Indian Patents. 217619:A PLASMID CONSTRUCT FOR TESTING THE RESISTANCE OF HIV-1 SUBTYPE C VIRUS ISOLATES TO ANTIRETROVIRAL DRUGS IN VITRO.

Title of Invention	A PLASMID CONSTRUCT FOR TESTING THE RESISTANCE OF HIV-1 SUBTYPE C VIRUS ISOLATES TO ANTIRETROVIRAL DRUGS IN VITRO.
Abstract	Recombinant plasmid pPS4.3HSAR-E- comprises HIV-1 subtype B genome with defective envelop gene, mutations in gag-pol gene to introduce Xmal and SacII restriction enzyme sites on either side of RT gene and HAS (Heat Stable Antigen) gene in the nef gene region mounted onto a plasmid vector.

Full Text	Field of Invention: This invention relates to a recombinant plasmid pPS4.3HSAR-E and the method of the constructing thereof. Background of Invention: AIDS is one of the most dreaded infections afflicting the human race today. Human Immunodeficiency virus (HIV) is the causative agent of Acquired Immunodeficiency Syndrome (AIDS). Several antiretroviral drugs have been introduced to combat this virus infection since last decade. Current therapy of HIV infection requires use of multiple drug combinations which resulted in dramatic improvements in the mortality and morbidity of HIV-disease. Despite all the therapeutic advantages achieved, eradication of the virus still remains impossible. In addition, new problems relating to the occurrence of resistance mutation in both circulating and transmitted viruses are emerging. Development of resistant HIV strains is one of the main reasons of failure of antiretroviral therapy. It is estimated that in many countries about one fourth of fresh infectious are due to resistant strains of HIV-1. In USA & Europe 10% of all fresh infections are due to resistant mutants. Therefore, it is desirable to screen fresh HIV isolates for their resistance to antiretroviral drugs. Currently there are two established assay systems available commercially for measuring resistance of HIV to antiretroviral drugs-the genotypic and the phenotypic resistance tests. The total cost ranges from $400 to $550 per test for genotyping and twice as high for phenotypic tests. Genotypic tests have a major drawback in that they only detect viral mutants comprising at least 20to 30% of the total population and provide indirect measurement of drug resistance. Phenotypic methods, though involve direct quantification of drug sensitivity, they are labour intensive, lengthy, dangerous (as they require virus isolation in cell cultures) and expensive. In the alternate approach employed in the present invention, the viral genome is mounted onto a carrier DNA molecule called a plasmid. This plasmid when transfected in an eukaryotic cell expresses the proteins of virus on the surface of the cell which can be easily visualized either under the immunofluorescent microscope or by flowcytometer or Enzyme Linked Immunosorbent Assay (ELISA). The expression of viral proteins on the cell surface is inhibited by antiretrovirals. Objects of Invention Object of this invention is to propose a plasmid containing the genome of Human Immunodeficiency Virus type 1 (HIV-1), which allows one replication of HIV-1 pol gene. Second object of this invention is to propose a plasmid containing the genome of HIV-1 for effectual testing of effectiveness of chemical compounds to inhibit HIV-1 replication in-vitro. Another object of this invention is to propose a plasmid containing genome of HIV-1 from which RT gene can be easily removed and replaced with RT of any other HIV-1 isolate. Brief Description of the Invention: According to this invention there is provided recombinant plasmid pPS4.3HSAR-E; comprising HIV-1 genome with defective envelope gene and mutated gag-pol gene wherein Xmal and SacII restriction enzyme sites have been introduced on either side of reverse transcriptase (RT) gene inserted in a plasmid vector and the method for constructing thereof. According to this invention there are also provided a method for constructing plasmid comprising isolating DNA segment of HIV-1, subjecting to the step of amplification using forward primer RTXmalF and a reverse primer RTSacllR in a polymerase chain reaction, restricting the amplified product with Xmal and SacII restriction enzymes, ligating to plasmid pPS4.3HSAR-E from which RT gene has been removed, wherein the plasmid pPS4.3HSAR-E is transected into eukaryotic T cells. Detailed Description of the Invention: 1. Construction of plasmid pGEM-NL4.SS by cloning HIV-1 subtype B gag-pol gene segment into pGEM-T Easy vector: Plasmid pNL4-HSAR-E-was obtained from the NIH AIDS Reagent Repository, NIH, Bethesda, MD, USA). This plasmid contains HIV-1 subtype B gene clone derived from two HIV-1 subtype B isolates (NY5 and LAV). Two frame shift mutations in the envelope gene (at position 5) and Vpr gene (at position 26) render this clone of HIV defective. However, presence of other genes including reverse-transcriptase (RT) gene induce single round of replication in mammalian cells. Using restriction enzymes Sail and SphI the gag-pol gene from this plasmid was removed and