Title of Invention	IMMUNOGENIC COMPOSITION FOR LYMPHATIC FILARIAL VACCINE
Abstract	The present invention provides methods and compositions for eliciting humoral immunity against lymphatic filariasis. In particular, the present invention discloses two peptide immunogens comprising immunodominant B epitopes from the filarial Secretory protein Abundant Larval Transcript (ALT-2) of Brugia Malayi. They were identified by monoclonal antibodies which were developed against purified recombinant ALT-2. Eight different peptides from ALT-2 protein were screened for dominant B epitopes and two peptide regions were identified. The peptides were immunized in BALB/ c (H-2d) mice and the antibody response was measured in terms of peak titers by ELISA. These two peptide immunogens induces a strong humoral response in mice models equivalent to recombinant ALT-2 whole protein. The two synthetic peptide immunogens is a highly immunogenic peptide vaccine for lymphatic filariasis.

Title of Invention

IMMUNOGENIC COMPOSITION FOR LYMPHATIC FILARIAL VACCINE

Abstract

The present invention provides methods and compositions for eliciting humoral immunity against lymphatic filariasis. In particular, the present invention discloses two peptide immunogens comprising immunodominant B epitopes from the filarial Secretory protein Abundant Larval Transcript (ALT-2) of Brugia Malayi. They were identified by monoclonal antibodies which were developed against purified recombinant ALT-2. Eight different peptides from ALT-2 protein were screened for dominant B epitopes and two peptide regions were identified. The peptides were immunized in BALB/ c (H-2d) mice and the antibody response was measured in terms of peak titers by ELISA. These two peptide immunogens induces a strong humoral response in mice models equivalent to recombinant ALT-2 whole protein. The two synthetic peptide immunogens is a highly immunogenic peptide vaccine for lymphatic filariasis.

