FORM 2
THE PATENTS ACT, 1970
(Act 39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(SECTION 10 & Rule 13)
1. TITLE OF THE INVENTION
MONOCLONAL ANTIBODIES AGAINST CHIKUNGUNYA
2. Applicant(s)

NAME

NATIONALITY

ADDRESS

TECHINVENTION LIFECARE PVT. LTD.

India

1004, The Summit Business Park,
Andheri East, Mumbai 400093, India

3. PREAMBLE TO THE DESCRIPTION
COMPLETE SPECIFICATION
The following specification particularly describes the invention and the manner in which
it is to be performed.

FIELD OF THE INVENTION
[0001] The present invention relates to Bio-pharmaceuticals. Specifically, the present invention relates to an isolated fully human monoclonal antibody or monoclonal antibody fragment or an immuno conjugate against Chikungunya. Further, the present invention relates to monoclonal antibodies against Chikungunya virus (CHIKV) or an antigen-binding fragment of the monoclonal antibody, its composition and uses thereof. The present invention particularly relates to binding molecules against Chikungunya virus. The said monoclonal antibodies are capable of neutralizing Chikungunya virus infectivity. The present invention further relates to the use of said monoclonal antibodies in the treatment or prevention or diagnosis of Chikungunya fever. The invention also relates to a pharmaceutical composition comprising said monoclonal antibodies for use in the treatment or prevention or diagnosis of Chikungunya fever.
BACKGROUND OF THE INVENTION
[0002] Background description includes information that may be useful in
understanding the present invention. It is not an admission that any of the information
provided herein is prior art or relevant to the presently claimed invention, or that any
publication specifically or implicitly referenced is prior art.
[0003] Chikungunya is an infection caused by the Chikungunya virus. It features
sudden onset fever usually lasting two to seven days, and joint pains typically lasting
weeks or months but sometimes years. The mortality rate is a little less than 1 in 1000,
with the elderly most likely to die. The virus is passed to humans by two species of
mosquito of the genus Aedes: A. albopictus and A. aegypti. Animal reservoirs of the
virus include monkeys, birds, cattle, and rodents. This is in contrast to dengue, for
which only primates are hosts.
[0004] Chikungunya has emerged as epidemic threat over the past two decades
leading to serious global health problems. As on today there are no therapies or
vaccines available for the treatment of Chikungunya virus.
[0005] There is no specific antiviral drug treatment for Chikungunya. The clinical
management targets primarily to relieving the symptoms, including the joint pain
using anti-pyretic, optimal analgesics, drinking plenty of fluids and general rest. There
is no commercial vaccine available to protect against Chikungunya virus infection.
While there are several vaccine strategies being pursued (as of mid-2020), of which

some are in various stages of clinical trials, they are still several years away from being licensed and available to the public.
[0006] There is increasing need for the discovery of the specific monoclonal antibodies which can neutralize the virus (Kumar et.al. Appl. Microbiol. Biotechnol. 2020 Apr; 104 (8):3209-3228). Monoclonal antibody phage display (APD) is based on genetic engineering of bacteriophages (viruses that infect bacteria) and repeated rounds of antigen-guided selection and phage propagation (Barbas CF. Phage display : a laboratory manual). This technique allows in vitro selection of mAbs of virtually any specificity which can be used for therapeutic purpose. Adalimumab is the first fully human APD-derived mAb (Lee et. al. Nat Protoc. 2007; 2(11):3001-83). Various types of libraries have been utilized for selection of specific antibodies. The single-pot universal libraries are extremely assorted and allow the selection of monoclonal antibody fragments with binding affinities to a wide range of antigens due to the size of their repertoire (Rajput et. al, Mater Methods. 2014;4: 873; Hoogenboom et.al. Immunotechnology. 1998; 4: 1-20).
[0007] Phage display based in vitro affinity selection of monoclonal antibodies resembles the clonal selection of monoclonal antibodies in vivo. The target antigen is immobilized on surfaces based on the choice of the researcher and the library is applied onto it, enabling affinity-based selection of highly reactive antibodies. For a further selection of monoclonal antibodies, the phage clones obtained after final round are propagated individually and characterized for desired binding specificities (Rodi et. al, Curr Opin Biotechnol. 1999;10: 87-93.; Willats, Plant Mol Biol. 2002;50: 837-54).
[0008] It is reported that two alpha virus glycoproteins viz E1 & E2, both containing a single transmembrane domain are responsible for virus attachment (Feng et. al, Proc Natl Acad Sci USA. 2015 Nov 10; 112(45):13898-903; Sun et.al. eLife. 2013; 2: e00435).
[0009] Chikungunya, a debilitating zoonotic disease caused by Chikungunya virus (CHIKV) does not have any approved antiviral treatment. So far, there is no approved vaccine or monoclonal antibody for the treatment of Chikungunya, this disease is a big threat to mankind globally. The development of single-chain variable fragments (scFv) monoclonal antibodies or full IgG mAbs against E1 & E2 of the virus could be a potential antiviral drug, which can effectively neutralize Chikungunya virus. Novel & efficacious monoclonal antibodies against CHIKV antigens E1 and E2 can be

potential target for disease prevention, therapeutic as well for diagnostic purposes against chikungunya infection.
[00010] Although there are different companies working on Chikungunya treatment methods, there is still need to explore the novel ways and compositions of treating Chikungunya that could be more economical, easily manufactured and scalable and can overcome deficiencies associated with the known arts. The present invention provides a novel, specific and industrially advantageous monoclonal antibodies that are capable of neutralizing Chikungunya virus infectivity. These monoclonal antibodies shall be fulfilling the unmet medical need for Chikungunya prophylaxis and treatment.
OBJECTS OF THE INVENTION
[00011] An object of the present invention is to provide an isolated fully human
monoclonal antibody or monoclonal antibody fragment or an immuno conjugate
against Chikungunya.
[00012] Another object of the present invention is to provide an isolated fully human
monoclonal antibody or monoclonal antibody fragment or an immuno conjugate
against Chikungunya.
[00013] Yet another object of the present invention is to provide a monoclonal
antibody comprising of heavy chain selected from Sequence ID:1- 17 or light chain
selected from Sequence ID: 18- 40.
[00014] In another object of the present invention is to provide a monoclonal antibody
comprising of heavy chain selected from Sequence ID: 1, 2, 11, 15, 17 or light chain
selected from Sequence ID: 18, 19, 28, 35, 37, 39.
[00015] In another object of the present invention is to provide a monoclonal
antibody, wherein the antibody is selected from the group of:
a. Antibody 1 (Heavy Chain Sequence ID: 2, Light Chain sequence ID: 19);
b. Antibody 2 (Heavy Chain Sequence ID: 17, Light Chain sequence ID: 39);
c. Antibody 3 (Heavy Chain Sequence ID: 2, Light Chain sequence ID: 35);
d. Antibody 4 (Heavy Chain Sequence ID: 2, Light Chain sequence ID: 37);
e. Antibody 5 (Heavy Chain Sequence ID: 15, Light Chain sequence ID: 19);
f. Antibody 6 (Heavy Chain Sequence ID: 1, Light Chain sequence ID: 18); or
g. Antibody 7 (Heavy Chain Sequence ID: 11, Light Chain sequence ID: 28).