them cloned in P-GEM T vector (which has also been restricted with Sail and SphI) with ligase enzyme. This resulted in formation of plasmid pGEM-NL4.SS. 2. Modification of HIV-1 subtype B gag-pol gene segment by PCR based site directed mutagenesis: Plasmid pGEM-NL4.SS was grown in E.coli strain DH-5a at 37°C and its DNA extracted and purified. The plasmid DNA was then subjected to PCR based on site directed mutagenesis at nucleotide positions 2078, 2079, 2080, 2081, 3197 and 3200 where nucleotides T, T, T, A, T and T were replaced by nucleotides C, C, G, C, C and G respectively. This introduced restriction sites for Sacll and Xmal restriction enzymes in HIV-1 subtype B gag-pol gene segment present in plasmid pGEM-NL4.SS. This modified plasmid was named aspGEM-PS4.3SS. 3. Confirmation of the mutated gene sequence ofpGEM-PS4.3.SS. The sequence of the mutated gene was confirmed by sequencing in an automated sequencer using Ml3 primers. The sequence was then blast confirmed for the mutations introduced at the above-mentioned nucleotide positions. Further confirmation was done by restriction digestion of the plasmid pGEM-PS4.3. SS. 4. Construction of plasmid pPS4.3HSAR-E- Plasmid pGEM-PS4.3SS was amplified in E. coli strain DH5-a. The plasmid DNA was extracted, purified and cut with restriction enzymes Sail and Sphl to isolate the mutated HIV-1 subtype B pol-gene. This mutated gene was cloned into Sail and Sphl restricted plasmid pNL4-HSAR-E- to result in plasmid pPS4.3HSAR-E-. The sequence of the plasmid pPS4.3HSAR-E- was confirmed by sequencing and restriction digestion. Transfection of Eukaiyotic cells, 293T cells, with plasmid PPS4.3HSAR-E-. 293T cell line was obtained from National Centre for Cell Science (Pune, Maharashtra). It is a human renal epithelial cell line transformed by adenovirus El A gene product. This cell line is used for studying the expression of recombinant DNA plasmid constructs. We used it for the expression of recombinant plasmid PPS4.3HSAR-E- construct. 293T cells were transfected with the plasmid PPS4.3HSAR-E- and incubated at 37°C for 72 hours. Expression of HAS gene (CD24) on transfected cells was monitored by flowcytometry using fluorescene isothiocyanate (FITC) conjugated anti-CD24 monoclonal antibodies. Antiretroviral drugs inhibited this expression. Suggesting thereby, this plasmid has immense potential for evaluating the effectiveness of chemical compounds for their antiretroviral activities against HIV-1 in vitro. Replacement of Reverse Transcriptase (RT) gene on plasmid PPS4.3HSAR-E- with RT gene from otherHIV-1 isolates: Reverse transcriptase (RT) gene is required for replication of HIV-1. Using primers, RTXmalF 5"-TTAAAGCCCGGGATGGATGG-3"; RTSacIIR 5"-GTAATTTAAACCGCGGAGTCTTTC-3"; RT gene of HIV-1 isolate was amplified by Reverse transcriptase polymerase chain reaction (RTPCR). The specificity of this RT gene was confirmed by sequencing in an automated sequence analyzer. The amplified product was purified and restricted with Xmal and Sacll restriction enzymes. It was ligated to Xmal and Sacll restricted plasmid pPS4.3HSAR-E- using ligase enzymes. This replaced RT gene of the plasmid pPS4.3HSAR-E- with RT gene of HIV-1 isolate resulting in a plasmid pPS4.3HSAR-E- (R). This plasmid was then used for transfection of 293T cells and expression of CD24 was monitored by flowcy tome try in the presence and absence of antiretro viral drugs. The expression of CD24 was inhibited in the presence of antiretroviral drugs. Meaning thereby, that the HIV-1 isolate was sensitive to antiretroviral drugs. Had the antiretroviral drugs not inhibited the expression of CD24, the HIV-1 isolate would have been resistant to antiretroviral drugs. These data suggest that this plasmid pPS4.3HSAR-E- has immense potential for testing the resistance of HIV-1 virus isolates to antiretroviral drugs in vitro. We CIAIM: 1. Recombinant plasmid pPS4.3HSAR-E; comprising HIV-1 genome with defective envelope gene as herein described, Heat stable Antigen gene CD24 (HAS) and mutated gag-pol gene wherein Xmal and SacII restriction enzyme sites have been introduced on either side of reverse transcriptase (RT) gene inserted in a plasmid. 2. Recombinant plasmid pPS4.3HSAR-E as claimed in claim 1 wherein method for constructing plasmid comprising isolating DNA segment of HIV-1, subjecting to the step of amplification using forward primer RTXmalF and a reverse primer RTSacllR in a polymerase chain reaction, restricting the amplified product with Xmal and SacII restriction enaymes, ligating to plasmid pPS4.3HSAR-E from which RT gene has been removed, wherein the plasmid is pPS4.3HSAR-E. 3. Recombinant plasmid pPS4.3HSAR-E as claimed in claim 2 wherein the said set of forward primer comprises 5"-TTAAAGCCCGGGATGGATGG-3" and a reverse primer 5"-GTAATTTAAACCGCGGAGTCTTTC-3". 4. The recombinant plasmid as claimed in claim 1 expresses HAS; Heat stable Antigen gene CD24. 5. Recombinant plasmid pPS4.3HSAR-E and the method of construction thereof substantially as herein described.

Full Text

Field of Invention:
This invention relates to a recombinant plasmid pPS4.3HSAR-E and the method of the constructing thereof.
Background of Invention:
AIDS is one of the most dreaded infections afflicting the human race today. Human Immunodeficiency virus (HIV) is the causative agent of Acquired Immunodeficiency Syndrome (AIDS).
Several antiretroviral drugs have been introduced to combat this virus infection since last decade. Current therapy of HIV infection requires use of multiple drug combinations which resulted in dramatic improvements in the mortality and morbidity of HIV-disease. Despite all the therapeutic advantages achieved, eradication of the virus still remains impossible. In addition, new problems relating to the occurrence of resistance mutation in both circulating and transmitted viruses are emerging.
Development of resistant HIV strains is one of the main reasons of failure of antiretroviral therapy. It is estimated that in many countries about one fourth of fresh infectious are due to resistant strains of HIV-1. In USA & Europe 10% of all fresh infections are due to resistant mutants. Therefore, it is desirable to screen fresh HIV isolates for their resistance to antiretroviral drugs. Currently there are two established assay systems available commercially for measuring resistance of HIV to antiretroviral drugs-the genotypic and the phenotypic resistance tests. The total cost ranges from $400 to $550 per test for genotyping and twice as high for phenotypic tests.
Genotypic tests have a major drawback in that they only detect viral mutants comprising at least 20to 30% of the total population and provide indirect measurement of drug resistance.
Phenotypic methods, though involve direct quantification of drug sensitivity, they are labour intensive, lengthy, dangerous (as they require virus isolation in cell cultures) and expensive. In the alternate approach employed in the present invention, the viral genome is mounted onto a carrier DNA molecule called a plasmid. This plasmid when transfected in an eukaryotic cell expresses the proteins of virus on the surface of the cell which can be easily visualized either under the immunofluorescent microscope or by flowcytometer or Enzyme Linked Immunosorbent Assay (ELISA). The expression of viral proteins on the cell surface is inhibited by antiretrovirals.
Objects of Invention
Object of this invention is to propose a plasmid containing the genome of Human Immunodeficiency Virus type 1 (HIV-1), which allows one replication of HIV-1 pol gene. Second object of this invention is to propose a plasmid containing the genome of HIV-1 for effectual testing of effectiveness of chemical compounds to inhibit HIV-1 replication in-vitro. Another object of this invention is to propose a plasmid containing genome of HIV-1 from which RT gene can be easily removed and replaced with RT of any other HIV-1 isolate.
Brief Description of the Invention:
According to this invention there is provided recombinant plasmid pPS4.3HSAR-E; comprising HIV-1 genome with defective envelope gene and mutated gag-pol gene wherein Xmal and SacII restriction enzyme sites have been introduced on either side of reverse transcriptase (RT) gene inserted in a plasmid vector and the method for constructing thereof.
According to this invention there are also provided a method for
constructing plasmid comprising isolating DNA segment of HIV-1,
subjecting to the step of amplification using forward primer RTXmalF
and a reverse primer RTSacllR in a polymerase chain reaction,
restricting the amplified product with Xmal and SacII restriction
enzymes, ligating to plasmid pPS4.3HSAR-E from which RT gene has
been removed, wherein the plasmid pPS4.3HSAR-E is transected into
eukaryotic T cells.
Detailed Description of the Invention:
1. Construction of plasmid pGEM-NL4.SS by cloning HIV-1 subtype B gag-pol gene segment into pGEM-T Easy vector: Plasmid pNL4-HSAR-E-was obtained from the NIH AIDS Reagent
Repository, NIH, Bethesda, MD, USA). This plasmid contains HIV-1 subtype B gene clone derived from two HIV-1 subtype B isolates (NY5 and LAV). Two frame shift mutations in the envelope gene (at position 5) and Vpr gene (at position 26) render this clone of HIV defective. However, presence of other genes including reverse-transcriptase (RT) gene induce single round of replication in mammalian cells. Using restriction enzymes Sail and SphI the gag-pol gene from this plasmid was removed and them cloned in P-GEM T vector (which has also been restricted with Sail and SphI) with ligase enzyme. This resulted in formation of plasmid pGEM-NL4.SS.
2. Modification of HIV-1 subtype B gag-pol gene segment by PCR based site directed mutagenesis:
Plasmid pGEM-NL4.SS was grown in E.coli strain DH-5a at 37°C and its DNA extracted and purified. The plasmid DNA was then subjected to PCR based on site directed mutagenesis at nucleotide positions 2078, 2079, 2080, 2081, 3197 and 3200 where nucleotides T, T, T, A, T and T were replaced by nucleotides C, C, G, C, C and G respectively. This introduced restriction sites for Sacll and Xmal restriction enzymes in HIV-1 subtype B gag-pol gene segment present in plasmid pGEM-NL4.SS. This modified plasmid was named aspGEM-PS4.3SS.
3. Confirmation of the mutated gene sequence ofpGEM-PS4.3.SS.
The sequence of the mutated gene was confirmed by sequencing in an automated sequencer using Ml3 primers. The sequence was then blast confirmed for the mutations introduced at the above-mentioned nucleotide positions. Further confirmation was done by restriction digestion of the plasmid pGEM-PS4.3. SS.
4. Construction of plasmid pPS4.3HSAR-E-
Plasmid pGEM-PS4.3SS was amplified in E. coli strain DH5-a. The plasmid DNA was extracted, purified and cut with restriction enzymes Sail and Sphl to isolate the mutated HIV-1 subtype B pol-gene. This mutated gene was cloned into Sail and Sphl restricted plasmid pNL4-HSAR-E- to result in plasmid pPS4.3HSAR-E-. The sequence of the plasmid pPS4.3HSAR-E- was confirmed by sequencing and restriction digestion.
Transfection of Eukaiyotic cells, 293T cells, with plasmid PPS4.3HSAR-E-. 293T cell line was obtained from National Centre for Cell Science (Pune, Maharashtra). It is a human renal epithelial cell line transformed by adenovirus El A gene product. This cell line is used for studying the expression of recombinant DNA plasmid constructs. We used it for the expression of recombinant plasmid PPS4.3HSAR-E- construct.
293T cells were transfected with the plasmid PPS4.3HSAR-E- and incubated at 37°C for 72 hours. Expression of HAS gene (CD24) on transfected cells was monitored by flowcytometry using fluorescene isothiocyanate (FITC) conjugated anti-CD24 monoclonal antibodies. Antiretroviral drugs inhibited this expression. Suggesting thereby, this plasmid has immense potential for evaluating the effectiveness of chemical compounds for their antiretroviral activities against HIV-1 in vitro.
Replacement of Reverse Transcriptase (RT) gene on plasmid PPS4.3HSAR-E- with RT gene from otherHIV-1 isolates:
Reverse transcriptase (RT) gene is required for replication of HIV-1. Using primers, RTXmalF 5"-TTAAAGCCCGGGATGGATGG-3"; RTSacIIR 5"-GTAATTTAAACCGCGGAGTCTTTC-3"; RT gene of HIV-1 isolate was amplified by Reverse transcriptase polymerase chain reaction (RTPCR). The specificity of this RT gene was confirmed by sequencing in an automated sequence analyzer. The amplified product was purified and restricted with Xmal and Sacll restriction