Full Text	COMPLETE SPECIFICATION "PEPTIDE IMMUNOGENS OF LYMPHATIC FILARIAL ALT-2 AND LYMPHATIC FILARIAL VACCINE COMPOSITION COMPRISING THE SAME" Field of the invention and use of invention:- This invention relates to the identification of highly potent peptide immunogens from the filarial antigen Brugia malayi abundant larval transcript (ALT-2). More particularly it relates to the effective vaccine in eliciting protective immunity against lymphatic filariasis. The peptide immunogens can be used to develop epitope based vaccines for lymphatic filariasis. Background of the invention:- Parasitic worm (helminth) infections are the most common infections in poor people living in the developing world known as the neglected tropical diseases (NTDs). Lymphatic filariasis causes millions of cases of lymphedema and hydrocele. The three parasites, Wuchereria bancrofti, Brugia malayi and Brugia timori, all of which produce disease in the lymphatic vessels, are the most widespread and abundant of all the human filarial worms. As global elimination of human lymphatic filariasis is based on Mass Drug Administration of DEC and Albendazole, the control of this disease relies heavily on availability of the drugs. The filarial parasites infect about 128 million people (Hotez 2009), in 83 coimtries (WHO 2006) of the world causing lymphatic filariasis and over 40 million of them are seriously incapacitated and disfigured by the disease. One of the most critical issues in human parasitic diseases is the need to develop new drugs and effective vaccines. Traditional vaccines have involved the use of killed microorganisms, live attenuated cultures or antigenic extracts. Despite extensive research, potential vaccine for filarial parasite has been a long struggle (Nutman and Kumaraswamy 2001). Although multiple antigen strategy shows promising results, the response appears to be unique with each antigen combination and thus is impredictable. The responses vary with different combinations which one of the reasons for the failure of crude undefined nuxture of antigens that are often toxic or allergic. With advances in parasite genomics, immunoinformatics, new vaccine strategies and delivery systems, there are now excellent opportunities for new antihelminth vaccines (Maizels 1999). The use of synthetic peptides for immunization is a very attractive strategy for antigen delivery, since they are relatively easy to obtain in large quantities, well defined with high purity and offers practical advantages such as relative ease of construction and production, chemical stability, and a lack of infectious or oncogenic potential (Disis et al 1998). The L3 infective larvae is known to be a key player involved in early modulation of host immune responses. These infective larvae secrete the highly stage-specific novel protein, ALT-2 which is the most abundant of the L3-expressed cDNAs in the EST dataset (Gregory et at., 2000). Abundant larval transcript (ALT) proteins are stock-piled in the oesophageal glands of infective larvae as 'magic bullets' and are secreted when they encounter mammalian culture conditions. They are reported to be involved in maintaining infection by their immuno-modulatory function (Gomez-Escobar et al 2005) and to be a potential vaccine candidate (Ramachandran et al., 2004). However there is no effective vaccine against adult worms and clears only microfilaria. The treatment period is for Long period for 6 years. Some parasites develop resistance against drugs. The present invention is the identification of the potent immunogenic epitopes using ALT specific monoclonal antibodies for developing novel peptide vaccines with improved efficacy is a significant contribution in the prophylaxis of lymphatic filariasis. Prior art and problem to be solved:- Lympliatic filariasis is a major tropical disease caused by the mosquito-born nematodes Brugia and Wuchereria. About 120 million people are infected and at risk of lymphatic pathology such as acute lymphangitis and elephantiasis. No such closest prior art is related to the present invention. However, epitopes from antigens of other diseases have been reported and used as vaccine immunogens. A US 6,669,945 provides methods and compositions for eliciting protective immunity against malaria. In particular, the invention relates to universal T-cell epitopes that elicit T-cell responses in individuals of differing genetic backgrounnds. Immunogenic compositions and vaccines including malaria-specific universal T-cell epitopes are disclosed. Another U.S patent No. 4915942 discloses the malarial circumsporozoite epitope where the Synthetic peptides containing non-repeating epitopes of circumsporozoite derived protein antigen and which are substantially shorter in length than the intact antigen are disclosed. The peptides when administered to a host raise antibodies in that host that will bind to the circumsporozoite antigen on the parasite. Vaccines based upon these peptides, as well as means of raising antibodies to circmnsporozoite antigens using the synthetic peptides are disclosed. An Indian Patent No.226614 describes an immunogenic hybrid polypeptide comprising a mimetic peptide of a B cell epitope of apolipoprotein B-l00 and a helper T cell epitope, the mimetic .peptide being fused at its C-terminus to an N-terminus of the helper T cell epitope is used as a vaccine composition for preventing or treating obesity comprising the polypeptide. There are no vaccines available for lymphatic filariasis till date. The recombinant proteins which pose several problems in manufacturing like difficulty in expression of soluble proteins, purification in non denaturing conditions, refolding, cold storage and transport, expensive production system and poor stability. Moreover, protein preparations are not reproducible and well defined due to the presence of different forms. The present invention consists of peptide immunogens which can be synthesized chemically. The peptide immunogens are stable, easily transported and does not require cold storage. The present invention describes the peptide immunogens showing high reactivity with monoclonal antibodies developed against ALT-2 protein and high immunogenicity determined by antibody titer. Objects of the invention:- The main objective of this invention is to identify highly potent peptide immunogens from the filarial antigen Brugia malayi abundant larval transcript (ALT-2). The second objective of the invention is to analyze the immunogenic potential of the peptides in animal models. A further objective of the invention is to provide peptides as vaccine immunogens for lymphatic Filariasis. Summary of the invention:- The present invention discloses immunogenic composition that provides immune responses against lymphatic filariasis diseases. BmALT-2 protein sequence is selected for the immunogenic production. The Eight peptide regions were identified in the BmALT-2 protein sequence, predicted to carry B epitopes is chosen based on silico tools such as lEDB and BcePRED. The peptides were synthesized by solid phase technique, using f-moc chemistry and purified by high performance liquid chromatography. Three monoclonal antibodies (Mabs) were developed against purified recombinant ALT-2. The Mabs were used to screen