Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1995-05-18
2003-03-25
Scheiner, Laurie (Department: 1648)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C424S208100, C424S188100
Reexamination Certificate
active
06538127
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to recombinant HIV (Human Immunodeficiency Virus) antigens. Recombinant antigens derived from the molecular cloning and expression in a heterologous expression system of the synthetic DNA sequences of the various HIV antigens can be used as reagents for the detection of antibodies and antigen in body fluids from individuals exposed to various HIV isolates.
The nucleotide sequence of the proviral genome has been determined for several HIV isolates, including HIV-1 strains HTLV-III (Ratner et al.,
Nature
(1985) 313:277); ARV-2 (Sanchez-Pescador et al.,
Science
(1985) 227:484); LAV (Wain-Hobson et al.,
Cell
(1985) 40:9); and CDC-451 (Desai et al.,
Proc. Natl. Acad. Sci. USA
(1986) 83:8380). The nucleotide sequence of the HIV-2 ROD isolate was reported by Guyader et al. (
Nature
(1987) 326:662).
HIV antigens have been obtained from the virus grown in tissue culture, or from a molecularly cloned genomic fragment expressed in heterologous hosts such as
Escherichia coli
. The tissue culture derived virus involves the cumbersome and often difficult process of growing virus infected cells in stringent sterile conditions. Further, the virus derived from tissue culture is infectious, and, therefore is hazardous to the health of individuals involved in propagation and purification. The expression of molecularly cloned HIV genomic fragments overcomes the biohazard problem. Generally, an HIV genomic fragment from a single HIV isolate with mammalian codons is expressed in a heterologous system, such as, bacteria or yeast, and is limited to the use of available restriction sites present in the viral genome for cloning and expression.
It has been difficult to obtain expression in heterologous systems of some of the HIV proteins, such as the HIV-1 envelope antigen gp41. Several researchers have tried deleting the hydrophobic regions of the HIV-1 gp41 to increase expression levels. UK Patent Application GB 2188639 discloses an HTLV-III gag/env gene protein wherein the env fragment of the DNA sequence deleted codons corresponds to the first hydrophobic region of the gp41 protein. U.S. Pat. No. 4,753,873 discloses a peptide fragment that is encoded by a nucleotide sequence wherein the nucleotides coding for a first and second hydrophobic region of HTLV-III gp41 are deleted.
Poor expression can be the result of many factors, including the specific nucleic acid sequence of the gene to be expressed, the fact that the mammalian codons of the gene sequence to be expressed may not be efficiently transcribed and translated in a particular heterologous system, and the secondary structure of the transcribed messenger RNA. The use of synthetic DNA fragments can increase expression in heterologous systems.
SUMMARY OF THE INVENTION
Recombinant antigens which are derived from the molecular cloning and expression of synthetic DNA sequences in heterologous hosts are provided. Synthetic DNA sequences coding for the recombinant antigens of the invention are further provided. The synthetic DNA sequences selected for expression of various HIV antigens are based on the amino acid sequence of either a single isolate or several isolates, optimized for expression in
Escherichia coli
by specific codon selection. The synthetic DNA sequence gives higher expression of the particular antigen encoded. These antigens can be substituted for viral antigens derived from tissue culture for use as diagnostic and therapeutic reagents.
The present invention can be utilized to synthesize full length HIV transmembrane envelope gene using bacterial codons. Another aspect of the invention involves the linkage of sequences which are poorly expressed as individual proteins, to sequences which are expressed with high efficiency. The combination of the sequence of the entire coding region of a gene of one virus with coding sequences of another gene from a different virus to produce a fusion protein can be achieved. The fusion proteins thus expressed have a unique advantage of antigenic epitopes of two viral antigens.
The present invention includes full length synthetic genes (FSG) for HIV-1 and HIV-2 transmembrane glycoprotein (TMP).
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Casey James M.
Desai Suresh M.
Devare Sushil G.
Abbott Laboratories
Casuto Dianne
Parkin Jeffrey S.
Scheiner Laurie
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