[00016] Another object of invention is to provide a monoclonal antibody or
monoclonal antibody fragment or an immuno conjugate against Chikungunya that
bind to antigen envelope protein E1 and E2.
[00017] Further another object of invention is to provide monoclonal antibodies with
Chikungunya virus neutralizing properties which make them particularly useful for
the treatment or prevention or diagnosis of Chikungunya fever.
[00018] An object of the present invention is to provide composition comprising
monoclonal antibodies against Chikungunya.
[00019] An object of the present invention is to provide composition comprising
monoclonal antibodies against Chikungunya further containing a pharmaceutically
acceptable carrier, diluent or adjuvant.
[00020] Another object of the present invention is to provide a method of production
of a monoclonal antibody against Chikungunya.
[00021] Another object of the present invention is to provide a monoclonal antibody
potently neutralizes Chikungunya virus by binding on E1 and E2 envelope protein.
[00022] Still another object of invention is to provide composition comprising
monoclonal antibodies selected from SEQ ID Nos:. 1 -40.
[00023] In still another object of invention is to provide composition comprising
monoclonal antibody wherein the antibody is selected from the group of:
a. Antibody 1 (Heavy Chain Sequence ID: 2, Light Chain sequence ID: 19);
b. Antibody 2 (Heavy Chain Sequence ID: 17, Light Chain sequence ID: 39);
c. Antibody 3 (Heavy Chain Sequence ID: 2, Light Chain sequence ID: 35);
d. Antibody 4 (Heavy Chain Sequence ID: 2, Light Chain sequence ID: 37);
e. Antibody 5 (Heavy Chain Sequence ID: 15, Light Chain sequence ID: 19);
f. Antibody 6 (Heavy Chain Sequence ID: 1, Light Chain sequence ID: 18); or
g. Antibody 7 (Heavy Chain Sequence ID: 11, Light Chain sequence ID: 28).
[00024] In one of the object of the present invention is to provide an isolated fully
human monoclonal antibody or monoclonal antibody fragment or an immuno
conjugate of the invention as a medicament. Moreover, the present invention relates
in a further aspect to an isolated fully human monoclonal antibody or monoclonal
antibody fragment, an immuno conjugate and/or the pharmaceutical composition of
the invention for use in the prevention or treatment or diagnosis of the infection of the
Chikungunya virus.

[00025] Another object of the present invention is to provide a fully human monoclonal antibody composition having the above detailed monoclonal antibody in combination with a second monoclonal antibody which binds simultaneously to the spike for neutralizing the Chikungunya virus.
SUMMARY OF THE INVENTION
[00026] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in Detailed Description section. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.
[00027] The present invention relates to a monoclonal antibody comprising of heavy chain selected from Sequence ID:1- 17 or light chain selected from Sequence ID: 18-40.
[00028] The present invention relates to a monoclonal antibody comprising of heavy chain selected from Sequence ID: 1, 2, 11, 15, 17 or light chain selected from Sequence ID: 18, 19, 28, 35, 37, 39.
[00029] The present invention provides a monoclonal antibody, wherein the antibody is selected from the group of:
a. Antibody 1 (Heavy Chain Sequence ID: 2, Light Chain sequence ID: 19);
b. Antibody 2 (Heavy Chain Sequence ID: 17, Light Chain sequence ID: 39);
c. Antibody 3 (Heavy Chain Sequence ID: 2, Light Chain sequence ID: 35);
d. Antibody 4 (Heavy Chain Sequence ID: 2, Light Chain sequence ID: 37);
e. Antibody 5 (Heavy Chain Sequence ID: 15, Light Chain sequence ID: 19);
f. Antibody 6 (Heavy Chain Sequence ID: 1, Light Chain sequence ID: 18); or
g. Antibody 7 (Heavy Chain Sequence ID: 11, Light Chain sequence ID: 28).
[00030] The present invention relates to an isolated fully human monoclonal antibody
or monoclonal antibody fragment or an immuno conjugate against Chikungunya.
[00031] In one aspect, the present invention relates to an isolated fully human
monoclonal antibody or monoclonal antibody fragment or an immuno conjugate
against Chikungunya that bind to antigen envelope protein E1 and E2.
[00032] In another aspect, the present invention relates to fully human monoclonal antibodies with Chikungunya virus neutralizing properties which make them particularly useful for the treatment or prevention or diagnosis of Chikungunya fever.

[00033] In one aspect, the present invention relates to composition comprising
monoclonal antibodies against Chikungunya.
[00034] In yet another aspect, the present invention relates to composition comprising
monoclonal antibodies against Chikungunya further containing a pharmaceutically
acceptable carrier, diluent or adjuvant.
[00035] In one aspect, the present invention relates to a method of production of a
fully human monoclonal antibody against Chikungunya.
[00036] In one aspect, the present invention relates to a fully human therapeutic
monoclonal antibody potently neutralizes Chikungunya virus by binding on E1 and
E2 envelope protein.
[00037] Still another object of invention is to provide composition comprising
monoclonal antibodies selected from SEQ ID Nos: 1 -40
[00038] In further one aspect, the present invention relates to an isolated fully human
monoclonal antibody or monoclonal antibody fragment or an immuno conjugate of
the invention as a medicament. Moreover, the present invention relates in a further
aspect to an isolated fully human monoclonal antibody or monoclonal antibody
fragment, an immuno conjugate and/or the pharmaceutical composition of the
invention for use in the prevention or treatment or diagnosis of the infection of the
Chikungunya virus.
[00039] In another one aspect, the present invention relates to a fully human
monoclonal antibody composition having the above detailed monoclonal antibody in
combination with a second monoclonal antibody which binds simultaneously to the
envelope protein (E1 & E2) for neutralizing the Chikungunya virus.
DRAWINGS
[00040] The following drawings form part of the present specification and are
included to further illustrate aspects of the present disclosure. The disclosure may be
better understood by reference to the drawings in combination with the detailed
description of the specific embodiments presented herein.
Figure 1: Gel Electrophoresis results of Total RNA Isolation
Figure 2: Gel Electrophoresis results of Primary PCR amplification
Figure 3: Gel Electrophoresis results of Secondary PCR amplification
Figure 4 (a-f): Novel Antibodies against E 1 & E2 antigens
Figure 5(a-b): Gel Screening results for E1 and E2 clones

Figure 6: RMSD of the E1 protein - antibody complex during simulation
Figure 7: RMSD of the E2 protein - antibody complex during simulation
Figure 8 (a-f): Chromatogram results after purification
Figure 9: PAGE Analysis Results
Figure 10: ELISA showing binding of chikungunya IgG to antigen
Figure 11: CHIKV micro PRNT plate neutralization results
DETAILED DESCRIPTION OF THE INVENTION
[00041] The following is a detailed description of embodiments of the disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.
[00042] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.
[00043] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
[00044] In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term "about." Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters

should be construed in light of the number of reported significant digits and by
applying ordinary rounding techniques. Notwithstanding that the numerical ranges
and parameters setting forth the broad scope of some embodiments of the invention
are approximations, the numerical values set forth in the specific examples are
reported as precisely as practicable. The numerical values presented in some
embodiments of the invention may contain certain errors necessarily resulting from
the standard deviation found in their respective testing measurements.
[00045] As used in the description herein and throughout the claims that follow, the
meaning of "a," "an," and "the" includes plural reference unless the context clearly
dictates otherwise.
[00046] Also, as used in the description herein, the meaning of "in" includes "in" and
"on" unless the context clearly dictates otherwise.
[00047] Unless the context requires otherwise, throughout the specification which
follow, the word "comprise" and variations thereof, such as, "comprises" and
"comprising" are to be construed in an open, inclusive sense that is as "including, but
not limited to."
[00048] The recitation of ranges of values herein is merely intended to serve as a
shorthand method of referring individually to each separate value falling within the
range. Unless otherwise indicated herein, each individual value is incorporated into
the specification as if it were individually recited herein. All methods described herein
can be performed in any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples, or exemplary
language (e.g. "such as") provided with respect to certain embodiments herein is
intended merely to better illuminate the invention and does not pose a limitation on
the scope of the invention otherwise claimed. No language in the specification should
be construed as indicating any unclaimed element essential to the practice of the
invention.
[00049] The description that follows, and the embodiments described therein, is
provided by way of illustration of an example, or examples, of particular embodiments
of the principles and aspects of the present disclosure. These examples are provided
for the purposes of explanation, and not of limitation, of those principles and of the
disclosure.
[00050] The headings and abstract of the invention provided herein are for
convenience only and do not interpret the scope or meaning of the embodiments.

[00051] Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.
[00052] The term "monoclonal antibody” ,”antibody” or “mAb “ according to the present invention means, but not limited to, the antibody that the colony of the antibody of basically homology obtains, that is, each antibody forming described colony is all identical except the possible naturally occurring sudden change that may exist with small quantity. Monoclonal antibody has high degree of specificity for single antigen. In addition, different from the polyclonal antibody preparations. [00053] Monoclonal antibodies of the invention include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific monoclonal antibodies (including bi-specific antibodies), human antibodies, fully human antibodies, chimeric antibodies, intra-bodies, single-chain Fvs (scFv) (e.g., including monospecific, bispecific, etc.), camelized antibodies, Fab fragments, F(ab′) fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. The monoclonal antibodies of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), or any subclass (e.g., IgG2a and IgG2b) of immuno globulin molecule.
[00054] The variable heavy chain portion of the Fab is coded for by a combination of 3 genes, called VH (variable heavy), DH (diversity heavy), and JH (joining heavy). The variable light chain portion of the Fab consists of either a kappa chain or a lambda chain coded for by a combination of 2 genes, VL (variable light) and JL (joining light). In the DNA of each B-lymphocyte there are multiple forms of each one of these variable determinant genes. Although the exact number of each gene isn't known and varies from person, there are approximately 38-46 VH genes; 23 DH genes; 6 JH genes; 34-38 kappa VL genes; 5 kappa JL genes; 29-33 lambda VL genes; and 4-5 lambda JL genes.
[00055] The term “fully human monoclonal antibody” or “human monoclonal antibody” according to the present invention means, but not limited to, refers to an monoclonal antibody that comprises a human variable region and, most preferably a human constant region. In specific embodiments, the terms refer to an monoclonal antibody that comprises a variable region and constant region of human origin.

[00056] The term “isolated” according to the present invention means, but not limited to, refers to nucleic acid molecule which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In a specific embodiment, a nucleic acid molecule(s) encoding an monoclonal antibody of the invention is isolated or purified. [00057] The term "complementarity determining regions" (CDR) according to the present invention means, but not limited to, refers to sequences within the variable regions of binding proteins, such as immunoglobulins, that usually contribute to a large extent to the antigen binding site which is complementary in shape and charge distribution to the epitope recognized on the antigen. The CDR regions can be specific for linear epitopes, discontinuous epitopes, or conformational epitopes of proteins or protein fragments, either as present on the protein in its native conformation or, in some cases, as present on the proteins as denatured, e. g., by solubilisation in SDS. Epitopes may also consist of posttranslational modifications of proteins.
[00058] The term “Bio-panning” according to the present invention means, but not limited to, refers to an affinity selection technique which selects for peptides that bind to a given target. This technique is often used for the selection of antibodies. Monoclonal antibody:
[00059] The present invention relates to a monoclonal antibody or monoclonal antibody fragment or an immuno conjugate.
[00060] In one embodiment, the present invention provides a monoclonal antibody comprising of heavy chain selected from Sequence ID:1- 17 or light chain selected from Sequence ID: 18- 40.
[00061] In another embodiment, the present invention provides a monoclonal antibody comprising of heavy chain selected from Sequence ID: 1, 2, 11, 15, 17 or light chain selected from Sequence ID: 18, 19, 28, 35, 37, 39.
[00062] In still another embodiment the present invention provides a monoclonal antibody, wherein the antibody is selected from the group of:
a. Antibody 1 (Heavy Chain Sequence ID: 2, Light Chain sequence ID: 19);
b. Antibody 2 (Heavy Chain Sequence ID: 17, Light Chain sequence ID: 39);
c. Antibody 3 (Heavy Chain Sequence ID: 2, Light Chain sequence ID: 35);

d. Antibody 4 (Heavy Chain Sequence ID: 2, Light Chain sequence ID: 37);
e. Antibody 5 (Heavy Chain Sequence ID: 15, Light Chain sequence ID: 19);
f. Antibody 6 (Heavy Chain Sequence ID: 1, Light Chain sequence ID: 18); or
g. Antibody 7 (Heavy Chain Sequence ID: 11, Light Chain sequence ID: 28).
[00063] In one embodiment, the present invention provides an isolated fully human
monoclonal antibody or monoclonal antibody fragment or an immuno conjugate
against Chikungunya that bind to antigen envelope protein E1 and E2.
[00064] In one embodiments, the present invention provides fully human monoclonal antibodies with Chikungunya virus neutralizing properties which make them particularly useful for the treatment or prevention or diagnosis of Chikungunya fever. [00065] In one embodiment, the present invention provides a fully human therapeutic monoclonal antibody potently neutralizes Chikungunya virus by binding on E1 and E2 envelope protein.
[00066] Still another embodiment the present invention provide composition comprising monoclonal antibodies with sequences selected from SEQ ID Nos: 1-27. [00067] In further embodiment, the present invention provides an isolated fully human monoclonal antibody or monoclonal antibody fragment or an immuno conjugate of the invention as a medicament. Moreover, the present invention relates in a further aspect to an isolated fully human monoclonal antibody or monoclonal antibody fragment, an immuno conjugate and/or the pharmaceutical composition of the invention for use in the prevention or treatment or diagnosis of the infection of the Chikungunya virus. Host:
[00068] In one of the embodiment the present invention provides a method of production of a fully human monoclonal antibody against Chikungunya, said method comprises use of host cells selected from, but not limited to, cells of mammalian, plant, insect, fungal or bacterial origin. Host cells of the present invention include but are not limited to eukaryotic cells such as mammalian cells, e.g. hamster, rabbit, rat, pig, mouse, etc.; avian cells, e.g. duck, chicken, quail, etc.; insect cells or other animal cells; plant cells and fungal cells, e.g. corn, tobacco, Saccharomyces cerevisiae, Pichia pastoris; prokaryotic cells such as E. coli; and other cells used in the art for the production of monoclonal antibodies and other binding proteins.

Process:
[00069] In one embodiment, the present invention provides a method of production
of a fully human monoclonal antibody against Chikungunya.
[00070] In one of the embodiments the present invention provides a method of
production of a fully human monoclonal antibody against Chikungunya, said method
comprising at least one of the step of;
a) Blood Collection
b) RNA Isolation
c) Monoclonal antibody Genes Amplification by PCR
d) Monoclonal antibody Genes Cloning and scFv library banking
e) Monoclonal antibody Selection by Bio-panning
f) Screening by ELISA and Restriction Digestion
g) DNA sequencing
h) Identification & In-silico validation of antibodies
i) Antibody engineering & conversion of potential single chain antibody into IgG
j) Expression & Purification in IgG Format
Composition/Formulation:
[00071] In one embodiment, the present invention provides composition comprising
at least one of an isolated fully human monoclonal antibody or monoclonal antibody
fragment or an immuno conjugate of the invention.
[00072] In one embodiment, the present invention provides composition comprising
monoclonal antibody wherein the antibody is selected from the group of:
a. Antibody 1 (Heavy Chain Sequence ID: 2, Light Chain sequence ID: 19);
b. Antibody 2 (Heavy Chain Sequence ID: 17, Light Chain sequence ID: 39);
c. Antibody 3 (Heavy Chain Sequence ID: 2, Light Chain sequence ID: 35);
d. Antibody 4 (Heavy Chain Sequence ID: 2, Light Chain sequence ID: 37);
e. Antibody 5 (Heavy Chain Sequence ID: 15, Light Chain sequence ID: 19);
f. Antibody 6 (Heavy Chain Sequence ID: 1, Light Chain sequence ID: 18); or
g. Antibody 7 (Heavy Chain Sequence ID: 11, Light Chain sequence ID: 28).
[00073] In one embodiment, the present invention provides composition comprising
at least one of an isolated fully human monoclonal antibody or monoclonal antibody
fragment or an immuno conjugate of the invention useful for the treatment or
prevention or diagnosis of Chikungunya fever.

[00074] In yet another embodiment, the present invention provides composition
comprising at least one of an isolated fully human monoclonal antibody or monoclonal
antibody fragment or an immuno conjugate of the invention further containing a
pharmaceutically acceptable carrier, diluent or adjuvant.
[00075] In yet another embodiment, the present invention provides composition
comprising at least one of an isolated fully human monoclonal antibody or monoclonal
antibody fragment or an immuno conjugate of the invention as active ingredient and
further comprising pharmaceutically acceptable carrier selected from but not limited
to, buffer, antioxidant, preservative, isotonic agent and chelating agent.
[00076] In another one the present invention, the present invention provides a fully
human monoclonal antibody composition having the above detailed monoclonal
antibody in combination with a second monoclonal antibody which binds
simultaneously to the spike for neutralizing the Chikungunya virus.
[00077] Another object of the present invention is to provide a fully human
monoclonal antibody cocktail comprising monoclonal antibodies with sequence
selected from SEQ ID Nos: 1-40 in combination with other antibodies.
Diagnostics
[00078] In another one the present invention, the present invention provides a
diagnostic kit containing at least one of an isolated fully human monoclonal antibody
or monoclonal antibody fragment or an immuno conjugate of the invention.
[00079] In another one the present invention, the present invention provides a
diagnostic kit containing at least one of an isolated fully human monoclonal antibody
or monoclonal antibody fragment or an immuno conjugate of the invention which are
packed in suitable containers and labelled for diagnosis, prevention and/or treatment
of Chikungunya.
[00080] In further embodiment, the present invention provides method for detecting
the presence or absence of a Chikungunya virus (CHIKV) strain in a sample,
comprising the steps of:
a) contacting the sample with an monoclonal antibody of the present invention or with a combination of anti-CHIKV monoclonal antibodies of the invention to form an immune complex; and
b) detecting the presence or absence of the immune complex formed in a).

[00081] Yet another object of the present invention is to provide the potent combination or cocktail of monoclonal antibodies comprising monoclonal antibodies with sequence selected from SEQ ID Nos: 1-40 with other monoclonal antibodies as the therapeutic or diagnostic reagents and methods of making the same for the treatment of Chikungunya virus (CHIKV).
[00082] EXAMPLES
[00083] The present invention is further explained in the form of following examples. However, it is to be understood that the following examples are merely illustrative and are not to be taken as limitations upon the scope of the invention.
[00084] Step 1: Blood Collection
10 Volunteers and 20 heath workers were selected as donors for blood collection. Thirty (30) ml of whole blood was collected in vacutainers and was processed immediately for isolation of lymphocytes by Ficoll Paque separation method. The isolated lymphocytes were counted using Neubauer chamber under inverted microscope and the lymphocyte were stored at -80oC, if not used immediately, for further use. The lymphocyte count details are as per table 1 and 2
a) FROM VOLUNTEERS.
[00085] Table 1: Lymphocytes Count For Individual Donor

Sr. No Donor ID Lymphocyte count
1 IK ~ 25 Million
2 SS ~27 Million
3 SHS ~24 Million
4 TS ~26 Million
5 TP ~27 Million
6 SA ~23 Million
7 MR ~28 Million
8 AA ~ 32 Million
9 RN ~31 Million
10
NS
~26 Million

b) FROM HEALTH CARE WORKER’S FOR ANTIBODY NAÏVE LIBRARY Table 2: Lymphocytes Count for Individual Donor

SR.NO DONOR's INITIALS Lymphocyte Count
1 RB-1 24 million
2 SA-2 27 million
3 RAB-3 28 million
4 FA-4 31 million
5 CT-5 18 million
6 IS-6 31 million
7 RC-7 25 million
8 WT-8 29 million
9 AS-9 22 million
10 AN-10 31 million
11 PS-11 36 million
12 SI-12 28 million
13 RS-13 28 million
14 AMS-14 28 million
15 BD-15 26 million
16 SS-16 32 million
17 KK-17 28 million
18 YM-18 22 million
19 SN-19 26 million
20 MP-20 31 million
[00086] Step 2: RNA Isolation
[00087] The isolated lymphocyte in step 1 were further processed for Total RNA isolation. The Trizol solubilization and extraction method was employed. The isolated samples were further checked by Gel electrophoresis on 1 % agarose gel (Fig.1). All the RNA samples were stored at -80oC, RNAse inhibitor were added in each RNA sample before storing at -80oC.

[00088] Step 3: Monoclonal antibody Genes Amplification by PCR
[00089] The isolated RNA samples of step 2 were used for cDNA preparation using One Step RT PCR Kit, (Qiagen, Germany) followed by PCR amplification of monoclonal antibody genes viz: Heavy chain and Light chain by using specific primer sets. The kit contains optimized components that allow both reverse transcription and PCR amplification to take place.
Nested PCR was used to increase the sensitivity and/or specificity of PCR. The primary PCR was performed by using the first set of primers which resulted in products of about 450 bp and of about 350bp (Fig.2). Secondary PCR amplification was performed with different set of primer on the DNA sequence obtained as a result of primary PCR amplification. The result of secondary PCR amplification are captured in (Fig 3). The amplified product was used for further cloning steps.
[00090] Step 4: Monoclonal antibody Genes Cloning and scFv library banking
The PCR amplified genes of step 3 were cloned in phagemid vector (pSEX, Progen) which were then transformed into E. coli XL1B strain. The transformants were selected by growing on 2YT media with antibiotic resistance. The bacteriophage M13K07 (Invitrogen, Germany) was used as helper phage for the expression of different monoclonal antibody clones. The pool of these transformants was used to prepare pool of recombinant phage particles which can be used for bio-panning. The pool were constructed into scFv format.
[00091] Step 5: Monoclonal antibody Selection by Bio-panning
[00092] Bio-panning process was used for the selection of monoclonal antibodies with desired specificities. The antigens from Chikungunya virus viz: E1 & E2 (Sino Biological Inc), the target proteins coated on immune tubes and recombinant phages prepared from above pool of step 4 were allowed to bind to coated target antigens. After removal of unbound phage with various washing steps, the bound phages were eluted and used for next round of bio-panning. The enrichment of bound phages was done by three rounds of bio-panning with reduced concentrations of target antigens. The antigen-specific phages can then be amplified in a proper strain of E. coli for their downstream applications or for the next selection cycle facilitating further enrichment.

[00093] Step 6: Screening by ELISA and Restriction Digestion
[00094] The recombinant clones obtained after three rounds of bio-panning in step 5 were screened (more than 2000) for their antigen specificity by soluble ELISA. In this screening procedure the target antigens (E1 & E2) were coated on ELISA plate against which the periplasmic preparations of the clones expressing scFv were allowed to bind (Fig 4 (a-f)). The un-bound periplasms were removed by multiple washing steps. The bound scFv were detected using anti cMYC-HRP monoclonal antibody. After ELISA screening, 60 clones were found positive for E1 and E2 proteins. Gel electrophoresis was carried out to visualize the bands of these positive clones (Fig 5(a-b)).
[00095] Step 7: DNA sequencing
[00096] ELISA positive clones obtained in step 6 were processed for DNA sequencing. The novel sequences obtained were 60. The sequences were further analyzed using insilico method so as to rank antibody candidates based on their affinity to E1 and E2 antigen. The sequences are reported were Sequences ID 1 to 40.
[00097] Step 8: Identification & In-silico validation of antibodies
[00098] Some of the novel sequences were then designed to form antibodies and validated for their affinity for E1 and E2 envelop glycoproteins using computational method. Modelling of variable regions of heavy chain and light chain was done using the SAbPred server's ABodyBuilder-ML. The antibodies were then docked with the E1 and E2 glycoprotein using HDOCK server. The binding strength and specificity of antibodies was determined by identifying the CDR regions using AbRSA tool and an epitope prediction was carried out to enhance the antibody antigen interaction using Immune Epitope Database (IEDB). A detailed analysis was carried out using SASA tool to identify the residues within the antibodies that are critical for binding. Using molecular dynamic stimulation, stable conformation of the antibody were chosen based on the modelling of antigen antibody complex and identification of the complex that gives the lowest binding free energy and RMSD (Fig.6 and 7).
Results: This study lead to the identification of the most favorable antibody systems (Antibody 1-7). These systems, characterized by their low binding energy and structural stability, could be further investigated for potential therapeutic use against Chikungunya virus infections. The comprehensive method, ranging from computational modelling to dynamic simulations, highlights the effectiveness of

combining computational and experimental methodologies to speed up the development and enhancement of antibody treatments.
[00099] Step 9: Antibody engineering & conversion of potential single chain antibody into IgG
[000100] Selected high affinity antibodies in step 8, were then codon optimized for expression into CHO cell line. These codon optimized sequences were converted into full IgG by cloning into pTRIOZ expression vector. Heavy chain is cloned into AgeI & NheI restriction site while light chain is cloned into SgrAI & BsiW1 restriction site. Specific signal sequences were included before heavy & light chain gene sequences, which direct expressed antibody molecules outside the cells. Final construct was validated by gene sequencing & restriction digestion method.
[000101] Step 10: Expression & Purification in IgG Format
[000102] On the basis of insilico validation, we selected 7 novel antibody genes which are reformatted into full IgG format named as Antibody 1 to Antibody7. These were cloned into pTRIOZ vector and then transfected and expressed into CHO cells. Four out of seven potential novel mAbs were purified using MABSELECT resin by cytiva, all the purified samples shown more than 98 % purity. (Fig 8 (a-f)). Protein samples were run on SDS-PAGE gel (Fig 9). ELISA of purified antibodies (Antibody3 ,4, 5 &7) were performed (Fig 10). ELISA was performed for the validation of the potent affinity binders . These purified samples were used to determine neutralization potential against live chikungunya viruses. For remaining 3 mAbs, purification optimization is in progress.
[000103] Step 11: Virus Neutralization Assay
Neutralizing ability of these clones against Chikungunya virus was determined by Plaque Reduction Neutralization Test (PRNT). The appropriate concentration of purified protein was mixed with constant volume of virus which was plated on Vero cells (CCL-81, ATCC). Twenty-four hours after seeding, the protein (antibody) samples were diluted 2-fold in MEM with 2% FBS and equal volume of 400-800 PFU/ml CHIKV was mixed with diluted samples and incubated for 1 h at 37 °C in incubator with 5% CO2. The antibody mix was added to Vero cell monolayer and incubated for 90 min at 37 °C in incubator with 5% CO2. After 1 hour, the mixture

was removed and overlay medium consisting of MEM with 2% FBS, 1% Aquacide-II (Merck, Germany) and antibiotics was added to Vero cell monolayer, and plates were incubated for 22 h at 37°C in incubator with 5% CO2. Overlay medium was discarded and virus-infected cells were stained as foci (plaques) using anti Chikungunya specific E1 antibody. Plaques were counted using CTL Plaque Counter and PRNT50 titers were calculated (Fig 11).
Results: Antibody 3 shows highest neutralization potential, therefore it is considered as most potential candidate for further studies in which the neutralization activity can be increased by concentrating the purified protein.
[000104] SEQUENCE LISTING

S.NO AMINO ACID SEQUENCE SEQ ID NO
1. QVQLVQSGAEVKRPGASVKVSCEASGYTFKNYAIHWVRQVPGQRLEWMGWI IAGSGNTKFSQRFQGRVTITTDTSANTAYMELRSLRSEDTALYFCARSDSLPYF DSWDHG 1
2. QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGITWVRQAPGQGLEWMWS AYNGYKSYAQNFQGRVTMTTDTFTSTAYMELRTLRSDDTAVYYCVRLVPRT ATLHYYIDVWGKGTTVTVSS 2
3. QMQLVQSGAEVKKPGASVKVSCKASGYTFNSYDINWVRQATGQGLEWMGI
INPVVGTTNYAQKSQDRVTVTRDTSTSTVYIELSSLRSEDTAVYYCARGFYG
QALDYWGQGTTVTVS 3
4. EVQLVQSGAEVKKPGSSVKVSCKPSGGTFSSFAVSWVRQAPGQGLEWMGRI SPILGIPEYAQKFQGRVTISADKSTNTVYMELSSLRSEDTAFYFCARSDSLPYF DSWGQ 4
5. QMQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMG
WISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDMAVYYCARV
ANSSGWSYFDYWGQGTLVTVS 5
6. PKRTATLSGAEVKKPGSLVKVSCKPSGGTFSSFAVSWVRQAPGQGLEWMGR ISPILGIPEYAQKFQGRVTISADKSTNTVYMELSSLRSEDTAFYFCATTLSAAG LQGYFDLWGRGTLVTVS 6
7. QMQLVQSGAEVKKPGESLRISCKGSGYSFTSYWISWVRQMPGKGLEWMGR
IDPSDSYTNYSPSFQGHVTISADKSISTAYLQWSSLKASDTAMYYCARHPSQY
YDILTGLRPHYYYYYGMDVWGQGTTVTVS 7
8. QMQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGI
IYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARLLERL
DWFDPWGQGTLVTVS 8

9. QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAMHWVRQAPGQRLEWMG
WINAGNGNTKYSQKFQGRVTVTRDTSASTAYMELSSLRSEDTAVYYCARDH
IAAGYWYFDLWGRGTLVTVS 9
10. EVQLVQSGAEMRKPGSSVKVSCQVSGGTFNSFSIHWMRQAPGQGPEWIGAII
PGFGTANYAQKFQGRVTITADEPTTTAYMELSSLRSDDTAIYYCARENWVY
DYWGQGTLVTVS 10
11. QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRI IPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDPREMA TIRTLYGMDVWGQGTTVTVS 11
12. QVQLVQSGAEVKKPGASVKVSCKASGYTFVSYAMHWVRQAPGQRLEWMR
IIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARAGEDT
GVIPSLYWYFDLWGRGTLVTVS 12
13. QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGW ISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARELR YFDWLKGYYYGMDVWGQGTTVTVS 13
14. QVQLVQSGAEVKKPGSSVRVSCKASGGSLSSYSFIWVRQAPGQGLEWMGRII PILYISDYGQKFQGRVTLSADTSTNTAYMDLRSLTSQDTAVYYCASARRDNS RYLYQYGLDVWGQGTTVTVS 14
15. QVQLVQSGAEVKKPGSSVKVSCKPSGGTFSSFAVSWVRQAPGQGLEWMGRI SPILGIPEYAQKFQGRVTISADKSTNTVYMELSSLRSEDTAFYFCATTLSAAGL QGCFDLWGRGTLVTVS 15
16. QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAITWVRQAPGQGLEWMGW ISAYNGYKSYAQNFQGRVTMTTDTFTSTAYMELRTLRSDDTAVYYCVRLVP KRTATLHYYIDVWGKGTTVTVSS 16
17. QVQLLESGAEVKKPGASVKVSCKTSGYTFTSYDINWVRQATGQRLEWMGW MMPDSGSTGFDQRFQGRVTMTRDTSTSTAYMELRDLTSEDTAVYYCARIRS GTTDLDYWGQGALVTVSS 17
Light Chain
1 DIQLTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASS
LERGVPSRFSGSGSGTDFTLTIRGLQPEDVATYYCQKYNSAPWAFGQGTKVE
IK 18
2 NIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASS LQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQSYSTPLFGQGTKVEIK 19
3 VIWMTQSPSLLSASTGDRVTISCRMSQGISSYLAWYQQKPGKAPKLLIYAAST LQSAVPSRFSGSGSGTDFTLTISSLQSEDLATYYCQQYYSYPPTFGGGTKVEIK 20
4 AIQLTQSPSSLSASVGDRVTITCRASQGISKNLAWYQQKIGTAPKLLISSASTL QSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYYSFPYTFGQGTKVDIK 21

5 AIQLTQSPSSLSASVGDRVTITCRTSQDIGNDLGWYQQQPGQAPKLLIYGSSS LQSGVPSRFSGSGSGTDFTLSISSLQPEDVATYYCLQDYTYPFTFGGGTKLEI 22
6 DIQLTQSPASLSASVGDRVTITCQASQHISHYLNWYQQKPGKAPKLLIYDASN LETGVPSRFSGSGCGTDFTLNIRSLKTEDIETYYCKEYENLLVNLRGGKKVEM K 23
7 AIQMTQSPSLLSASTGDRVTITCRASQYISNYLAWYQQKPGQAPKLLIYGAST LHSGVPSRFSGSGSGTDFTLTISCLQSEDFATYYCQQYYTYPYTCGQGTKVEI K 24
8 AIRMTQSPSLLSASTGDRVTISCRMSQGISSYLAWYQQKPGKAPKLLIYAAST LQSGVPSRFSGSGSGTDFTLTISRLQSEDFATYYCQQYYSFPFTFGPGTKLEIK 25
9 AIQMTQSPSSFSASTGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAAST LQSGVPSRFSGSGSGTDFTLSMSCLQSEDSATDYCQQSYGFPQHSWTGDQSG YERC 26
10 AIQMTQSPSSFSASTGDRVTITCRASQGISSYLAWYQQKPGKAPELLIYAAST LQSGVPSRFSGSGSGTDFTLTISCLQSEDFATYYCQQYYSFLCRPWDQG 27
11 EIVMTQSPATLSLSPGGTATLSCRASQNIRDYLVWYQQKPGQAPRLLIYDVS
NRAAGIPARFSGSGSGTDFTLTISSLEPEDVAVYYCQQRVNWPLTFGPGTKVE
IK 28
12 DIQMTQSPSSLSASVGDRVTITCRASQSISNYLSWYQQKP
GKAPKLLMYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQ
SYSIPLTFGGGTKVEIK 29
13 NIQMTQSPSSLSESVGDRVTITCQASQDISNSLNWYQQKP
GKAPKLLISDASNLKTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQ
DLNYPIDLGKGTRLEIK 30
14 AIRMTQSPSSLSASTGDRVTISCRMSQGISSYLAWYQQKP
GKAPELLIYAASTLQSGVPSRFSGSGSGTDFTLTISCLQSEDFATYYCQQ
YYSFPPTFGQGTKVEIK 31
15 EIVMTQSPATLSVSPGERATLSCRASQGISSYLAWYQQIP
GQAPRLLIYVASTRATGIPARFSGSGSGTEFTLTISSLQPEDSAVYYCQQ
YVKWPLTFGGGTKVEIK 32
16 NIQMTQSPSSLSESVGDRVTITCQASQDISNSLNWYQQKP
GKAPKLLISDASNLKTGVPSRFSGSGCGTDLTLNMRRLKTEDFENYCIKD
LNPMGLEGKGMEIK 33
17 AIRMTQSPSLLSASTGDRVTISCRMSQGISSYLAWYQQKP
GKAPELLIYAASTLQSGVPSRFSGSGSGTDFTLTISCLQSEDFATYYCQQ
YYSFPLVRPRDQGGNQTCGRWIQRYQSNCKLFSKIPYRKFIYRLE 34

18 EIVMTQSPATLSLSPGQRATLSCRASQSFYSYLAWYQQKP
GQAPRLLIYDVSHRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQ
RGNTFGGGTKVEIK 35
19 AIRMTQSPSLLSASTGDRVTISCRMSQGISSYLAWYQQKP
GKAPELLIYAASTLQSGVPSRFSGSGSGTDFTLTISCLQSEDFATYYCQQ
YYSFPLVRPRDQG 36
20 SYELTQPHSVSESPGKTVTISCTRSSGSIANNYVQWYQQRPGSAPTTVI
YEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSSNW
VLGGGTKLTVL 37
21 SELTQPASVSESPGKTVTISCTRGSGSIASNSVQWYQQRPGSAPTTVIY
EDNQRPSGVTDRFSGSIDSSSNSASLTISGLKTDDEADYYCQSYDSSNHN
WVFGGGTKLTVL 38
22 QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYVQLPGTAPKLLI
YDNNKRFSGVPDRFSGSKSGTSATLGITGLQTGDEADYYCGAWDGSLREA
VFGGGTKVTVL 39
23 EIVMTQSPATLSLSPGERATLSCRASQSIDRYLAWYQQRPGQAPRLLIY
DTSHRATGAPARFSGSGSGTDFTLTISNLETEDFAVYYCQQRYNWPVTFG
GGTKVEIR 40

Antibody No.
1

Seq No
HC – 2
LC – 19

Sequence
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGITWVRQAPGQGLEWMGWISA YNGYKSYAQNFQGRVTMTTDTFTSTAYMELRTLRSDDTAVYYCVRLVPKRTATL HYYIDVWGKGTTVTVSS
NIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQSYSTPLFGQGTKVEIK

2
3

HC – 17
LC – 39
HC -2
LC -35

QVQLLESGAEVKKPGASVKVSCKTSGYTFTSYDINWVRQATGQRL
EWMGWMMPDSGSTGFDQRFQGRVTMTRDTSTSTAYMELRDLTSEDTAVYYCARIRSGTTD
DYWGQGALVTVSS
QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYVQLPGTAPKLLIYDNNKR
FSGVPDRFSGSKSGTSATLGITGLQTGDEADYYCGAWDGSLREAVFGGGTKVT
VL
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAITWVRQAPGQGLEWMGWISAY NGYKSYAQNFQGRVTMTTDTFTSTAYMELRTLRSDDTAVYYCVRLVPKRTATLH YYIDVWGKGTTVTVSS
EIVMTQSPATLSLSPGQRATLSCRASQSFYSYLAWYQQKPGQAPRLLIYDVSHRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRGNTFGGGTKVEIK

4 HC – 2 QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGITWVRQAPGQGLEWMGWISA YNGYKSYAQNFQGRVTMTTDTFTSTAYMELRTLRSDDTAVYYCVRLVPKRTATL HYYIDVWGKGTTVTVSS

LC- 37 SYELTQPHSVSESPGKTVTISCTRSSGSIANNYVQWYQQRPGSAPTTVIYEDNQRPS GVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSSNWVLGGGTKLTVL
5 HC – 15 QVQLVQSGAEVKKPGSSVKVSCKPSGGTFSSFAVSWVRQAPGQGLEWMGRISPIL GIPEYAQKFQGRVTISADKSTNTVYMELSSLRSEDTAFYFCATTLSAAGLQGCFDL WGRGTLVTVS

LC – 19 NIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSG VPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQSYSTPLFGQGTKVEIK
6 HC- 1 QVQLVQSGAEVKRPGASVKVSCEASGYTFKNYAIHWVRQVPGQRLEWMGWIIA GSGNTKFSQRFQGRVTITTDTSANTAYMELRSLRSEDTALYFCARSDSLPYFDSW DHG

LC -18 DIQLTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKP
GKAPKLLIYKASSLERGVPSRFSGSGSGTDFTLTIRGLQPEDVATYYCQKYNSAPW
AFGQGTKVEIK
7 HC -11 QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPIL GIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDPREMATIRTLY GMDVWGQGTTVTVS

LC -28 EIVMTQSPATLSLSPGGTATLSCRASQNIRDYLVWYQQKP
GQAPRLLIYDVSNRAAGIPARFSGSGSGTDFTLTISSLEPEDVAVYYCQQRVNWPL
TFGPGTKVEIK

ADVANTAGES OF THE PRESENT INVENTION
[000105] The present invention provides an isolated fully human monoclonal
antibody or monoclonal antibody fragment or an immuno conjugate against
Chikungunya that satisfies existing need of Chikungunya treatment.
[000106] The present invention provides composition comprising at least one of an
isolated fully human monoclonal antibody or monoclonal antibody fragment or an
immuno conjugate of the current invention that satisfies existing need of Chikungunya
treatment.
[000107] The present invention provides a novel, specific and industrially
advantageous monoclonal antibodies that are capable of neutralizing Chikungunya
virus infectivity. These monoclonal antibodies shall be fulfilling the unmet medical
need for Chikungunya prophylaxis and treatment.

Claims:
We claim:
1. A monoclonal antibody comprising of heavy chain selected from Sequence ID:1- 17 or light chain selected from Sequence ID: 18- 40.
2. The human monoclonal antibody as claimed in claim1, wherein the heavy chain selected from Sequence ID: 1, 2, 11, 15, 17 or light chain selected from Sequence ID: 18, 19, 28, 35, 37, 39.
3. The monoclonal antibody as claimed in claim1, wherein the antibody is selected from the group of:
i. Antibody 1 (Heavy Chain Sequence ID: 2, Light Chain sequence ID: 19);
ii. Antibody 2 (Heavy Chain Sequence ID: 17, Light Chain sequence ID: 39);
iii. Antibody 3 (Heavy Chain Sequence ID: 2, Light Chain sequence ID: 35);
iv. Antibody 4 (Heavy Chain Sequence ID: 2, Light Chain sequence ID: 37);
v. Antibody 5 (Heavy Chain Sequence ID: 15, Light Chain sequence ID: 19);
vi. Antibody 6 (Heavy Chain Sequence ID: 1, Light Chain sequence ID: 18); or
vii. Antibody 7 (Heavy Chain Sequence ID: 11, Light Chain sequence ID: 28).
4. The monoclonal antibody as claimed in claim 1-4, wherein the antibody binds to antigen envelope protein E1 or E2.
5. The monoclonal antibody as claimed in any of the preceding claim 1-4 for the manufacture of pharmaceutical composition against Chikungunya.
6. The monoclonal antibody as claimed in any of the preceding claim 1-4 useful for the treatment, prevention or diagnosis of Chikungunya.
7. A pharmaceutical composition comprising the antibody as claimed in any of the claims 1-3 and a pharmaceutically acceptable carrier.
8. The composition as claimed in claim 6, wherein the pharmaceutically acceptable carrier is selected from but not limited to, buffer, antioxidant, preservative, isotonic agent and chelating agent.
9. The monoclonal antibody as claimed in any of the preceding claim 1-4, wherein the process comprising of at least one of the following steps:

a) Blood Collection
b) RNA Isolation
c) cDNA Conversion and Monoclonal antibody Genes Amplification by PCR

d) Monoclonal antibody Genes Cloning and scFv library banking
e) Monoclonal antibody Selection by Bio-panning
f) Screening by ELISA and Restriction Digestion
g) DNA sequencing
h) Identification & In-silico validation of antibodies
i) Antibody engineering & conversion of potential single chain antibody into IgG j) Expression & Purification in IgG Format 10. A kit for detecting in vitro the presence of Chikungunya in a sample comprising
- a monoclonal antibody of any one of claims 1 to 3; and
- instructions for use of the kit.