the peptides carrying putative epitopes by indirect ELISA. The peptides were immunized in BALB/c (H-2d) mice and the antibody response was measured in terms of peak titers by ELISA. Antibody titers were assessed and the peptides (PI to P8) which shows high titers they are highly immunogenic and contains dominant B epitopes. The peptide regions of B. malayi ALT-2 protein are potent peptide immunogens carrying dominant B epitopes that could be exploited filarial vaccine development and therapy. The peptide immunogens are cost effective, reproducible and well defined compared to recombinant ALT-2 protein vaccine. They are more stable, easy to manufacture, store and transport than recombinant ALT-2 protein vaccine. Detailed description of the invention with respect to drawings:- The B.malayi ALT-2 consists of 128 residues and 387 bp of mRNA. Acidic domain is highly variable and May have immunomodulatory role. The Conserved domain is highly rich in Cysteine. It constians 9.4% Aspartic acid and 14.1% Glutamic acid residues and pi is 4.18. Eight peptide regions were identified in the BmALT-2 protein sequence, predicted to carry B epitopes by in silico tools such as lEDB and BcePRED and they were designated as PI, P2, P3, P4, P5, P6, P7 and P8. The peptides were synthesized by solid phase technique, using f-moc chemistry and assembled on wang resin. The peptides were purified and the purity was analyzed by high performance liquid chromatography. Three monoclonal antibodies (Mabs) were developed against purified recombinant ALT-2 in IISc, Bangalore by Dr.Anjali Karande in Sp2/0 mouse myeloma cells and the Mabs were used to screen the peptides carrying putative epitopes by indirect ELISA. The peptides PI, P7 and P8 were reactive with any two of the Mab's while peptide P5 was detected by one Mab. Other peptides did not show significant reactivity. This confirms that PI, P7 and P8 carry B epitopes whereas P5 may carry a less dominant epitope not detected by other Mabs (fig 1). The peptides were encapsulated in PLGA microparticles for efficient delivery and immunized in BALB/c (H-2d) mice at 50 pg per mice by intranasal route on lst, 14th and 28th days. Blood was collected by tail vein at weekly intervals and sera were separated. The antibody response was measured in terms of peak titers by ELISA. Briefly, 96-well microtiter plates (Immunlon2, Dynatech, VA, USA) were coated with 100 μl of peptide (500 ng/ well) and left overnight at 4°C. After washing and blocking with 5% skimmed milk powder, a serial two-fold dilution (1:500-1:1,28,000) of antisera was used. After three washings with PBST, ALP conjugated anti-mouse IgG was incubated for 1 h at 37°C. The color was developed using p-nitrophenyl phosphate substrate (1 mg/ml) in substrate buffer (100 mM Tris-Cl, pH 9.5,100 mM NaCl, 5 mM MgC12) substrate and absorbance was read at 405nm. Antibody titers were assessed as the highest serum dilution giving an absorbance (0.15) higher than that of preimmune sera. The peptides PI, P7 and P8 induced high peak titers of 18,000, 14,000, and 22,000 respectively in mice at 35th day after immunization while other peptides did not show significant antibody levels (fig 2). This shows that the peptide regions PI (2-29), P7 (90-110) and P8 (112-128) of ALT-2 are highly immunogenic and contains dominant B epitopes. Thus, the regions 1-29 (Seq.No.l- MNKLUAFGLVILLVTLPCASESDEEFDDG) and 90-128(Seq.No.2-WTDKGCFCEDKLHSCVIERKNNGKLEYSYCAPEAGWQCA) of B.malayi ALT-2 protein are potent peptide immunogens carrying dominant B epitopes that could be exploited filarial vaccine development and therapy. Hence, the peptide immunogen 1 and 2 containing 29 (Seq.No 1: 1-29) and 39 (Seq.No 2: 90-128) amino acids respectively were chemically synthesized, immunized in mice and the antibody response elicited by them were compared to the recombinant ALT-2 whole antigen. The peptide 2 elicited a very high titer of 32,000 which was equal to ALT-2 whole protein whereas the immunogen 1 induced a titer of 20,000 (fig 3). Hence, the two synthetic peptides can be used as potent immunogens either individually, in a mixed form or as a single polypeptide conjugates for management of filariasis. Brief description of the drawings:- Fig 1 is a bar chart showing the reactivity of three monoclonal antibodies (Mabl, Mab2 and Mab3) with peptide immimogen (PI to P8) and its absorbance at 450 nm. Fig 2 is a bar chart showing the production of antibodies by peptide regions in mice and estimation of antibody titre (*100). Fig 3 is a bar chart showing the antibody titer of peptide immunogen 1 (PI) and peptide immunogen 2 (P7 and P8). Claims:- 1. An immunogenic composition comprising: a) Selection of potent synthetic peptide immunogens from eight different peptide regions of ALT-2, b) Three monoclonal antibodies (Mabs) were developed against purified recombinant ALT-2, c) Peptides were encapsulated in PLGA microparticles and immunized in BALB/c (H- 2d) mice and d) Estimation of antibody production in mice is detected by ELISA, Whereby, potent synthetic peptide immunogens could be used as peptide vaccines to prevent lymphatic filariasis disease. 2. The immunogenic composition as claimed in claim 1, comprising a potent synthetic peptide immunogens is derived from Brugia Malayia filarial ALT-2. 3. The immunogenic composition as claimed in claim 1, wherein the synthetic peptide immunogens were selected from eight different peptide regions of ALT-2 (PI, P2, P3, P4, P5, P6, P7 and P8) chosen based on in-silico immunoinformatic prediction tools, synthesized chemically by solid phase synthesis and purified by high performance liquid chromatography. 4. The immunogenic composition as claimed in claim 1, wherein the above said Mabs were used to screen the peptides carrying putative epitopes by indirect ELISA. 5. The immunogenic composition as claimed in claim 1, wherein the peptides PI, P7 and P8 were reactive with any two of the Mab's and carries B epitopes. 6. The immunogenic composition as claimed in claim 1, wherein the peptides PI, P7 and PS were immunized in BALB/c (H-2d) mice and the antibody response was measured in terms of peak titers by ELISA. 7. The immunogenic composition as claimed in claim 1, wherein the above said peptides PI (2-29), P7 (90-110) and PS (112-128) of ALT-2 induced high peak titers of 1S,000, 14,000, and 22,000 respectively in mice at 35th day; and they are highly immunogenic and contains dominant B epitopes. 8. The immimogenic composition as claimed in claim 1, wherein the above said peptides regions 1-29 (Seq.No.l-MNKLLIAFGLVILLVTLPCASESDEEFDDG) and 90- 12S (Seq.No.2- WTDKGCFCEDKLHSCVIERKNNGKLEYSYCAPEAGWQCA) of B. malayi filarial ALT-2 protein are potent peptide immunogens carrying dominant B epitopes that could be exploited for filarial vaccine development and therapy. 9. The immunogenic composition as claimed in claim 1, wherein the above said peptides immunogen 1 and 2 containing 29 (Seq.No 1: 1-29) and 39 (Seq.No 2: 90-128) amino acids elicited a very high titer of 20,000 and 32,000 respectively. 10. The immunogenic composition as claimed in claim 1, wherein, the above said two synthetic peptides can be used as potent immunogens either individually, in a mixed form or as a single polypeptide conjugate to prevent filariasis.

Full Text

COMPLETE SPECIFICATION

"PEPTIDE IMMUNOGENS OF LYMPHATIC FILARIAL ALT-2 AND LYMPHATIC FILARIAL VACCINE COMPOSITION COMPRISING THE SAME"

Field of the invention and use of invention:-

This invention relates to the identification of highly potent peptide immunogens from the filarial antigen Brugia malayi abundant larval transcript (ALT-2). More particularly it relates to the effective vaccine in eliciting protective immunity against lymphatic filariasis.

The peptide immunogens can be used to develop epitope based vaccines for lymphatic filariasis.

Background of the invention:-

Parasitic worm (helminth) infections are the most common infections in poor people living in the developing world known as the neglected tropical diseases (NTDs). Lymphatic filariasis causes millions of cases of lymphedema and hydrocele. The three parasites, Wuchereria bancrofti, Brugia malayi and Brugia timori, all of which produce disease in the lymphatic vessels, are the most widespread and abundant of all the human filarial worms.

As global elimination of human lymphatic filariasis is based on Mass Drug Administration of DEC and Albendazole, the control of this disease relies heavily on availability of the drugs. The filarial parasites infect about 128 million people (Hotez 2009), in 83 coimtries (WHO 2006) of the world causing lymphatic filariasis and over 40 million of them are seriously incapacitated and disfigured by the disease.

One of the most critical issues in human parasitic diseases is the need to develop new drugs and effective vaccines. Traditional vaccines have involved the use of killed microorganisms, live attenuated cultures or antigenic extracts. Despite extensive
research, potential vaccine for filarial parasite has been a long struggle (Nutman and Kumaraswamy 2001). Although multiple antigen strategy shows promising results, the response appears to be unique with each antigen combination and thus is impredictable. The responses vary with different combinations which one of the reasons for the failure of crude undefined nuxture of antigens that are often toxic or allergic.

With advances in parasite genomics, immunoinformatics, new vaccine strategies and delivery systems, there are now excellent opportunities for new antihelminth vaccines (Maizels 1999). The use of synthetic peptides for immunization is a very attractive strategy for antigen delivery, since they are relatively easy to obtain in large quantities, well defined with high purity and offers practical advantages such as relative ease of construction and production, chemical stability, and a lack of infectious or oncogenic potential (Disis et al 1998).

The L3 infective larvae is known to be a key player involved in early modulation of host immune responses. These infective larvae secrete the highly stage-specific novel protein, ALT-2 which is the most abundant of the L3-expressed cDNAs in the EST dataset (Gregory et at., 2000). Abundant larval transcript (ALT) proteins are stock-piled in the oesophageal glands of infective larvae as 'magic bullets' and are secreted when they encounter mammalian culture conditions. They are reported to be involved in maintaining infection by their immuno-modulatory function (Gomez-Escobar et al 2005) and to be a potential vaccine candidate (Ramachandran et al., 2004).

However there is no effective vaccine against adult worms and clears only microfilaria. The treatment period is for Long period for 6 years. Some parasites develop resistance against drugs.

The present invention is the identification of the potent immunogenic epitopes using ALT specific monoclonal antibodies for developing novel peptide vaccines with improved efficacy is a significant contribution in the prophylaxis of lymphatic filariasis.
Prior art and problem to be solved:-

Lympliatic filariasis is a major tropical disease caused by the mosquito-born nematodes Brugia and Wuchereria. About 120 million people are infected and at risk of lymphatic pathology such as acute lymphangitis and elephantiasis. No such closest prior art is related to the present invention. However, epitopes from antigens of other diseases have been reported and used as vaccine immunogens.

A US 6,669,945 provides methods and compositions for eliciting protective immunity against malaria. In particular, the invention relates to universal T-cell epitopes that elicit T-cell responses in individuals of differing genetic backgrounnds. Immunogenic compositions and vaccines including malaria-specific universal T-cell epitopes are disclosed.

Another U.S patent No. 4915942 discloses the malarial circumsporozoite epitope where the Synthetic peptides containing non-repeating epitopes of circumsporozoite derived protein antigen and which are substantially shorter in length than the intact antigen are disclosed. The peptides when administered to a host raise antibodies in that host that will bind to the circumsporozoite antigen on the parasite. Vaccines based upon these peptides, as well as means of raising antibodies to circmnsporozoite antigens using the synthetic peptides are disclosed.

An Indian Patent No.226614 describes an immunogenic hybrid polypeptide comprising a mimetic peptide of a B cell epitope of apolipoprotein B-l00 and a helper T cell epitope, the mimetic .peptide being fused at its C-terminus to an N-terminus of the helper T cell epitope is used as a vaccine composition for preventing or treating obesity comprising the polypeptide.

There are no vaccines available for lymphatic filariasis till date. The recombinant proteins which pose several problems in manufacturing like difficulty in expression of soluble proteins, purification in non denaturing conditions, refolding, cold storage and transport, expensive production system and poor stability. Moreover, protein preparations are not reproducible and well defined due to the presence of different forms. The present invention consists of peptide immunogens which can be synthesized chemically. The peptide immunogens are stable, easily transported and does not require cold storage. The present invention describes the peptide immunogens showing high reactivity with monoclonal antibodies developed against ALT-2 protein and high immunogenicity determined by antibody titer.

Objects of the invention:-

The main objective of this invention is to identify highly potent peptide immunogens from the filarial antigen Brugia malayi abundant larval transcript (ALT-2).

The second objective of the invention is to analyze the immunogenic potential of the peptides in animal models.

A further objective of the invention is to provide peptides as vaccine immunogens for lymphatic Filariasis.

Summary of the invention:-

The present invention discloses immunogenic composition that provides immune responses against lymphatic filariasis diseases. BmALT-2 protein sequence is selected for the immunogenic production. The Eight peptide regions were identified in the BmALT-2 protein sequence, predicted to carry B epitopes is chosen based on silico tools such as lEDB and BcePRED.

The peptides were synthesized by solid phase technique, using f-moc chemistry and purified by high performance liquid chromatography. Three monoclonal antibodies (Mabs) were developed against purified recombinant ALT-2. The Mabs were used to screen the peptides carrying putative epitopes by indirect ELISA. The peptides were immunized in BALB/c (H-2d) mice and the antibody response was measured in terms of peak titers by ELISA. Antibody titers were assessed and the peptides (PI to P8) which shows high titers they are highly immunogenic and contains dominant B epitopes.

The peptide regions of B. malayi ALT-2 protein are potent peptide immunogens carrying dominant B epitopes that could be exploited filarial vaccine development and therapy. The peptide immunogens are cost effective, reproducible and well defined compared to recombinant ALT-2 protein vaccine. They are more stable, easy to manufacture, store and transport than recombinant ALT-2 protein vaccine.

Detailed description of the invention with respect to drawings:-

The B.malayi ALT-2 consists of 128 residues and 387 bp of mRNA. Acidic domain is highly variable and May have immunomodulatory role. The Conserved domain is highly rich in Cysteine. It constians 9.4% Aspartic acid and 14.1% Glutamic acid residues and pi is 4.18. Eight peptide regions were identified in the BmALT-2 protein sequence, predicted to carry B epitopes by in silico tools such as lEDB and BcePRED and they were designated as PI, P2, P3, P4, P5, P6, P7 and P8.

The peptides were synthesized by solid phase technique, using f-moc chemistry and assembled on wang resin. The peptides were purified and the purity was analyzed by high performance liquid chromatography. Three monoclonal antibodies (Mabs) were developed against purified recombinant ALT-2 in IISc, Bangalore by Dr.Anjali Karande in Sp2/0 mouse myeloma cells and the Mabs were used to screen the peptides carrying putative epitopes by indirect ELISA.

The peptides PI, P7 and P8 were reactive with any two of the Mab's while peptide P5 was detected by one Mab. Other peptides did not show significant reactivity. This confirms that PI, P7 and P8 carry B epitopes whereas P5 may carry a less dominant epitope not detected by other Mabs (fig 1).

The peptides were encapsulated in PLGA microparticles for efficient delivery and immunized in BALB/c (H-2d) mice at 50 pg per mice by intranasal route on lst, 14th and 28th days. Blood was collected by tail vein at weekly intervals and sera were separated. The antibody response was measured in terms of peak titers by ELISA. Briefly, 96-well microtiter plates (Immunlon2, Dynatech, VA, USA) were coated with 100 μl of peptide (500 ng/ well) and left overnight at 4°C. After washing and blocking with 5% skimmed milk powder, a serial two-fold dilution (1:500-1:1,28,000) of antisera was used.

After three washings with PBST, ALP conjugated anti-mouse IgG was incubated for 1 h at 37°C. The color was developed using p-nitrophenyl phosphate substrate (1 mg/ml) in substrate buffer (100 mM Tris-Cl, pH 9.5,100 mM NaCl, 5 mM MgC12) substrate and absorbance was read at 405nm. Antibody titers were assessed as the highest serum dilution giving an absorbance (0.15) higher than that of preimmune sera.

The peptides PI, P7 and P8 induced high peak titers of 18,000, 14,000, and 22,000 respectively in mice at 35th day after immunization while other peptides did not show significant antibody levels (fig 2). This shows that the peptide regions PI (2-29), P7 (90-110) and P8 (112-128) of ALT-2 are highly immunogenic and contains dominant B epitopes.

Thus, the regions

1-29 (Seq.No.l- MNKLUAFGLVILLVTLPCASESDEEFDDG) and

90-128(Seq.No.2-WTDKGCFCEDKLHSCVIERKNNGKLEYSYCAPEAGWQCA) of B.malayi ALT-2 protein are potent peptide immunogens carrying dominant B epitopes that could be exploited filarial vaccine development and therapy.

Hence, the peptide immunogen 1 and 2 containing 29 (Seq.No 1: 1-29) and 39 (Seq.No 2: 90-128) amino acids respectively were chemically synthesized, immunized in mice and the antibody response elicited by them were compared to the recombinant ALT-2 whole antigen. The peptide 2 elicited a very high titer of 32,000 which was equal to ALT-2 whole protein whereas the immunogen 1 induced a titer of 20,000 (fig 3). Hence, the two synthetic peptides can be used as potent immunogens either individually, in a mixed form or as a single polypeptide conjugates for management of filariasis.