enzymes. It was ligated to Xmal and Sacll restricted plasmid pPS4.3HSAR-E- using ligase enzymes. This replaced RT gene of the plasmid pPS4.3HSAR-E- with RT gene of HIV-1 isolate resulting in a plasmid pPS4.3HSAR-E- (R). This plasmid was then used for transfection of 293T cells and expression of CD24 was monitored by flowcy tome try in the presence and absence of antiretro viral drugs. The expression of CD24 was inhibited in the presence of antiretroviral drugs. Meaning thereby, that the HIV-1 isolate was sensitive to antiretroviral drugs. Had the antiretroviral drugs not inhibited the expression of CD24, the HIV-1 isolate would have been resistant to antiretroviral drugs.
These data suggest that this plasmid pPS4.3HSAR-E- has immense potential for testing the resistance of HIV-1 virus isolates to antiretroviral drugs in vitro.

We CIAIM:
1. Recombinant plasmid pPS4.3HSAR-E; comprising HIV-1 genome with defective envelope gene as herein described, Heat stable Antigen gene CD24 (HAS) and mutated gag-pol gene wherein Xmal and SacII restriction enzyme sites have been introduced on either side of reverse transcriptase (RT) gene inserted in a plasmid.
2. Recombinant plasmid pPS4.3HSAR-E as claimed in claim 1 wherein method for constructing plasmid comprising isolating DNA segment of HIV-1, subjecting to the step of amplification using forward primer RTXmalF and a reverse primer RTSacllR in a polymerase chain reaction, restricting the amplified product with Xmal and SacII restriction enaymes, ligating to plasmid pPS4.3HSAR-E from which RT gene has been removed, wherein the plasmid is pPS4.3HSAR-E.

3. Recombinant plasmid pPS4.3HSAR-E as claimed in claim 2 wherein the said set of forward primer comprises 5"-TTAAAGCCCGGGATGGATGG-3" and a reverse primer 5"-GTAATTTAAACCGCGGAGTCTTTC-3".
4. The recombinant plasmid as claimed in claim 1 expresses HAS; Heat stable Antigen gene CD24.
5. Recombinant plasmid pPS4.3HSAR-E and the method of construction thereof substantially as herein described.

Documents:

504-del-2004-abstract.pdf

504-del-2004-claims-cancelled.pdf

504-del-2004-claims.pdf

504-del-2004-complete specification (granted).pdf

504-DEL-2004-Correspondence-Others-(22-10-2009).pdf

504-del-2004-correspondence-others.pdf

504-del-2004-correspondence-po.pdf

504-del-2004-description (complete).pdf

504-del-2004-form-1.pdf

504-DEL-2004-Form-15-(22-10-2009).pdf

504-del-2004-form-18.pdf

504-del-2004-form-2.pdf

504-del-2004-form-26.pdf

504-del-2004-form-3.pdf

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Patent Number

217619

Indian Patent Application Number

504/DEL/2004

PG Journal Number

15/2008

Publication Date

11-Apr-2008

Grant Date

27-Mar-2008

Date of Filing

18-Mar-2004

Name of Patentee

SETH PRADEEP

Applicant Address

C/o All India Institute of Medical Sciences, Ansari Nagar, New Delhi

Inventors:

#	Inventor's Name	Inventor's Address
1	PRADEEP SETH	C/o. All India Institute of Medical Sciences, Ansari Nagar, New Delhi-110 029

PCT International Classification Number

A 61 K 48/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA