coli HB2151 for production of soluble Fab fragments. The purified protein was checked for their antigen binding activity against the rabies virus was found to be active. The present invention discloses the use of recombinant human antibody Fab fragments generated from naive human antibody phage library. These antibody fragments can be used in neutralization of rabies virus in vitro or in vivo.

In one of the aspect of the present invention there is provided an isolated human anti-Rabies monoclonal antibody comprising a heavy chain polypeptide having the amino acid sequence having at least 80% homology to the amino acid sequence as set forth in SEQ ID NO: 45 and a light chain polypeptide having the amino acid sequence having at least 80% homology to the amino acid sequence as set forth in SEQ ID NO: 43.

In another aspect of the present invention there is provided an isolated polynucleotide encoding human anti-Rabies monoclonal antibody heavy chain having nucleotide sequence as set forth in SEQ ID NO: 44.

In yet another aspect of the present invention there is provided an isolated human anti-Rabies monoclonal antibody heavy chain polypeptide having amino acid sequence as set forth in SEQ ID NO: 45.

In yet another aspect of the present invention there is provided an isolated polynucleotide encoding human anti-Rabies monoclonal antibody light chain having nucleotide sequence as set forth in SEQ ID NO: 42.

In further aspect of the present invention there is provided an isolated human anti-Rabies monoclonal antibody heavy chain polypeptide having amino acid sequence as set forth in SEQ ID NO: 43.
In another aspect of the present invention there is provided an isolated polynucleotide encoding
human anti-Rabies monoclonal antibody Fab fragment having nucleotide sequence as set forth in SEQ ID NO: 46.

In another aspect of the present invention there is provided ant-Rabies monoclonal antibody having polypeptide sequence as set forth in SQ ID NO: 47.

In yet another aspect of the present invention there is provided a recombinant vector comprising the polynucleotide as set forth in SEQ ID NO: 44 and SEQ ID NO: 42; or the polynucleotide as set forth in SEQ ID NO: 46.

In yet another aspect of the present invention there is provided a recombinant host cell comprising the recombinant vector comprising the polynucleotide as set forth in SEQ ID NO: 44 and SEQ ID NO: 42; or the polynucleotide as set forth in SEQ ID NO: 46.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

Figure 1 shows binding activity of the selected Fab fragments to the rabies virus glycoprotein

Figure 2 shows western blot analysis of periplasmic preparations of HB2151 containing Fabs. The periplasmic preparation was electrophoresed through 10 % non-reducing SDS-polyacrylamide gel and transferred onto PVDF membrane. The Fab was detected with the HRP-conjugated goat anti-human IgG which is Fab specific as evidenced by band position approximately 47 kDa indicated by the arrow. Figure 3 shows analysis of rabies virus protein with which human Fabs interact was performed by western blot. Figure 4 shows the competitive binding assay

DETAILED DESCRIPTION OF THE INVENTION

In the present invention, the recombinant human antibody Fab fragments were generated using a naive human antibody library using the phage display technology. These fragments can be used in detecting rabies virus in vitro as in diagnostic methods, in-vitro potency assays and also for therapeutic purposes intended for neutralization of rabies virus in vivo. The invention provides methods for generating immunoglobulin libraries by isolating RNA from a subset of B cells. The immunoglobulin libraries are used to identify and obtain immunoglobulins having a specific functionality of interest.

The term "host," as used herein, is intended to refer to an organism or a cell into which a vector such as a cloning vector or an expression vector has been introduced. The organism or cell can be prokaryotic or eukaryotic. It should be understood that this term is intended to refer not only to the particular subject organism or cell, but to the progeny of such an organism or cell as well. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent organism or cell, but are still included within the scope of the term "host" as used herein.

Human: The term "human," when applied to antibody/binding molecule as defined herein, refers to molecules that are either directly derived from a human or based upon a human sequence. When a binding molecule is derived from or based on a human
sequence and subsequently modified, it is still to be considered human as used throughout the specification. In other words, the term human, when applied to binding molecules is intended to include binding molecules having variable and constant regions derived from human germline immunoglobulin sequences based on variable or constant regions either or not occurring in a human or human lymphocyte or in modified form. Thus, the human binding molecules may include amino acid residues not encoded by human gremline immunoglobulin sequences; comprise substitutions and/or deletions (e.g., mutations introduced by, for instance, random or site-specific mutagenesis in vitro or by somatic mutation in-vivo).

Monoclonal antibody: The term "monoclonal antibody" as used herein refers to a preparation of antibody molecules of single molecular composition, i.e., primary structure, i.e., having a single amino acid sequence. A monoclonal antibody displays a single binding specificity and affinity for a particular epitope. Accordingly, the term "human monoclonal antibody" refers to an antibody displaying a single binding specificity which has variable and constant regions derived from, or based on, human germ line immunoglobulin sequences or derived from completely synthetic sequences. The method of preparing the monoclonal antibody is not relevant.

Post exposure prophylaxis: "Post exposure prophylaxis" (PEP) is indicated for persons possibly exposed to a rabid animal. Possible exposures include bite exposure (i.e., any penetration of the skin by teeth) including animal bites, and non-bite exposure. Non-bite exposures include exposure to large amounts of aerosolized rabies virus in laboratories or caves and surgical recipients of corneas transplanted from patients who died of rabies. The contamination of open woimds, abrasions, mucous membranes, or theoretically, scratches, with saliva or other potentially infectious material (such as neural tissue) from a rabid animal also constitutes a non-bite exposure. Other contact by itself, such as petting a rabid animal and contact with blood, urine, or feces of a rabid animal, does not constitute an exposure and is not an indication for prophylaxis. PEP should begin as soon as possible after an exposure. If no exposure has occurred
post exposure prophylaxis is not necessary. In all post exposure prophylaxis regimens, except for persons previously immunized, active and passive immunizations should be used concurrently.

The term "vector" denotes a nucleic acid molecule into which a second nucleic acid molecule can be inserted for introduction into a host where it will be replicated, and in some cases expressed. In other words, a vector is capable of transporting a nucleic acid molecule to which it has been linked. Cloning as well as expression vectors are contemplated by the term "vector," as used herein. Vectors include, but are not limited to, plasmids, cosmids, bacterial artificial chromosomes (BAC) and yeast artificial chromosomes (YAC) and vectors derived from phages or plant or animal (including human) viruses. Vectors comprise an origin of replication recognized by the proposed host and in case of expression vectors, promoter and other regulatory regions recognized by the host. A vector containing a second nucleic acid molecule is introduced into a cell, for example, by transformation, transfection, or by making use of bacterial or viral entry mechanisms. Other ways of introducing nucleic acid into cells are known, such as electroporation or particle bombardment often used with plant cells, and the like. The method of introducing nucleic acid into cells depends among other things on the type of cells, and so forth. This is not critical to the invention. Certain vectors are capable of autonomous replication in a host into which they are introduced (e.g., vectors having a bacterial origin of replication can replicate in bacteria). Other vectors can be integrated into the genome of a host upon introduction into the host, and thereby are replicated along with the host genome.

Even though significant advances have been made in the development of human MAbs, there still exists inherent problem in the technology that makes further development and clinical application of these MAbs difficult. Some of these problems include the instability of human hybridoma cell lines especially after cryopreservation and the use of Epstein Barr virus to transform B cells which introduces the risk of viral contamination in a MAb preparation. One approach to overcome these problems is to express the immunoglobulin domain in a heterologous expression system.
To overcome the above mentioned drawback the present invention describes use of recombinant antibody Fab fragments consisting of variable heavy and light chains selected against rabies virus. These fragments may provide a substitute for the currently available polyclonal rabies immunoglobulins. Monoclonal antibodies against the virus glycoprotein are useful in detecting the virus and also in neutralization of virus in-vivo. As compared to the conventional technologies, the human phage antibody technology has more advantages where in large number of high affinity antibodies can be produced with out prior immunization (Brekke O.H., Loset G.A. 2003. New technologies in therapeutic antibody development. Curr. Opin. Pharmacol. 3, 544-550).

All currently available immunoglobulins used are plasma derived polyclonal products obtained from vaccinated human donors or horses are produced in low quantities and suffer from batch to batch variation which is difficuh to maintain in a quality assured manner and in the quantities required. Comparatively, in the present invention, the recombinant human Fab fragments can be over-expressed in bacteria and produced in large quantities at low cost to guarantee the supply of a consistent and well characterized biological. The production of recombinant antibody fragments does not require the maintenance of viable hybridoma cell lines. In the present invention, recombinant phage displayed antibodies may potentially replace the available antibodies.

The present invention provides a method for generating recombinant antibody Fab fragment against rabies virus glycoprotein, the major immunogenic surface protein known to harbor virus neutralizing epitopes by using human naive library. The invention further provides Fab fragment capable of specifically binding to and their use in neutralization of rabies virus by in vitro or in vivo. Furthermore, the invention pertains to nucleic acid and amino acid sequences encoding the Fab fragment.

The invention further provides a method to enrich for and isolate phage which contain cloned library sequences that encode a desired protein, and thus to uUimately isolate the nucleic acid sequences themselves, phage harvested from the bacterial debris.

One embodiment of the present invention is to provide a method for generating an immunoglobulin library by isolating RNA from a subset of B cells and preparing the immunoglobulin library from the RNA, characterized in that the subset of B cells consists essentially of IgM memory B cells.

Yet another embodiment of the present invention provides a method of production of recombinant Fab fragment, wherein the immunoglobulin library is selected from the group consisting of a naive antibody Fab library.

Yet another embodiment of the present invention provides a method of identifying an immunoglobulin having a functionality of interest, the method comprises generating the immunoglobulin library and screening the immunoglobulin library for an immunoglobulin having the functionality of interest.

Further embodiment of the present invention provides a method of production of recombinant Fab fragment, wherein screening the immunoglobulin comprising a) contacting the immunoglobulin library with an antigen under conditions conducive to binding, b) separating and recovering immunoglobulins that bind to the antigen from immunoglobulins that do not bind, c) isolating at least one recovered immunoglobulin, d) screening if the immunoglobulin isolated has a functionality of interest, and e) isolating an immunoglobulin having the functionality of interest.
Construction of human Fab antibody library

B lymphocytes from the peripheral blood lymphocytes from healthy donors were isolated on a Ficoll-Plaque gradient by centrifugation. The cell pellet was dissolved in 50 ml of Qiazol (Qiagen). Total RNA was isolated using RNeasy mini Kit with optional step to eliminate the genomic DNA. In order to completely recover the entire antibody gene repertoire RNA was reverse transcribed using both random hexamers and Oligo dT as primer.

Oligonucleotide primers used for primary PCR amplification of human heavy and light chains and linkers are described in Table 1. IgM-derived heavy chain variable region (354 bps) was obtained by a primary PCR using HuIgM Reverse (SEQ ID NO: 1) and separate VH FOR (SEQ ID NO: 39) primers. The VH fi-agments were re-amplified with a combination of JHR primers (SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32 and SEQ ID NO: 33) annealing to the 3' end of VH and separate VH FOR (SEQ ID NO: 39) primers annealing to the 5' end of corresponding purified PCR product (354bps). CHI fragment of IgGl were amplified using CHIF (SEQ ID NO: 35) and primer FlagR (SEQ ID NO: 36) in the PCR (261bps). Fd fi-agment was obtained by overlap PCR with primer VH FOR (SEQ ID NO: 39) and FlagR (SEQ ID NO: 36) (615bps).
Light chains (kappa and lambda families) were amplified by PCR with a set of CK reverse (SEQ ID NO: 2) or CX reverse primer (SEQ ID NO: 3) annealing to the 3' end of the constant domain and Sfi-tagged FOR (SEQ ID NO: 34) primers priming at the 5' end of the VL-regions. For the light chain families, 6 different kappa chain products and 11 lambda chain products were obtained (648bps) and mixed from each family and gel-purified.

For the preparation of linker between light chain and heavy chain, primer linkerF (SEQ ID NO: 37) and primer linkerR (SEQ ID NO: 38) were used. All the amplified products were purified fi-om agarose gel with the QIAquick Gel Extraction Kit (Qiagen) and the DNA concentration was determined.

Preparation of Fab fragment insert through overlap PCR
Heavy chain (615 bps), light chain (648 bps) and linker (24 bps) were fused by three-fi-agment overlap PCR. The full length Fab fragment was pre-amplified without flanking primers and then amplified with the flanking primers SfiF (SEQ ID NO: 34) and FlagR (SEQ ID NO: 36) under the same conditions.

Construction of Fab library

For construction of the Fab library, the gel-purified overlapped PCR products (1500 bps) containing the light chain and heavy chains were appended with Sfil on both sides prior to and after digestion with Sfil. The insert and vector were digested with Sfil restriction enzyme. The gel-purified filaments and gel-purified phonemic vector pComb3X were ligated. The desalted ligation mixtures are electroporated into competent TGI E. coli cells. The transformed bacteria were spread on 2YT agar and incubated at 37°C overnight.
Phage display Fab library

The library comprises over 5x10^ different Fab filaments cloned in an ampicillin resistant phagemid vector pComb3x and transformed into TGI E. coli cells. Fab fragment comprises one constant and one variable domain of each of the heavy and the light chain.

Production of the phage library
Production of the phage was performed essentially as described (Marks, J. D., Hoogenboom, H. R., Bonnert, T. P.,McCafferty, J., Griffiths, A. D. &Winter, G. (1991). Bypassing immunization. Human antibodies from Vgene libraries displayed on phage. J. Mol. Biol. 222, 581-597). Briefly, E. coli TGI cultures in the exponential phase were infected with helper phage M13K07 at multiplicity of infection of 10 to the bacteria. The bacteria infected with helper phage were grown over night for phage production. The phages were PEG precipitated and used for Biopanning.

Selection of recombinant antibodies against rabies virus glycoprotein from a human naive antibody library by biopanning
Phages displaying individual human Fab antibody fragment were selected by three rounds of biopanning on immunotubes coated with a decreasing amount of purified rabies glycoprotein antigen for the first, second and third round of panning. Eluted phage titers showed an increase after each of the three rounds of panning, indicating successful enrichment (Table 2). Eluted phage from round 3 was used to infect E. coli TGI and randomly selected clones were tested by phage ELISA against rabies glycoprotein.
Analysis of Fab sequences

Specific clones from each round of biopanning were selected for sequencing analysis. Phagemid DNA was purified according to the manufacturer protocol and sequenced at Ocimum Biosolutions Ltd, India. The sequencing primers, VH pelB (SEQ ID NO: 40) and VL Bomp (SEQ ID NO: 41) were used respectively for variable heavy and light gene analysis. The entire sequences was submitted to IMGT and verified as the variable domains of the antibody. The obtained 1287 base pairs sequences of heavy and light chain with constant regions as set forth in SEQ ID NO: 46.

Production of soluble Fab fragments and reactivity with rabies virus glycoprotein

Positive Fab clones identified by phage ELISA were transformed into non-suppressor Escherichia coli HB2151 for production of soluble Fabs. Individually selected clones were grown and production of Fab was induced by addition of IMm isopropyl-P-D-thiogalactopyranoside (IPTG). Both supernatant and periplasmic fractions were screened for antigen binding by ELISA on high affinity microtiter plates coated with purified rabies virus glycoprotein (Figure 1). The periplasmic fraction of Fab fragments were analyzed by SDS-PAGE analysis under non-reducing conditions after staining with coomassie brilliant blue and immunoblot analysis under non-reducing conditions revealed a major band with an apparent molecular weight of about 47 kDa
(Figure 2) which was probed with HRP-conjugated goat anti-human IgG (Fab specific).

Reactivity of the Fab fragment with the purified rabies virus glycoprotein
A western blot assay was used to determine the nature of the nature viral antigen recognized by the human Fab fragments. Rabies virus proteins were separated under non reducing conditions, in 10 % SDS-PAGE and transferred onto PVDF membrane. The membrane was probed with the selected Fab which is designated as FabB6. The Fabs showed a specific binding to a protein band of 65 kDa, which corresponds to the molecular weight of rabies virus glycoprotein (Figure 3).

Competitive ELISA

The competitive ELISA was performed by coating rabies purified antigen to check for competition between FabB6 and mouse derived monoclonal antibody (M5B4). Competition was done by incubating the immunized mouse sera with varying dilutions of the Fab and vice-versa. No decrease in the optical density values following the dilution of the Fab indicating that the Fab is not competing wdth mouse monoclonal antibody (M5B4) for the same site on rabies virus (Figure 4).

In accordance with the present invention one embodiment of the present invention provides a recombinant human anti-Rabies monoclonal antibody or antigen binding portion thereof that specifically binds to rabies virus and inhibits the ability of the virus to infect cells, wherein the polypeptide of the monoclonal antibody comprises the amino acid sequence as set forth in SEQ ID NO: 43 and SEQ ID NO: 43.

Yet another embodiment of the present invention provides recombinant human anti-Rabies antibody or antigen binding portion, wherein the antibody or antigen binding portion thereof neutralizes rabies virus in a Rapid Fluorescent Focus Inhibition Test, wherein the polypeptide of the monoclonal antibody comprises the amino acid sequence as set forth in SEQ ID NO: 43 and SEQ ID NO: 43.

Yet another embodiment of the present invention provides an antibody or antigen binding portion thereof, wherein the antibody inhibits rabies virus in vivo in a subject, wherein the polypeptide of the monoclonal antibody comprises the amino acid sequence as set forth in SEQ ID NO: 43 and SEQ ID NO: 43.
Yet another embodiment of the present invention provides an antibody or antigen binding portion thereof, wherein the antibody or antigen binding portion thereof, protects from or inhibits rabies virus-mediated neuronal pathology in a subject; protects from or inhibits rabies virus-mediated encephalomyelitis in a subject; or protects from or inhibits rabies virus-mediated paralysis in a subject, wherein the polypeptide of the monoclonal antibody comprises the amino acid sequence as set forth in SEQ ID NO: 43 and SEQ ID NO: 43.

Further embodiment of the present invention is to provide a recombinant monoclonal antibody, wherein the antibody is human antibody, wherein the polypeptide of the monoclonal antibody comprises the amino acid sequence as set forth in SEQ ID NO: 43 and SEQ ID NO: 43.

Yet another embodiment of the present invention provide a method of diagnosing, prophylaxing, treating, or any combination thereof, of exposure to or infection by a rabies virus in a subject, the method comprises utilizing the antibody of the present invention in the method of diagnosis, prophylaxis, treatment, or combination thereof, of rabies virus.

In yet another embodiment, the desired protein is also transported to an extra-cytoplasm compartment of the host cell, such as the bacterial periplasm. In this embodiment the desired protein (or one of its polypeptide chains if it is a multi-chain protein, such as an antibody) is expressed separately and forms Fab fragment in the cell inner membrane.

In one embodiment of the present invention there is provided a recombinant human anti-Rabies monoclonal antibody comprising a heavy chain polypeptide having the amino acid sequence having at least 80% homology to the amino acid sequence as set forth in SEQ ID NO: 45 and a light chain polypeptide having the amino acid sequence having at least 80% homology to the amino acid sequence as set forth in SEQ ID NO:
43.

In another embodiment of the present invention there is provided recombinant human anti-Rabies monoclonal antibody comprising a heavy chain polypeptide having the amino acid sequence having at least 80% homology to the amino acid sequence as set forth in SEQ ID NO: 45 and a light chain polypeptide having the amino acid sequence having at least 80% homology to the amino acid sequence as set forth in SEQ ID NO: 43, wherein the amino acid sequence of the heavy chain polypeptide is as set forth in SEQ ID NO: 45 and the amino acid sequence of the light chain polypeptide is as set forth in SEQ ID NO: 43.

In another embodiment of the present invention there is provided a recombinant human anti-Rabies monoclonal antibody comprising a heavy chain polypeptide having the amino acid sequence having at least 80% homology to the amino acid sequence as set forth in SEQ ID NO: 45 and a light chain polypeptide having the amino acid sequence having at least 80% homology to the amino acid sequence as set forth in SEQ ID NO: 43, wherein the amino acid sequence of the monoclonal antibody is as set forth in SEQ ID NO: 47.
In another embodiment of the present invention there is provided a recombinant human anti-Rabies monoclonal antibody comprising a heavy chain polypeptide having the amino acid sequence having at least 80% homology to the amino acid sequence as set forth in SEQ ID NO: 45 and a light chain polypeptide having the amino acid sequence having at least 80% homology to the amino acid sequence as set forth in SEQ ID NO: 43, wherein the amino acid sequence of the monoclonal antibody is as set forth in SEQ ID NO: 47 is encoded by the nucleotide sequence as set forth in SEQ ID NO: 46.

In another embodiment of the present invention there is provided a recombinant human anti-Rabies monoclonal antibody comprising a heavy chain polypeptide having the amino acid sequence having at least 80% homology to the amino acid sequence as set forth in SEQ ID NO: 45 and a light chain polypeptide having the amino acid sequence having at least 80% homology to the amino acid sequence as set forth in SEQ ID NO: 43, wherein the antibody is antibody Fab fragment.

In yet another embodiment of the present invention there is provided an isolated polynucleotide encoding human anti-Rabies monoclonal antibody heavy chain having nucleotide sequence as set forth in SEQ ID NO: 44.

In still yet another embodiment of the present invention there is provided an isolated human anti-Rabies monoclonal antibody heavy chain polypeptide having amino acid sequence as set forth in SEQ ID NO: 45.

In further embodiment of the present invention there is provided an isolated polynucleotide encoding human anti-Rabies monoclonal antibody light chain having nucleotide sequence as set forth in SEQ ID NO: 42.

In still another embodiment of the present invention there is provided an isolated human anti-Rabies monoclonal antibody heavy chain polypeptide having amino acid sequence as set forth in SEQ ID NO: 43.

The present invention also provides an isolated polynucleotide encoding human anti-Rabies monoclonal antibody Fab fragment having nucleotide sequence as set forth in SEQ ID NO: 46.

A recombinant human anti-Rabies monoclonal antibody having polypeptide sequence as set forth in SEQ ID NO: 47.

In another embodiment of the present invention there is provided a recombinant vector comprising the polynucleotide as set forth in SEQ ID NO: 44 and SEQ ID NO: 42; or the polynucleotide as set forth in SEQ ID NO: 46.

The present invention further provides a recombinant host cell comprising the recombinant vector as claimed in claim 12.

The invention also provides use of the Fab fragment of the invention in the diagnosis, treating and post exposure prophylaxis of a subject at risk of developing a condition resuhing from rabies virus.

It should be understood that the following examples described herein are only for illustrative purposes and all the possible modifications or changes suggested to a person skilled in the art are included within the scope and purview of this application and appended claims.

EXAMPLES

EXAMPLE 1

Construction of Fab naive library

RNA Isolation

Peripheral blood lymphocytes from healthy donors were used as the source for antibody gene cloning. B lymphocytes were isolated on a FicoU-Paque gradient from a total of 2 liters of blood from 10 donors. The cell pellet was dissolved immediately in 50 ml of Qiazol (Qiagen). Total RNA was isolated using RNeasy mini Kit with optional step to eliminate the genomic DNA. The total RNA was frozen in water at -80°C for short term storage and vacuum dried RNA was prepared for long term storage.

cDNA Synthesis

In order to completely recover the entire antibody gene repertoire, cDNA was prepared with both random hexamers and Oligo dT as primer. 250 jig of total RNA for each primer with Superscript TM III first-strand Synthesis System from Invitrogen was used. The total RNA was denatured for 5 min at 65°C in the presence of 20 |ig of random primer or oligo dT (Invitrogen); subsequently dithiothreitol was added, as well as 250 ^M dNTP's, 800 units of Rnase OUT (40 units/^l), and 2,000 units of Moloney murine leukemia virus reverse transcriptase (200 units/^l) in a total volimie of 500 \i\. After 50 min at 50°C, the reaction was terminated by heating at 85°C for 5 min. The cDNA from the two reactions was mixed and concentrated by filtering (filter with Millipore with molecular mass cut of size 10 kDa) and stored in 100 ^l of water at -80°C freezer.

Amplification of heavy chain variable regions

IgM-derived heavy chain variable regions were obtained by a primary PCR with a HuIgM Reverse (SEQ ID NO: 1) and separate VH FOR (SEQ ID NO: 39) primers. The VH fi-agments were amplified with a combination of JHR primers (SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33) annealing to the 3' end of VH and separate VH FOR primers (SEQ ID NO: 39) annealing to the 5' end of corresponding purified PCR product; 150 ng of purified DNA fragment was used as template in a 100-^l reaction volume. The size of the amplified product is 354bps.

Amplification of heavy chain constant regions

CHI fi-agment of 261 bp of IgGl was amplified for the overlap PCR from vector pComb3X using primer CHIF (SEQ ID NO: 35) and FlagR (SEQ ID NO: 36) primers in the PCR.

Amplification of Fd fragment

Fd fragment of 615 bp size was obtained by overlap PCR with 100 ng of purified VH and CHI fragments in 5x100 \il PCR reactions for 7 cycles of 30 seconds at 94°C, 30 seconds at 50°C, and 2 min at 72''C without primer and another 15 cycles with primer VHF and FlagR (SEQ ID NO: 36).

Amplification of light chains
Light chains (kappa and lambda families) were amplified by PCR with a set of CK reverse (SEQ ID NO: 2) or CX reverse (SEQ ID NO: 3) primer annealing to the 3' end of the constant domain and Sfi-tagged FOR primer (SEQ ID NO: 34), priming at the 5' end of the VL-regions. PCR was performed in a volume of 100 fil using High Fidelity PCR Master TM from Roche and 500 pM of each primer for 28 cycles for 1 min at 94°C, 1 min at 55°C, and 2 min at 72°C. Nine separate IgM-derived VH amplifications were generated with 3 ^1 cDNA (equivalent to 6 \ig of PBL RNA) as template for each reaction. Six different kappa chain products and 11 lambda chain products were obtained and mixed from each family and gel-purified. Size of the amplified product is 648bps

Amplification of linker

For the preparation of linker between light chain and heavy chain, primer linkerF (SEQ ID NO: 37) and primer linkerR (SEQ ID NO: 38) were used. Size of the linker region is 24 bp.

All amplified products (Light chain variable with constant region of size 648 bps and Heavy chain variable with constant region of size 615 bps) were purified from agarose gel with the QIA quick Gel Extraction Kit (Qiagen) and the DNA concentration was determined through spectrophotometer measurement. The heavy chain (SEQ ID NO: 44) and light chain (SEQ ID NO: 42) were fused by three-fragment overlap PCR using 100 ng of purified light chain and heavy chain each and the same molar amount of linker (SEQ ID NO: 37) in 5x100 ^1 reactions. The fiill length Fab insert was first pre-amplified without flanking primers for 9 cycles of 30 seconds at 94°C, 30 seconds at 50°C, and 3 min at 72°C, and then amplified for another 15 cycles with the flanking primers SfiF (SEQ ID NO: 34) and FlagR (SEQ ID NO: 36) under the same conditions. The length of amplified product i.e. final FabB6 fragment is 1287 bps having nucleotide sequence as set forth in SEQ ID NO: 46.

For the construction of the Fab library, gel-purified overlapping PCR products containing the light chain and heavy chains were appended with Sfil on both sides prior to and after digestion with Sfil. The insert and vector were digested with Sfi I restriction enzyme. The gel-purified fragments (8 ^g with kappa light chain and 6 (jg with lambda light chain) were ligated into 8 ng and 6 ^g of Sfil digested and gel-purified phagemid vector pCombSX in 500 \i\ reactions, respectively. The DNA buffer system was changed to pure water using Micron filter with a 50 KD cut off size, and further concentrated to appropriate volume though DNA vacuum concentrator. The two desalted ligation mixtures were electroporated into competent TGI bacteria (Stratagene) with the Gene Pulser System II (Biorad); 100 µ l of the total DNA ligated preparation was mixed with 1.5 ml of TGI competent cells, distributed into 60 aliquots and each one put into 0.1 cm cuvette, and electroporated using the pre-set program with setting at 1.8 kV/ 200 ohms/25 nF. The transformed bacteria were spread on 16 large 2YT agar plates (24 cm x 24 cm) supplied with 100 |ig/ml ampicillin and 1% glucose, and incubated at 37°C overnight.
Table 1: Primers used for amplification of heavy and light chains µ

Wherein R = AorG,Y = CorT,W = AorT,M = AorC,S = CorG
Table 2: Enrichment of Rabies Virus Glycoprotein Specific Fab Clones In Three
Rounds of Biopanning

SEQ ID NO: 43 Amino acid sequence encoded by the nucleotide sequence of light chain sequence (216 a. a.)

SEQ ID NO: 45 Amino acid sequence encoded by the nucleotide sequence of heavy chain sequence (205 a. a.)

SEQ ID NO: 46 nucleotide sequence encoding recombinant human anti-Rabies monoclonal antibody fragment- FabB6 (1287 nts) (Light chain: 1-648; Linker: 649-672 and heavy chain: 673-1287)

SEQ ID NO: 47 Amino acid sequence of recombinant human anti-Rabies monoclonal antibody fragment- FabB6 (Light chain: 1-216; 225-429)

SEQ ID NO: 48 (24bps) Nucleotide sequence of linker

Wherein R = AorG,Y = CorT,W = AorT,M = AorC,S = CorG
Table 2: Enrichment of Rabies Virus Glycoprotein Specific Fab Clones In Three
Rounds of Biopanning

(648 nts)
CAGTCTGTGCTGACGCAGCCGCCCTCAGTGTCTGCGGCCCCAGGACAGAG
GGTCGCCATCTCCTGCTTTGGAAGCACCTCCAACATTGCCAGTAATTTTGT
GTCGTGGTACCGAGTGCTCCCAGGAACAGCCCCCAAGCTCGTCATTTTTGA
CAATAATCAGCGACCCTCAGACATTCCTGACCGATTCTCTGTCTCCAAGTC
TGGCACGTCAGCCACCCTGGGCATCACCGGACTCCAGACTGGGGACGAGG
CCGACTACTATTGCGGAACATGGGATAGCGCCCTGAGTGCCTGGGTATTCG
GCGGAGGGACCAAGCTGACCGTCCTGAGTCAGCCCAAGGCTGCCCCCTCG
GTCACTCTGTTCCCACCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACA
CTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGG
AAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTC
CAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGC CTGAGCAGTGGAAGTCCCACAAAAGCTACAGCTGCCAGGTCACGCATGAA GGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA
SEQ ID NO: 43 Amino acid sequence encoded by the nucleotide sequence of light chain sequence (216 a. a.)
QSVLTQPPSVSAAPGQRVAISCFGSTSNIASNFVSWYRVLPGTAPKLVIFDNNQ
RPSDIPDRFSVSKSGTSATLGITGLQTGDEADYYCGTWDSALSAWVFGGGTKL
TVLSQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVK
AGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAP
TECS
SEQ ID NO: 44 Nucleotide sequence of heavy chain sequence with constant region
CTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCCTGAGACT
CTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATAGCATGAACTGGGT
CCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAGTAGTA
GTAGTAGTTACATATACTACGCAGACTCAGTGAAGGGCCGATTCACCATCT
CCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGA
GCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATAGGGTAGTAGTACC
ACCCTACTACATGGACGTCTGGGGCAAAGGGACCACGATCACCGTCTCCT
CAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGA
GCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTC
CCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGT
GCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAG
CGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAA
CGTGAATCAC
SEQ ID NO: 45 Amino acid sequence encoded by the nucleotide sequence of heavy chain sequence (205 a. a.)
LVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSS YIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRVVVPPYY MDVWGKGTTITVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
SEQ ID NO: 46 nucleotide sequence encoding recombinant human anti-Rabies monoclonal antibody fragment- FabB6 (1287 nts) (Light chain: 1-648; Linker: 649- 672 and heavy chain: 673-1287)
CAGTCTGTGCTGACGCAGCCGCCCTCAGTGTCTGCGGCCCCAGGACAGAG
GGTCGCCATCTCCTGCTTTGGAAGCACCTCCAACATTGCCAGTAATTTTGT
GTCGTGGTACCGAGTGCTCCCAGGAACAGCCCCCAAGCTCGTCATTTTTGA
CAATAATCAGCGACCCTCAGACATTCCTGACCGATTCTCTGTCTCCAAGTC
TGGCACGTCAGCCACCCTGGGCATCACCGGACTCCAGACTGGGGACGAGG
CCGACTACTATTGCGGAACATGGGATAGCGCCCTGAGTGCCTGGGTATTCG
GCGGAGGGACCAAGCTGACCGTCCTGAGTCAGCCCAAGGCTGCCCCCTCG
GTCACTCTGTTCCCACCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACA
CTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGG
AAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTC
CAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGC
CTGAGCAGTGGAAGTCCCACAAAAGCTACAGCTGCCAGGTCACGCATGAA
GGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCATAATTCTA
GATAATTAATTAGGAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTG
GGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCT
ATAGCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTC
TCATCCATTAGTAGTAGTAGTAGTTACATATACTACGCAGACTCAGTGAAG
GGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCA
AATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAG
ATAGGGTAGTAGTACCACCCTACTACATGGACGTCTGGGGCAAAGGGACC
ACGATCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTG
GCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCT
GGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCG
CCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGAC
TCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCC
AGACCTACATCTGCAACGTGAATCAC
SEQ ID NO: 47 Amino acid sequence of recombinant human anti-Rabies monoclonal antibody fragment- FabB6 (Light chain: 1-216; 225-429)
QSVLTQPPSVSAAPGQRVAISCFGSTSNIASNFVSWYRVLPGTAPKLVIFDNNQ
RPSDIPDRFSVSKSGTSATLGITGLQTGDEADYYCGTWDSALSAWVFGGGTKL
TVLSQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVK
AGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAP
TECS*F*IIN*ELVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGL
EWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR
DRVVVPPYYMDVWGKGTTITVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC
NVNH
SEQ ID NO: 48 (24bps) Nucleotide sequence of linker TAATTCTAGATAATTAATTAGGAG

We Claim:

1. A recombinant human anti-Rabies monoclonal antibody comprising a heavy chain polypeptide having the amino acid sequence having at least 80% homology to the amino acid sequence as set forth in SEQ ID NO: 45 and a light chain polypeptide having the amino acid sequence having at least 80% homology to the amino acid sequence as set forth in SEQ ID NO: 43.

2. The human anti-Rabies monoclonal antibody as claimed in claim 1, wherein the amino acid sequence of the heavy chain polypeptide is as set forth in SEQ ID NO: 45 and the amino acid sequence of the light chain polypeptide is as set forth in SEQ ID NO: 43.

3. The human anti-Rabies monoclonal antibody as claimed in claim 1, wherein the amino acid sequence of the monoclonal antibody is as set forth in SEQ ID NO: 47.

4. The human anti-Rabies monoclonal antibody as claimed in claim 3, wherein the amino acid sequence of the monoclonal antibody is as set forth in SEQ ID NO: 47 is encoded by the nucleotide sequence as set forth in SEQ ID NO: 46.

5. The human anti-Rabies monoclonal antibody as claimed in claim 1, wherein the antibody is antibody Fab fragment.

6. An isolated polynucleotide encoding human anti-Rabies monoclonal antibody heavy chain having nucleotide sequence as set forth in SEQ ID NO: 44.

7. An isolated human anti-Rabies monoclonal antibody heavy chain polypeptide having amino acid sequence as set forth in SEQ ID NO: 45.

8. An isolated polynucleotide encoding human anti-Rabies monoclonal antibody light chain having nucleotide sequence as set forth in SEQ ID NO: 42.

9. An isolated human anti-Rabies monoclonal antibody heavy chain polypeptide having amino acid sequence as set forth in SEQ ID NO: 43.

10. A recombinant polynucleotide encoding human anti-Rabies monoclonal antibody
Fab fragment having nucleotide sequence as set forth in SEQ ID NO: 46.

11. A recombinant human anti-Rabies monoclonal antibody having polypeptide
sequence as set forth in SEQ ID NO: 47.

12. A recombinant vector comprising the polynucleotide as set forth in SEQ ID NO: 44 and SEQ ID NO: 42; or the polynucleotide as set forth in SEQ ID NO: 46.

13. A recombinant host cell comprising the recombinant vector as claimed in claim