Brief description of the drawings:-

Fig 1 is a bar chart showing the reactivity of three monoclonal antibodies (Mabl, Mab2 and Mab3) with peptide immimogen (PI to P8) and its absorbance at 450 nm.

Fig 2 is a bar chart showing the production of antibodies by peptide regions in mice and estimation of antibody titre (*100).

Fig 3 is a bar chart showing the antibody titer of peptide immunogen 1 (PI) and peptide immunogen 2 (P7 and P8).

Claims:-

1. An immunogenic composition comprising:

a) Selection of potent synthetic peptide immunogens from eight different peptide regions of ALT-2,

b) Three monoclonal antibodies (Mabs) were developed against purified recombinant ALT-2,

c) Peptides were encapsulated in PLGA microparticles and immunized in BALB/c (H- 2d) mice and

d) Estimation of antibody production in mice is detected by ELISA,

Whereby, potent synthetic peptide immunogens could be used as peptide vaccines to prevent lymphatic filariasis disease.

2. The immunogenic composition as claimed in claim 1, comprising a potent synthetic peptide immunogens is derived from Brugia Malayia filarial ALT-2.

3. The immunogenic composition as claimed in claim 1, wherein the synthetic peptide immunogens were selected from eight different peptide regions of ALT-2 (PI, P2, P3, P4, P5, P6, P7 and P8) chosen based on in-silico immunoinformatic prediction tools, synthesized chemically by solid phase synthesis and purified by high performance liquid chromatography.

4. The immunogenic composition as claimed in claim 1, wherein the above said Mabs were used to screen the peptides carrying putative epitopes by indirect ELISA.

5. The immunogenic composition as claimed in claim 1, wherein the peptides PI, P7 and P8 were reactive with any two of the Mab's and carries B epitopes.

6. The immunogenic composition as claimed in claim 1, wherein the peptides PI, P7 and PS were immunized in BALB/c (H-2d) mice and the antibody response was measured in terms of peak titers by ELISA.

7. The immunogenic composition as claimed in claim 1, wherein the above said peptides PI (2-29), P7 (90-110) and PS (112-128) of ALT-2 induced high peak titers of 1S,000, 14,000, and 22,000 respectively in mice at 35th day; and they are highly immunogenic and contains dominant B epitopes.

8. The immimogenic composition as claimed in claim 1, wherein the above said peptides regions 1-29 (Seq.No.l-MNKLLIAFGLVILLVTLPCASESDEEFDDG) and 90- 12S (Seq.No.2- WTDKGCFCEDKLHSCVIERKNNGKLEYSYCAPEAGWQCA) of B. malayi filarial ALT-2 protein are potent peptide immunogens carrying dominant B epitopes that could be exploited for filarial vaccine development and therapy.

9. The immunogenic composition as claimed in claim 1, wherein the above said peptides immunogen 1 and 2 containing 29 (Seq.No 1: 1-29) and 39 (Seq.No 2: 90-128) amino acids elicited a very high titer of 20,000 and 32,000 respectively.

10. The immunogenic composition as claimed in claim 1, wherein, the above said two synthetic peptides can be used as potent immunogens either individually, in a mixed form or as a single polypeptide conjugate to prevent filariasis.

Documents:

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Patent Number

278637

Indian Patent Application Number

1367/CHE/2011

PG Journal Number

54/2016

Publication Date

30-Dec-2016

Grant Date

27-Dec-2016

Date of Filing

20-Apr-2011

Name of Patentee

REGISTRAR, ANNA UNIVERSITY, CHENNAI

Applicant Address

THE DIRECTOR, CIPR, CPDE BUILDING, ANNA UNIVERSITY, CHENNAI, CHENNAI - 600 025

Inventors:

#	Inventor's Name	Inventor's Address
1	DR. P. KALIRAJ	DIRECTOR, HOD, PROFESSOR, DEPARTMENT OF BIOTECHNOLOGY, ANNA UNIVERSITY, CHENNAI.
2	MADHUMATHI. J	RESEARCH SCHOLAR, ANNA UNIVERSITY, CHENNAI
3	G. ANUGRAHA	RESEARCH SCHOLAR, ANNA UNIVERISTY, CHENNAI
4	PRINCE.R.PRABHU	RESEARCH SCHOLAR, ANNA UNIVERSITY, CHENNAI

PCT International Classification Number

A61K39/00, C07K16/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA