Acquired immune deficiency syndrome (AIDS) viral envelope protein and method of testing for AIDS

An envelope protein of the etiologic agent of acquired immune deficiency syndrome (AIDS) and a method for its preparation are disclosed. Proviral DNA is transferred into a host cell after engineering into an expression vector which produces the envelope protein. A method of testing human blood for the presence of antibodies to the AIDS virus using the AIDS envelope protein is disclosed.

FIELD OF THE INVENTION 
This invention relates to a protein, designated env AIDS, which is the 
viral envelope protein of the etiologic agent for acquired immune 
deficiency syndrome, the expression vector which encodes env AIDS, the 
production of env AIDS using recombinant DNA technology and a method for 
detecting the presence of AIDS antibodies in human blood. 
BACKGROUND OF THE INVENTION 
From 1981 to date, there have been eight thousand (8,000) people diagnosed 
as having acquired immune deficiency syndrome (AIDS). N. Y. Times, A-11 
Jan. 11, 1985. AIDS has been characterized by the onset of severe 
opportunistic infections secondary to an effect on the body's immune 
system. Gottlieb, M. S. et al., Pneumocystis Carinic Pneumonia and Mucosal 
Candidiasis in previously healthy homosexual men: evidence of a new 
acquired cellular immuno-deficiency, N. Eng. J. Med. 305, 1426-1431 
(1981). The disease has been found in male homosexuals, patients receiving 
blood products, intravenous drug addicts, and individuals originating from 
Haiti and Central Africa. Piot, P. et al. Acquired immunodeficiency 
syndrome in a heterosexual population in Zaire. Lancet 11, 65-69 (1984). 
The causative agent was suspected to be of viral origin as the 
epidemiological pattern of AIDS was consistent with a transmissable 
disease. At least three (3) retroviruses have been isolated from cultured 
T-cells of several patients with AIDS, or from white blood cells of 
persons at risk for the disease. A novel human retrovirus called 
lymphadenopathy-associated virus (LAV) was discovered and its properties 
were consistent with its etiological role in AIDS. That virus was isolated 
from a patient with lymphadenopathy and hence the name. Montagnier, L. et 
al. A New Human T-lymphotropic retrovirus: characterization and possible 
role in lymphadenopathy and acquired immune deficiency syndromes. In Human 
T-Cell Leukemia/Lymphoma Virus, R. C. Gallo, M. Essex and L. Gross, eds. 
(Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory) pp. 363-370. 
Other human retroviruses, specifically two subgroups of the human T-cell 
leukemia/lymphoma/lymphotropic virus, types I and III have been isolated. 
(HTLV I: Poicsz, B. J. et al. PNAS (USA) 77, 7415 (1980)): (HTLV-III: 
Popovic, M. et al. Detection, isolation and continuous production of 
cytopathic retroviruses (HTLV-III) from patients with AIDS and pre-AIDS. 
Science 224, 797-500 (1984)). Still another virus, the AIDS-associated 
retrovirus (ARV), was proposed as the causative agent. Levy, J. A. et al. 
Isolation of lymphocytopathic retroviruses from San Francisco patients 
with AIDS. Science 225, 840-842 (1984)). Both the HTLV-III and ARV 
retroviruses display biological and sero-eidemiological properties similar 
to LAV. Levy et al., supra, Popovic et al. supra. As seen from the above, 
at least three (3) retroviruses have been postulated as the etiologic 
agent or AIDS: LAV; ARV; and, HTLV subtypes I and III. 
LAV, HTLV III and ARV-II genomes have been molecularly cloned. Schupbach, 
G. M. et al., Serological analysis of a subgroup of human T-lymphotropic 
retroviruses (HTLV III) associated with AIDS. Science 224, 503-505 (1984). 
Alizon, M. et al. Molecular Cloning of lymphadenopathy--associated virus. 
Nature, in press. The complete nucleotide sequence of the proviral genome 
of LAV, ARV and HTLV III has been determined. Ratner, L. et al. Complete 
nucleotide sequence of the AIDS virus, HTLV III. Nature 313, 277-284 
(1985); Sanchez-Pescador, R. et al. Nucleotide sequence and expression of 
an AIDS-associated retrovirus (ARV-2). Science 227, 484-492 (1985); and, 
Wain-Hobson, S. et al. Nucleotide sequence of the AIDS virus, LAV. Cell 
40, 9-17 (1985). 
One reason for the difficulty in determining the etiologic agent of AIDS 
was due to the reactivity of various retroviral antigens with serum 
samples from AIDS patients. For example, serum samples from AIDS patients 
have been shown to react with antigens of HTLV I and HTLV III. (HTLV-I: 
Essex, M., et al., "Antibodies to Cell Membrane Antigens Associated with 
Human T-Cell Leukemia Virus in Patients with AIDS", Science 220, 
859(1983)); (HTLV-III: Sarngadharan, M. G. et al., "Antibodies Reactive 
With Human T-Lymphotropic Retroviruses (HTLV-III) in the Serum of Patients 
With AIDS", Science 224, 506-508 (1984)). Envelope gene products of HTLV 
demonstrated antigenicities cross-reactive with antibodies in sera from 
adult T-cell leukemia patients. Kiyokana, T. et al. Envelope proteins of 
human T-cell leukemia virus: Expression in Escherichia coli and its 
application to studies of env gene functions"PNAS (USA) 81, 6202-6206 
(1984). Adult T-cell leukemias (ATL) differ from acquired immune 
deficiency syndrome (AIDS) in that HTLV-I causes T-cell malignancies, that 
is uncontrolled growth of T-cell. In AIDS rather than cell growth there is 
cell death. In fact this cytopathic characteristic of HTLV III was 
critical to determining ultimately the specific retroviral origin of the 
disease. Thus the etiologic agent of AIDS was isolated by use of 
immortalized human neoplastic T cell lines (HT) infected with the 
cytopathic retrovirus characteristic of AIDS, isolated from AIDS afflicted 
patients. Seroepidemiological assays using this virus showed a complete 
correlation between AIDS and the presence of antibodies to HTLV III 
antigens. Gallo et al. supra 1984; Sarngadharan et al. supra 1984; 
Schupbach et al. Serological Analysis of a subgroup of human 
T-lymphotropic retroviruses (HTLV III) associated with AIDS, Science 224. 
503-505 (1984). In addition, nearly 85% of patients with lymphadenopathy 
syndrome and a significant proportion of asymptomatic homosexual men in 
AIDS endemic areas were also found to carry circulating antibodies to HTLV 
III. Taken together, all these data indicate HTLV III to be the etiologic 
agent for AIDS. 
Until the successful culturing of AIDS virus using H-9 cell line the env 
AIDS protein of the AIDS virus had not been isolated, characterized or 
synthesized. This in major part is due to the fact that the virus is 
cytopathic and thus isolation of the virus was not possible. Popovic, M. 
et al., Detection, Isolation, and Continuous Production of Cytopathic 
Retroviruses (HTLV III) From Patients With AIDS and Pre AIDS, Science 224, 
497-500 (1984). Once the human T-cell line resistant to the cytopathic 
effects of the virus was discovered, a molecular clone of proviral DNA 
could be achieved. 
The need for a sensitive and rapid method for the diagnosis of AIDS in 
human blood and its prevention by vaccination is very great. Virtually all 
the assays/tests presently available are fraught with errors. In fact the 
Center for Disease Control (CDC) has indicated that presently available 
tests be used solely for screening units of blood for antibody to HTLV 
III. The CDC went further by stating that the presently available ELISA 
tests not be used for general screening of high risk populations or as a 
diagnostic test for AIDS. Federal Register 50(48), 9909, Mar. 12, 1985. 
The errors have been traced to the failure to use a specific antigenic 
protein of the etiologic agent for AIDS. The previously used proteins were 
derived from a viral lysate. Since the lysate is made from human cells 
infected with the virus, i.e. the cells used to grow the virus, the lysate 
will contain human proteins as well as viral proteins. Thus preparation of 
a pure antigen of viral protein is very difficult. The antigen used 
produced both false positive and false negative results. Budiansky, S., 
AIDS Screening, False Test Results Raise Doubts, Nature 312, 583(1984). 
The errors caused by the use of such lysate proteins/peptides can be 
avoided by using a composition for binding AIDS antibodies which is 
substantially free of the non-AIDS specific proteins. Compositions that 
are substantially pure AIDS envelope protein can be used as antigens. The 
AIDS envelope protein of the instant invention has been established to 
have conserved epitopes which permit its use to screen for, diagnose 
and/or prevent by vaccination the AIDS virus. The instant invention 
demonstrates that the envelope protein with its conserved epitopes 
includes all the variants which have been claimed as the sole etiologic 
agent. 
The envelope AIDS protein of the present invention may be produced by 
conventionally known methods. The processes by which the novel protein may 
be produced can be divided into three groups: (1) chemical synthesis; (2) 
preparation of a gene prepared by chemical synthesis is inserted into a 
host and a protein is produced by the host; and (3) a gene obtained 
biotechnically is inserted into a host and a protein is produced by the 
host. 
In one embodiment of this invention, recombinant DNA techniques are 
utilized by which env AIDS DNA from a natural source is introduced into a 
cell to produce the env AIDS protein. One method of obtaining DNA which 
encodes env AIDS is to read the genetic code in reverse and synthesize an 
oligodeoxynucleotide which should encode the env AIDS amino acid sequence. 
As the env protein has not been isolated or characterized this approach 
cannot be pursued. 
Alternatively gene expression can be obtained using recombinant DNA 
technology if DNA isolated from natural sources is used instead of 
synthetic DNA. 
SUMMARY OF THE INVENTION 
This invention is directed to the engineering of HTLV III env gene into 
suitable expression vectors; transformation of host organisms with such 
expression vectors: and production of envelope AIDS protein (env AIDS) by 
culture of such transformed cells. Another aspect of the present invention 
relates to the isolation and use of the resulting recombinant env AIDS 
protein. 
Another aspect of the present invention is the identification and 
determination of the proviral DNA sequence. More specifically, this aspect 
of the invention relates to determination and comparison of the proviral 
nucleotide sequence of the envelope genes of the purported etiologic agent 
of AIDS i.e. lymphadenopathy-associated virus (LAV), AIDS-associated 
retrovirus (ARV) and the human T-cell leukemia/lymphoma/lymphotropic virus 
type III (HTLV III). 
A further aspect of this invention relates to a diagnostic method for 
testing human blood for the presence of antibodies to the env AIDS 
protein. This aspect of the invention overcomes the problems of all 
previously used blood tests for AIDS. One of the problems is the use of 
compositions to bind AIDS antibody which contain proteins or peptides 
which were not derived solely from the AIDS etiologic agent. A composition 
using homogeneous envelope AIDS protein of this invention overcomes the 
nonspecificity of the prior tests or assays. Yet another aspect of this 
invention is a diagnostic method for detecting and/or determining the 
presence of the antigen in human blood. 
Another aspect of this invention is to use the env AIDS proteins of the 
instant invention as antigens suitable for providing protective immunity 
against AIDS when incorporated into a vaccine.

DETAILED DESCRIPTION OF THE INVENTION 
In the description the following terms are employed: 
Nucleotide--A monomeric unit of DNA consisting of a sugar moiety (pentose), 
a phosphate, and either a purine or pyrimidine base (nitrogenous 
heterocyclic). The base is linked to the sugar moiety via the glycosidic 
carbon (1' carbon of the pentose). That combination of a base and a sugar 
is called a nucleoside. Each nucleotide is characterized by its base. The 
four DNA bases are adenine ("A"), guanine ("G"), cytosine ("C") and 
thymine ("T"). 
DNA Sequence--A linear array of nucleotides connected one to the other by 
phosphodiester bonds between the 3' and 5' carbons of adjacent pentoses. 
Codon--A DNA sequence of three nucleotides (a triplet) which encodes 
through mRNA an amino acid, a translation start signal or a translation 
termination signal. For example, the nucleotide triplets TTA, TTG, CTT, 
CTC, CTA and CTG encode for the amino acid leucine ("Leu"). TAG, TAA and 
TGA are translation stop signals and ATG is a translation start signal. 
Reading Frame--The grouping of codons during translation of mRNA into amino 
acid sequences. During translation the proper reading frame must be 
maintained. For example, the sequence GCTGGTTGTAAG may be translated in 
three reading frames or phases, each of which affords a different amino 
acid sequence: 
GCT GGT TGT AAG--Ala-Gly-Cys-Lys 
G CTG GTT GTA AG--Leu-Val-Val 
GC TGG TTG TAA G--Trp-Leu-(STOP) 
Polypeptide--A linear array of amino acids connected one to the other by 
peptide bonds between the -amino and carboxy groups of adjacent amino 
acids. 
Genome--The entire DNA of a cell or a virus. It includes inter alia the 
structural genes coding for the polypeptides of the substance, as well as 
operator, promoter and ribosome binding and interaction sequences, 
including sequences such as the Shine-Dalgarno sequences. 
Structural Gene--A DNA sequence which encodes through its template or 
messenger RNA ("mRNA") a sequence of amino acids characteristic of a 
specific polypeptide. 
Transcription--The process of producing mRNA from a structural gene. 
Translation--The process of producing a polypeptide from mRNA. 
Expression--The process undergone by a structural gene to produce a 
polypeptide. It is a combination of transcription and translation. 
Plasmid--A circular double-stranded DNA molecule that is not a part of the 
main chromosome of an organism containing genes that convey resistance to 
specific antibiotics. When the plasmid is placed within a unicellular 
organism, the characteristics of that organism may be changed or 
transformed as a result of the DNA of the plasmid. For example, a plasmid 
carrying the gene for tetracycline resistance (Tet.sup.R) transforms a 
cell previously sensitive to tetracycline into one which is resistant to 
it. A cell transformed by a plasmid is called a "transformant". 
Cloning Vehicle--A plasmid, phage DNA or other DNA sequences which are able 
to replicate in a host cell, which are characterized by one or a small 
number of endonuclease recognition sites at which such DNA sequences may 
be cut in a determinable fashion without attendant loss of an essential 
biological function of the DNA, e.g., replication, production of coat 
proteins or loss of promoter or binding sites, and which contain a marker 
suitable for use in the identification of transformed cells, e.g., 
tetracycline resistance or ampicillin resistance. A cloning vehicle is 
often called a vector. 
Cloning--The process of obtaining a population of organisms or DNA 
sequences derived from one such organism or sequence by asexual 
reproduction. 
Recombinant DNA Molecule or Hybrid DNA--A molecule consisting of segments 
of DNA from different genomes which have been joined end-to-end outside of 
living cells and have the capacity to infect some host cell and be 
maintained therein. 
The nomenclature used to define the peptides or proteins is that used in 
accordance with conventional representation such that the amino group at 
the N-terminus appears to the left and the carboxyl group at the 
C-terminus to the right. By natural amino acid is meant one of common, 
naturally occurring amino acids found in proteins comprising Gly, Ala, 
Val, Leu, Ile, Ser, Thr, Lys, Arg, Asp, Asn, Glu, Gln, Cys, Met, Phe, Tyr, 
Pro, Trp and His. By Nle is meant norleucine, and by Nva is meant 
norvaline. Where the amino acid residue has isomeric forms, it is the 
L-form of the amino acid that is represented unless otherwise expressly 
indicated. In addition, amino acids have been designated by specific 
letters of the alphabet such that: A-Alanine; B-Aspartic Acid-Asparagine; 
C-Cysteine; D-Aspartic Acid; E-Glutamic Acid; F-Phenylalanine; G-Glycine; 
H-Histidine; I-Isoleucine; K-Lysine; L-Leucine: M-Methionine; 
N-Asparagine: P-Proline: Q-Glutamine: R-Arginine; S-Serine: T-Threonine; 
V-Valine; W-Tryptophan; Y-Tyrosine; Z-Glutamine-Glutamic Acid. 
In accordance with the present invention, the search for the envelope 
protein of the etiologic agent for acquired immune deficiency syndrome 
(AIDS) has led to the isolation and sequencing of the proviral gene of the 
AIDS virus. It has now been discovered, for what is believed to be the 
first time that the postulated etiologic agents of AIDS, 
lymphadenopathy-associated virus (LAV), AIDS-Associated retrovirus (ARV) 
and human T-cell leukemia/lymphoma/ lymphotropic virus, HTLV III, are in 
fact variants of the same virus. For purposes of this invention and claims 
the virus causing AIDS will be referred to herein as AIDS virus. AIDS 
virus will be understood to include the variants which have been 
postulated as the causative agent of AIDS, namely LAV, ARV and HTLV III. 
The envelope protein of the AIDS virus (env AIDS) is a 97,200 dalton 
protein with 32 potential N-glycosylation sites. Nucleotide sequence 
analysis of the AIDS envelope gene of the putative etiologic agents of 
AIDS demonstrates that all the viruses are variants of the same virus. 
That is there is approximately 1 to 20% divergence or variation from the 
sequence of the envelope gene of HTLV III and the sequences of the 
envelope genes of the other viruses LAV and ARV-2. The amino acid sequence 
of the AIDS env protein is set forth in FIG. 6(a). The amino acid 
distribution is set forth in FIG. 6(b). 
The nucleotide sequence of the envelope gene is shown in FIG. 1. The 
proviral DNA sequence, using methods known to one of ordinary skill in the 
art such as the chemical degradation method of Maxam and Gilbert or the 
M13 sequencing system of Messing which is a modification of the dideoxy 
nucleotide chain termination method of Sanger, was analyzed to determine 
the location of the region coding for the envelope protein. The location 
of an open reading frame, i.e. a long stretch of triplet codons not 
interrupted by a translational stop codon, for the envelope gene was 
determined. The open reading frame coding for the env gene is 863 amino 
acids and contained an ATG codon at the eighth position from the 5' end of 
the reading frame. The ATG codon is known to be a universal 
translation-initiation codon. 
The integrated proviral genome of HTLV-III was cloned from the genomic DNA 
of H9 cells infected with HTLV-III. Shaw et al., 1984 Molecular 
characterization of Human T-cell leukemia (lymphotropic) virus type III in 
the acquired immune deficiency syndrome. Science 226, 1165-1171 (1984). 
Since the HTLV-III provirus was found to lack XbaI restriction sites a 
genomic library was constructed by using XbaI digested H9/HTLV-III DNA. 
There are several methods available to one of ordinary skill in the art 
for screening the bacterial clones containing the AIDS env protein CDNA. 
These include, for example, RNA selection hybridization, differential 
hybridization with a synthetic probe or screening for clones that produce 
the desired protein by immunological or biological assays. From the 
genomic library, colonies of cells transformed with DNA that contains the 
HTLV III sequences were selected by hybridization screening of the library 
with HTLV III cDNA. The DNA insert of the hybridization-positive clone, 
HXB-3, was excised from the plasmid DNA and sequenced. 
The predicted product of the env gene shares many features in common with 
the envelope gene products of other retroviruses. Thus, a hydrophobic 
region is seen in the middle of the protein (amino acids 519-534) which 
includes a processing site for the cleavage of the precursor protein into 
exterior and transmembrane proteins. Similarly, the amino terminal end 
contains a short stretch of hydrophobic amino acids (amino acids 17-37) 
which constitutes a potential signal sequence. The HTLV-III envelope 
precursor differs from the other retroviral envelope protein precursors in 
that it contains an additional stretch of 180 amino acids at the carboxy 
terminus. 
Polymorphism within the Envelope Region of AIDS Virus 
The recent publication of the nucleotide sequences of LAV, ARV-2 and 
HTLV-III (Ratner et al., supra, 1985; Sanchez-Pescador et al., supra, 
1985; Wain-Hobson et al., supra, 1985) allows a detailed comparison of 
these various isolates obtained from AIDS patients from different parts of 
the world. HTLV-III clones were isolated from AIDS patient lymphocytes 
obtained from the east coast of the United States, while LAV was isolated 
from a French man and ARV was isolated from a patient in California. A 
comparison of the sequence data confirms the earlier observations made 
using restriction enzyme site analysis which showed approximately 10% 
variation. The present analysis shows that the various isolates show the 
greatest amount of conservation in the gaq and pol regions while the most 
divergence occurs in the env region. A comparison of the four env 
sequences is presented in FIG. 2. With respect to the envelope gene, 
HTLV-III and LAV are more closely related to each other than the ARV 
clone. Approximately 1.6% divergence was observed between the HTLV-III 
(HXB-3) and LAV sequence. Among the HTLV sequences, the divergence was 
about 1.6%. However, approximately 17% divergence was observed between 
HTLV-III and ARV-II and this was more pronounced in the extracellular 
region of the envelope gene product. FIG. 2. This high rate of divergence 
could be due to the geographical location where the two isolates were 
derived or the time of isolation of these variants. ARV-2 was isolated 
from the west coast of the United States more recently. The HTLV-III 
isolates for which the nucleotide sequences have been determined were all 
obtained from the east coast of the United States a year earlier. LAV was 
obtained from a French patient who appears to have acquired the virus in 
New York about the same period. The observed differences in the sequence 
probably reflect divergent evolution of strains separated in time or 
geography or both. Within the env region, the highest level of divergence 
is in the extracellular portion of the protein. 
Expression Vector 
A wide variety of host/cloning vehicle combinations may be employed in 
cloning the double-stranded DNA. For example, useful cloning vehicles may 
consist of segments of chromosomal, nonchromosomal and synthetic DNA 
sequences, such as various known bacterial plasmids, e.g., plasmids from 
E. coli such as pBR322, phage DNA, and vectors derived from combinations 
of plasmids and phage DNAs such as plasmids which have been modified to 
employ phage DNA or other expression control sequences or yeast plasmids. 
Useful hosts may include microorganisms, mammalian cells, plant cells and 
the like. Among them microorganisms and mammalian cells are preferably 
employed. As preferable microorganisms, there may be mentioned yeast and 
bacteria such as Escherichia coli, Bacillus subtilis, Bacillus 
stearothermophilus and Actinomyces. The above-mentioned vectors and hosts 
may also be employed for the production of a protein from a gene obtained 
biologically as in the instant invention. Of course, not all host/vector 
combinations may be equally efficient. The particular selection of 
host/cloning vehicle combination may be made by those of skill in the art 
after due consideration of the principles set forth without departing from 
the scope of this invention. 
Furthermore, within each specific cloning vehicle, various sites may be 
selected for insertion of the double-stranded DNA. These sites are usually 
designated by the restriction endonuclease which cuts them. For example, 
in pBR322 the EcoRI site is located just outside the gene coding for 
ampicillin resistance. Various sites have been employed by others in their 
recombinant synthetic schemes. Several sites are well recognized by those 
of skill in the art. It is, of course, to be understood that a cloning 
vehicle useful in this invention need not have a restriction endonuclease 
site for insertion of the chosen DNA fragment. Instead, the vehicle could 
be joined to the fragment by alternative means. 
The vector or cloning vehicle and in particular the site chosen therein for 
attachment of a selected DNA fragment to form a recombinant DNA molecule 
is determined by a variety of factors, e.g., number of sites susceptible 
to a particular restriction enzyme, size of the protein to be expressed, 
susceptibility of the desired protein to proteolytic degradation by host 
cell enzymes, contamination of the protein to be expressed by host cell 
proteins difficult to remove during purification, expression 
characteristics, such as the location of start and stop codons relative to 
the vector sequences, and other factors recognized by those of skill in 
the art. The choice of a vector and an insertion site for a particular 
gene is determined by a balance of these factors, not all selections being 
equally effective for a given case. 
There are several known methods of inserting DNA sequences into cloning 
vehicles to form recombinant DNA molecules which are equally useful in 
this invention. These include, for example, direct ligation, synthetic 
linkers, exonuclease and polymerase-linked repair reactions followed by 
ligation. or extension of the DNA strand with DNA polymerase and an 
appropriate single stranded template followed by ligation. 
It should, of course, be understood that the nucleotide sequences of the 
DNA fragment inserted at the selected site of the cloning vehicle may 
include nucleotides which are not part of the actual structural gene for 
the desired polypeptide/protein or may include only a fragment of the 
complete structural gene for the desired protein. It is only required that 
whatever DNA sequence is inserted, a transformed host will produce a 
protein/peptide having an immunological activity to the AIDS env protein 
or that the DNA sequence itself is of use as a hybridization probe to 
select clones which contain DNA sequences useful in the production of 
polypeptides/proteins having an immunological activity to the AIDS env 
protein. 
The cloning vehicle or vector containing the foreign gene is employed to 
transform a host so as to permit that host to express the protein or 
portion thereof for which the hybrid DNA codes. The selection of an 
appropriate host is also controlled in a number of factors recognized by 
the art. These include, for example, compatibility with the chosen vector, 
toxicity of proteins encoded by the hybrid plasmid, ease of recovery of 
the desired protein, expression characteristics, biosafety and costs. A 
balance of these factors must be struck with the understanding that not 
all hosts may be equally effective for expression of a particular 
recombinant DNA molecule. 
A preferred embodiment of the instant invention was to express segments of 
the AIDS env protein in E. coli by inserting restriction fragments 
isolated from the cloned proviral genome into the versatile pEV-vrf 
(variable reading frame) expression plasmids (Lacal et al., Expression of 
Normal and Transforming H-ras Genes in E. coli and Purification of Their 
Encoded Proteins. Proc. Nat. Acad. Sci, USA 81, 5305-5309, 1984; Lomedico, 
P. et al., Cloning and Expression of Murine Interleukin-1 cDNA in E. coli, 
Nature 312, 458-462, 1984). Restriction fragments are fragments of 
proviral DNA resulting from the action of restriction enzymes. These 
versatile pEV-vrf plasmids are derivatives of pBR322 which contain the 
phage lambda P1 promoter, a synthetically-derived ribosome-binding site, 
and convenient cloning sites (EcoRI, BamHI, ClaI and HindIII) just 
downstream to the initiation codon. In the present synthesis the preferred 
initial cloning vehicle is the bacterial plasmid pBR322 (ATCC 37017) and 
the preferred initial restriction endonuclease sites therein are the EcoRI 
and HindIII sites. (FIG. 3). The plasmid is a small (molecular weight 
approximately 2.6 megadaltons) plasmid carrying a resistance gene to the 
antibiotic ampicillin (amp). The plasmid has been fully characterized (F. 
Bolivar et al., Construction And Characterization Of New Cloning vehicles 
II. A Multi-Purpose Cloning System, Gene. 2, 95-113 (1977); J. G. 
Sutcliffe, pBR322 Restriction Map Derived From The DNA Sequence: Accurate 
DNA Size Markers Up To 4361 Nucleotide Pairs Long, Nucleic Acids Research, 
5, pp. 2721-28 (1978)). Insertion of the DNA product in this site provides 
a large number of bacterial clones each of which contains one of the 
proviral DNA genes or fragments thereof present in the DNA product of Hg 
cells. Only a very few of these clones will contain the gene for env AIDS 
or fragments thereof. The preferred host for initial cloning in accordance 
with this invention is E. coli MC 1061. Casadaban, M. J. and Cohen, S. N., 
Analysis of Gene Control Signals by DNA Fusion and Cloning E. coli, J. 
Mol. Biol., 138, 179-207, 1980. A set of three pEF-vrf plasmids was 
constructed to accommodate all three translational reading frames. The P1 
promoter is regulated by a temperature-sensitive cI repressor encoded on 
the compatible plasmid pRK248cIts (ATCC 33766) (Bernard and Helinski, 
1979). These expression plasmids have been used to produce substantial 
amounts of several heterologous proteins in E. coli, including v-bas p21 
(Lacal et al., supra, 1984) and murine interleukin-1 (Lomedico et al., 
supra, 1984). 
The coding sequences for amino acid residues #44 to 640 of the env protein 
are located downstream of the p1 promoter between the KpnI and HindIII 
sites on the restriction map as shown in FIG. 3. Aside from the location 
of these convenient restriction sites, these sequences were chosen for 
bacterial expression experiments because they did not include the 
amino-terminal signal peptide as well or the hydrophobic transmembrane 
segment at the carboxyl end. These sequences were excluded to avoid 
possible toxicity problems which can occur when hydrophobic proteins are 
over-produced in bacterial cells. In a preferred embodiment of this 
invention an expression plasmid was constructed that would direct the 
synthesis of this segment of the env gene product (designated pEV/env 
44-640), an intermediate construction was first made by inserting a 2400 
bp EcoRI-HindIII fragment between the EcoRI and HindIII sites in the 
pEV-vrf plasmids. The HTLV-III sequences (600 bp) between the EcoRI and 
the KPnI site were then removed from the intermediate construction as 
shown in FIG. 3. These plasmid constructions were carried out with all 
three pEV-vrf plasmids so that subsequent deletions could be made and the 
correct reading frame maintained. In addition, the constructions made in 
the incorrect reading frames served as important controls in the 
expression experiments described below. 
In another embodiment of this invention, a second set of expression 
plasmids were constructed in a similar fashion by deleting sequences 
between EcoRI and StuI sites which occur 483 bp downstream to the site in 
the env gene. Again these deletions (designated pEV/env 205-640) were made 
in all three reading frames. The translation termination codon used in all 
of the env expression plasmids is presumably an in-frame TAA located 23 bp 
downstream of the HindIII site in the plasmid. Thus, 8 amino acid residues 
at the carboxyl terminus are encoded by pBR322. 
Expression of ENV AIDS 
There are several approaches to screen for bacterial clones containing env 
AIDS cDNA. These include, for example, RNA selection hybridization, 
differential hybridization, hybridization with a synthetic probe and 
screening for clones that produce the desired protein by immunological or 
biological assays. Two methods are available to screen using immunological 
assays: screening of bacterial colonies for the presence of protein using 
antibody; and, preferably, the bacterial lysates are electrophoresed, 
blotted onto a nitrocellulose paper and then probed with the antibody. 
In a preferred embodiment of this invention, cultures of the E. coli strain 
MC 1061 transformed with pRK248cIts and the pEV 1, 2, or 3/env 44-640 (or 
pEV 1, 2 or 3/env 205-640) were grown in M9 medium at 30.degree. C. to 
mid-log phase and then induced by shifting to 42.degree. C. for 2 hr. 
Samples of the bacterial cultures were then taken and subjected to 
SDS-polyacrylamide gel electrophoresis, followed by Western blot analysis 
to detect env proteins. The protein blots were treated with antisera to 
env AIDS proteins isolated either from immunized rabbits or from AIDS 
patients previously shown to contain high titer antibodies to AIDS 
antigens. This was followed by incubation with .sup.125 I-labelled 
Staphylococcus aureus protein A, washing and autoradiography. Similar 
results were obtained with both sera except that the human serum was found 
to contain much higher titers of anti-HTLV-III antibodies and was devoid 
of all background reactivity with the E. coli proteins. For this reason 
human antibodies were used in all subsequent characterization. 
FIG. 4 shows the pattern of reactivity of the env AIDS proteins synthesized 
in bacteria (recombinant proteins) with anti-HTLV-III antibodies. The open 
reading frame in pEV3/env 44-640 encodes a protein that should migrate as 
a 68 Kd band on the gel. In fact, a 68 Kd band is observed in the lane 
corresponding to the induced cells containing pEV3/env 44-640 (lane C). 
However, in addition to the 68 Kd band, these cells synthesized proteins 
of 35 Kd, 25 Kd and 18 Kd which specifically cross-reacted with 
anti-HTLV-III antibodies. No HTLV-III cross-reacting bands are evident in 
the uninduced control (Lane b) or in a second negative control sample 
(Lane a) of induced cells containing a plasmid that directs the synthesis 
of v-bas p21 oncogene product (Lacal et al., 1984). The appearance of 
multiple bands synthesized from the env gene sequences was an unexpected 
result. Another unexpected result was the synthesis of env gene products 
from the plasmid (pEV1/env 44-640) where the insert was placed in the 
wrong reading frame with respect to the initiator codon immediately 
downstream of the P1 promoter (Lane d). In this case, the E. coli 
containing these plasmids synthesized a 68 Kd protein in addition to the 
35 Kd, 25 Kd and 18 Kd proteins. These results could be readily explained 
when the nucleotide sequence of the envelope gene (FIG. 1) was examined. 
About 155 bases downstream to the KPnI site is an ATG codon which appeared 
to be utilized for the synthesis of env gene product by the two expression 
plasmids pEV1/env and pEV2/env 44-640. Internal translation initiation is 
also the likely explanation for the appearance of the 35 Kd, 25 Kd and 17 
Kd proteins. Initiation codons which are preceded by so-called 
Shine-Dalgarno sequences (AGGA) are found within the env coding region at 
locations that are consistent with the sites of the observed protein 
products. 
To confirm the above interpretation and to rule out the possibilities that 
the smaller proteins are not formed as a result of premature termination 
or from proteolytic cleavage of the larger product, we constructed another 
deletion mutant in which sequences between the KpnI and StuI sites were 
deleted. This expression plasmid contains the coding sequences from amino 
acid positions 205-640 which could code for a protein of 49 Kd. Analysis 
of the proteins induced from E. coli harboring this plasmid verified that, 
in fact, these cells synthesize a 49 Kd protein in addition to the 35 Kd, 
25 Kd and 17 Kd proteins (lane e. FIG. 4). From these results, we conclude 
that pEV3/env 44-640 expression plasmid directs the synthesis of a 68 Kd 
protein in addition to several additional smaller polypeptides (i.e., 35 
Kd, 25 Kd and 17 Kd) produced from all of the env expression plasmids 
resulting from internal translation initiation within the env gene. 
The nucleic acid sequences of the envelope gene subsequences described 
above and the corresponding protein amino acid sequences are shown in 
FIGS. 9 and 10, respectively. 
Screening of AIDS SERA 
Because anti-HTLV-III antibodies are found in more than 90% of the AIDS 
bacterially synthesis interest to see if the bacterially synthesized env 
gene products could be used as diagnostic tools for the detection of these 
antibodies. For this analysis, total cell protein from an induced 
bacterial culture was fractionated by SDS-PAGE and transferred to a 
nitrocellulose filter by Western blotting technique. Strips of the filter 
containing transferred proteins were reacted with 1000-fold diluted human 
sera, and the antigen-antibody complexes formed were detected by 
incubation of the strips with 125-I-labelled Staphylococus aureus protein 
A followed by autoradiography. Prominent bands corresponding to reaction 
of the antibody to the 68 Kd, 35 Kd, 25 Kd and 17 Kd proteins were 
consistently observed when the serum used was from patients with AIDS 
syndrome. The results of one such assay with 20 human sera are presented 
in FIG. 5. The negative controls used were normal human sera and serum 
from a patient with HTLV-I infection. No reaction was observed with sera 
from healthy individuals or from HTLV-I infected individuals. The patient 
sera were derived from all parts of the United States including California 
and all AIDS patient sera tested so far were found to be positive. The 
results suggest that these antibodies are mainly directed against the 
protein back bone or the molecule. 
It appears, therefore, that the env gene products constitute the best 
diagnostic reagents for the detection of AIDS associated antibodies. The 
env gene product of the instant invention encompasses a large portion of 
the protein molecule and contains both the conserved and divergent 
portions of the molecule. In spite of the divergence observed between 
HTLV-III and ARV-2 sequences the recombinant env proteins of the instant 
invention synthesized by the bacteria react with AIDS patient sera derived 
from both geographical locations of the United States. One hundred percent 
(100%) of AIDS patient sera (50 individual samples, 25 derived from the 
East Coast of the United States and 25 derived from California) tested 
showed high reactivity. This is strong evidence for the presence of 
conserved epitopes within the molecule against which the immune system 
could mount an antibody reaction. The human immune system may thus be 
mounting an immune response against conserved epitopes of the envelope 
molecule, as suggested by the reactivity of the AIDS patient sera. The 
observed divergence between various isolates of HTLV-III thus may not pose 
a problem for the use of recombinant protein as a vaccine. The 68 Kd 
protein is ideally suited for such a purpose since it encompasses a large 
portion of the gene product and has the unique structural feature of 
containing both the extracellular hydrophilic region and the membrane 
associated hydrophobic regions. This structural feature makes it well 
suited for encapsulation into liposomes which have been used as vehicles 
for vaccination against other vital envelope proteins. 
Based on these discoveries it is proposed that in the practice of screening 
blood for acquired immune deficiency syndrome (AIDS) only AIDS envelope 
protein or a variant of said protein be utilized. Utilizing the env AIDS 
protein of the instant invention, human blood can be screened for the 
presence of antibodies to the AIDS virus. This and other techniques are 
readily determined, once, as taught for the first time by the present 
invention that the envelope AIDS protein is the envelope protein of the 
etiologic agent of AIDS. The foregoing and other objects, features and 
advantages of the invention will be apparent from the following examples 
of preferred embodiments of the invention. 
EXAMPLE 1 
Molecular Cloning and Nucleotide Sequence Analysis 
The integrated proviral genome of HTLV-III was recently cloned from the 
genomic DNA of H9 cells infected with HTLV-III (Schupbach et al., supra, 
1984). The proviral genome which was obtained by using XbaI digested 
H9/HTLV-III DNA contained two internal EcoRi sites within the viral genome 
and two additional sites in the cloning vector .lambda.J1. These sites 
were used for further subcloning of the three DNA fragments of 5.5 Kbp, 
4.5 Kbp and 1.1 Kbp into pBR322. Nucleotide sequence analysis of the 
proviral genome was determined by the chemical degradation method of Maxam 
and Gilbert, supra, (1980). For the sequence analysis, DNA inserts from 
the three subclones were isolated by electroelution and further cleaved 
with appropriate restriction enzymes. The DNA fragments were labelled 
either with .lambda.-32P-ATP and polynucleotide kinase at their 5' ends or 
with .lambda.-32P-NTP, by filling with DNA polymerase (Klenow fragment), 
at their 3' ends. The DNA fragments labelled at the two ends were cleaved 
with a second enzyme and the fragments labelled at a single end were 
purified on 5% acrylamide gels and used for sequence analysis. For the 
sequence analysis of the env gene, a shotgun approach was utilized where 
the 4.5 EcoRI fragments were cleaved with one of the following enzymes: 
BgIII, HindIII, XhoI, AvaII, HinfI and Sau3A and the restriction fragments 
labelled and sequenced as described above. 
EXAMPLE 2 
Construction of pEV/env 44-640 
Expression plasmids pEV-vrf 1, 2, 3 have been described. Lacal et al., 
Expression of Normal and Transforming H-ras Genes in E.coli and 
Purification of Their Encoded Proteins. Pro. Natl. Acad. Sci. USA 31, 
5305-5309 (1984); Crowl, R. et al. Versatile Expression Vectors for High 
Level Synthesis of Cloned Gene Products in E. coli, Gene, 38, 31-38 
(1985). 
pRC2 is a derivative of pBR322 containing a unique BGlII site adjacent (on 
the amp.sup.R side) to the EcoRI site in the plasmid. This plasmid was 
constructed in the following manner. 20 .mu.g of pBR322 plasmid DNA was 
digested with EcoRI and then split into two reactions. In one, the 
protruding 5' single-stranded termini were removed with S1 nuclease; in 
the other reaction, the termini were filled-in by incorporating 
deoxynucleotides with the Klenow fragment of DNA polymerase I. Both 
reactions were terminated by phenol extraction followed by ethanol 
precipitation. Approximately 1 .mu.g of DNA from each reaction was mixed 
with 90 pmoles of phosphorylated BglII linkers (CAGATCTG, purchased from 
Collaborative Research) and incubated with T4 DNA ligase at 15.degree. for 
18 hours. The ligation products were then digested with BglII and PstI and 
subjected to gel electrophoresis in 1% agarose. The 3600 bp and 760 bp 
fragments from both reactions were recovered from the gel. For the 
construction of pRC2, the 3600 bp from the Klenow reaction was ligated to 
the 760 bp fragment from the S1 reaction. To construct a plasmid with the 
BglII site on the other side of EcoRI (tet.sup.R side), designated pRC1, 
the 3600 bp fragment from the S1 reaction was ligated to the 760 bp 
fragment from the Klenow reaction. E. coli strain RR1 was transformed with 
the ligation mixtures, and transformants were selected on LB agar plates 
containing 50 .mu.g/ml ampicillin. Transformants containing the expected 
plasmid constructions were identified by restriction analysis of the 
isolated plasmid DNA. DNA sequence analysis confirmed that the S1 nuclease 
treatment precisely removed the 5' single-stranded termini. 
pRC23 (see FIG. 7) was constructed by inserting into pRC2 a 250 bp 
BglII-HaeIII fragment containing the .lambda.P.sub.L promoter joined to a 
pair of complementary synthetic oligonucleotides comprising a model 
ribosome-binding site (RBS). The HaeIII site is located within the 5' 
non-coding region of the .lambda.N gene 115 downstream of the P.sub.L 
transcriptional initiation site. Approximately 1 .mu.g of a 450 bp 
BglII-HpaI fragment isolated from phage DNA was digested with HaeIII. 200 
ng of the resulting digestion products were mixed with 60 pmoles each of 
phosphorylated synthetic oligonucleotides containing the model RBS. The 
ligated molecules were digested with BglII and EcoRI and separated on a 5% 
polyacrylamide gel. The 270 bp ligation product was recovered from the 
gel, mixed with gel purified pRC2 vector that had been digested with BglII 
and EcoRI, and incubated with T4 DNA ligase at 15.degree. for 15 hours. 
The ligation mixture was used to transform strain RR1(pRK248cIts). 
Transformants selected on ampicillin-containing medium were screened by 
restriction analysis of the isolated plasmid DNA. The expected plasmid 
construction, pRC23, was confirmed by further restriction enzyme 
digestions and by DNA sequence analysis across the EcoRI junction (FIG. 
7). 
For the construction of the pEV-vrf set of plasmids (see FIG. 8), pRC23 was 
digested with EcoRI and HindIII and isolated by preparative agarose gel 
electrophoresis. The mixture of synthetic oligonucleotides (32, 33, and 34 
nucleotides) was combined with the mixture of the complementary sequences 
heated to 58.degree. for 5 minutes in 50 mM NaCl, and cooled slowly to 
allow annealing. 0.1 pmoles of the synthetic duplexes were added to 0.07 
pmoles of the pRC23/EcoRI-HindIII vector and incubated with T4 DNA ligase 
at 15.degree. for 15 hours. Strain RR1 (.lambda.cI857) was transformed 
with the ligation products. Six ampicillin resistant transformants were 
selected for DNA sequence analysis. Of the six, two contained the expected 
sequence for pEV-vrf1. one for pEV-vrf2, and three for pEV-vrf3 (FIG. 3). 
For the expression of the AIDS env gene, one .mu.g of a 2400 bp EcoRI - 
HindIII DNA fragment, which was isolated from the cloned HTLV-III proviral 
genome by preparative agarose gel electrophoresis. as mixed with 0.1 .mu.g 
of EcoRI - HindIII digested vector DNA (pEV-vrf 1. 2, or 3). After heating 
at 65.degree. C. for 3 minutes, the mixtures were chillled on ice, and 20 
.mu.l ligation reactions were assembled which contained 50 mM Tris-HCl (pH 
7.4), 10 mM MgCl.sub.2, 10 mM DTT, 0.3 mM ATP, and 200 units of T.sub.4 
DNA ligase. After incubation at 15.degree. C. for 4 hours, the reactions 
were terminated by heating at 65.degree. C. for 5 minutes. The ligation 
products were used to transform E. coli strain MC1061 (pRK248cIts). 
Transformants were selected on Luria broth agar containing 50 .mu.g/ml 
ampicillin at 30.degree. C. for 18 hours plasmid DNA was isolated from 1 
ml of each culture and subjected to restriction analysis. All 12 isolates 
contained the expected plasmid construction. These intermediate 
constructions were then used to make PEV1, 2, and 3/env 44-640 by deleting 
the 600 bp between the EcoRI and KpnI sites as described below. 
Approximately 0.5 .mu.g of plasmid DNA was digested with KpnI and EcoRI. 
The resulting termini were then treated with the Klenow fragment of DNA 
polymerase I in the presence of all four deoxyribonucleotides at (100 
.mu.M) at 37.degree. C. for 30 minutes. This step results in the 
"filling-in" of the 5' overhang of the EcoRI terminus and the removal of 
the 3' overhang of the KpnI terminus. Upon recirculization of the linear 
plasmid and blunt-end ligation of these termini, an EcoRI site is 
regenerated. Transformants containing plasmids with the expected deletion 
were identified by restriction analysis. 
A second set of deletion derivatives, designated pEV/env 205-640 was 
constructed in a similar fashion. A portion of the linear plasmid that had 
been digested with EcoRI and KpnI and treated with Klenow, as described 
above, was further digested with StaI. Again, upon recircularization and 
blunt-end ligation, the EcoRI site was regenerated; however, an additional 
483 bp of env coding sequences were removed. 
EXAMPLE 3 
Expression Plasmids 
Expression plasmids pEV-vrf 1,2, 3 have been described (Lacal et al., 
supra, 1984; Crowl et al., supra, 1985). For the expression of the 
HTLV-III envelope gene, a 2400 bp EcoRI-HindIII fragment was inserted into 
the three expression vectors between the EcoRI and HindIII sites (FIG. 3). 
This intermediate construct was then cleaved with EcoRI and KpnI and the 
vector DNA purified from the 600 bp fragment by agarose gel 
electrophoresis. The DNA fragment was then treated with the Klenow 
fragment of E. coli DNA polymerase I and recircularized using the T4 
ligase. Cultures of E. coli strain MC 1061 transformed with pRK248cIts and 
the pEV 1,2, or 3/env plasmids were grown in M9medium at 30.degree. C. to 
mid-log phase and then induced by shifting to 42.degree. C. for 2 hr. 
EXAMPLE 4 
Expression and Purification of Env AIDS 
A homogeneous recombinant viral env AIDS was purified according to the 
following procedure. The env AIDS protein expressed by a microbe tends to 
associate with the membrane fractions of the host microbe, principally the 
inner membrane of the microbe. The following purification method was 
designed to deal with this finding. 
This purification method comprises: 
(a) lysis of transformed microbial cells producing recombinant env AIDS 
protein; 
(b) separation of env AIDS associated cellular membranes from other 
cellular components; 
(c) extraction of env AIDS from associated membranes; and 
(d) chromatographic purification of the resultant extraction solution 
containing env AIDS to yield a substantially pure recombinant viral env 
protein. 
More specifically, the preferred purification method for the preparation of 
substantially pure recombinant viral env protein comprises: 
(a) cultivating a transformed organism containing a DNA sequence which 
codes for viral env protein; 
(b) causing a culture of the transformed organism of step (a) to accumulate 
the env protein; 
(c) lysing the culture of transformed organisms of step (b) to form a cell 
lysate mixture; 
(d) isolating the cell membrane components of the cell lysate mixture of 
step (c); 
(e) washing the isolated cell membrane components with an extraction 
solution to yield a wash solution containing env protein; and 
(f) chromatographically purifying the wash solution of step (e) to yield a 
substantially pure env AIDS protein. 
In carrying out this method it is preferred that the cells be lysed by 
sonication, although it is forseeable that other known methods such as 
enzyme or mechanical lysis could also be used. It is preferred that the 
cell membrane component, specifically the inner and outer membranes, be 
isolated from other cellular components by methods such as centrifugation. 
It has been found that env AIDS expressed by the transformed microorganism 
tends to become associated with the cellular membranes. Therefore, 
isolation of these membranes during the purification process ensures high 
purification levels and high purity env AIDS at the end of the 
purification procedure. 
Once the cell membranes are isolated from the lysate mixture, they are 
washed with an extraction solution, preferably salt solutions and a 
detergent to yield a second solution containing approximately 50% env AIDS 
protein. Preferably the cell membranes are washed in four separate steps 
with the salt solutions and detergent although it is forseeable that 
certain of these steps could be combined, rearranged or eliminated. The 
first step of washing the cell membrane may be done with a salt solution, 
preferably 1M NaCl. In the second step the cell membrane is washed with a 
detergent solution, preferably 1% Triton X-100. In the third step, the 
cell membrane is washed with another salt solution, 1.75M to 3.5M 
Guanidine HCl. The final wash is also with a salt solution preferably 
about 7M Guanidine HCl. The wash solution which results from the fourth 
and final wash comprises about 50% env AIDS. 
The final 50% env AIDS wash solution is then further purified by a 
chromatography step, preferably reverse phase high performance liquid 
chromatography (HPLC). The HPLC step yields env AIDS protein in a 
substantially 100% pure form. It is also foreseeable that monoclonal 
antibody affinity chromatography columns utilizing env AIDS polyclonal or 
monoclonal antibodies, could be used as an alternative to HPLC. 
EXAMPLE 5 
Polyacrylamide Gel Electrophoresis and Western Blot Analysis 
Cells were lysed by resuspending the cell pellets (approximately 10.sup.8 
cells) in TG buffer (10 mM Tris, pH 7.4, 10% glycerol), mixed with an 
equal volume of 2.times.sample buffer of Laemmli (Laemmli, U. K., Cleavage 
of Structural Proteins During the Assembly of the Head of Bacteriophage 
T4. Nature 227, 680-685, 1970) and incubated at 95.degree. C. for five (5) 
minutes. Debris was pelleted by centrifugation and the cleared lysates 
were subjected to SDS-PAGE analysis, Id. For Western blot analysis, the 
proteins from the acrylamide gel were electroblotted onto a 0.1 .mu.m 
nitrocellulose membrane (Schleicher and Schuell) for 16 hr at 50V, in 12.5 
mM Tris, 96 mM glycine, 20% methanol, 0.01% SDS at pH 7.5. Processing of 
the blot was carried out using the methods described by Towbin et al. 
Electrophoretic Transfer of Proteins From Polyacrylamide Gels to 
Nitrocellulose Sheet: Procedure and Some Applications, Proc. Natl. Acad. 
Sci. U.S.A., 76. 4350-4354, (1979). For treatment with the human sera, the 
blots were incubated with a 1000 fold dilution of the sera in antibody 
buffer (20 mM sodium phosphate buffer, pH 7.5 containing 0.5M NaCl, 1% BSA 
and 0.05% Tween 20) for 2-6 hr. The blots were then washed twice with 
phosphate buffered saline containing 0.05% Tween 20 and then incubated 
with .sup.125 -I-labelled Staphylococcus aureus protein A for an 
additional period of 1 hr. The blot was then washed twice in PBS-Tween 20 
buffer, dried and autoradiographed. 
EXAMPLE 6 
Immunization with Env Protein of AIDS Virus 
It is clear that in spite of the divergence observed between HTLVIII and 
ARV-2 sequences, the recombinant proteins synthesized by the bacteria 
react well with AIDS patient sera derived from both geographical locations 
of the United States. One hundred percent (100%) of the AIDS patient sera 
tested showed high reactivity (50 individual samples, 25 from the east 
coast of the United States and 25 from the west coast of the United 
States). Thus all the env proteins contain at least one conserved epitope. 
All of the human sera from AIDS patients tested contained antibodies to 
the env proteins of the instant invention. This strongly suggests that 
these env proteins with the conserved epitopes would be immunogenic in 
man. 
It will be readily appreciated that the env proteins of the instant 
invention can be incorporated into vaccines capable of inducing protective 
immunity against the AIDS virus. By methods known in the art, the specific 
amino acids comprising the epitopes of the env protein may be determined. 
Peptides may then be synthesized, comprising an amino acid sequence 
corresponding to an epitope of an env AIDS protein either in monomeric or 
multimeric form. These synthetic peptides may then be incorporated into 
vaccines capable of inducing protective immunity against AIDS virus. 
Techniques for enhancing the antigenicity of such repeated peptides 
include incorporation into a multimeric structure, binding to a highly 
immunogenic protein carrier, for example, keyhold limpet hemocyanin, or 
diptheria toxoid, and administration in combination with adjuvants or any 
other enhancers of immune response. In addition, the vaccine composition 
may comprise antigens to provide immunity against other diseases in 
addition to AIDS. 
An amino acid sequence corresponding to an epitope of a env protein 
(repeated peptide) may be obtained by chemical synthetic means or by 
purification from biological sources including genetically modified 
microorganisms or their culture media. The repeated peptide may be 
combined in an amino acid sequence with other peptides including fragments 
of other proteins, as for example, when synthesized as a fusion protein, 
or linked to other antigenic or non-antigenic peptides of synthetic or 
biological origin. The term "corresponding to an epitope of a env protein" 
will be understood to include the practical possibility that, in some 
instances, amino acid sequence variations of a naturally occurring 
repeated peptide may be antigenic and confer protective immunity against 
AIDS infection. Possible sequence variations include, without limitation, 
amino acid substitutions, extensions, deletions, interpolations and 
combinations thereof. Such variations fall within the contemplated scope 
of the invention provided the peptide containing them is antigenic and 
antibodies elicited by such peptide cross-react with naturally occurring 
env protein or non-variant repeated peptides of env protein, to an extent 
sufficient to provide protective immunity when administered as a vaccine. 
Such vaccine compositions will be combined with a physiologically 
acceptable medium. The size and shape of epitopes found in carbohydrate 
antigens have been extensively studied, but less is known about the 
structure of epitopes from protein molecules. Some epitopes of protein 
antigens have been defined at the level of their tertiary structure. In 
every instance, the epitopes were formed not by the primary sequences 
alone, but by the juxtaposition of residues brought together by the 
folding of the polypeptide chain(s) of the native molecule. In addition, 
the structure of the 68 Kd env protein of the instant invention makes it 
particularly well suited for use as a vaccine. The 68 Kd env protein 
comprises a large portion of the gene product which: was shown to be 
reactive with all the AIDS sera tested; and, has the unique structural 
feature of containing both an extracellular hydrophilic region and the 
transmembrane hydrophobic regions. The latter structural feature makes it 
well suited for use as a vaccine using liposome encapsulation to create a 
vehicle for administration. 
Routes of administration, antigen dose, number and frequency of injections 
are all matters of optimization within the scope of ordinary skill in the 
art, particularly in view of the fact that there is experience in the art 
in providing protective immunity by the injection of other related 
antigens to provide immunity in other viral infections. It is anticipated 
that the principal value of providing immunity to AIDS infection will be 
for those individuals who have had no previous exposure to AIDS, e.g., 
individuals who are in the high risk population, such as homosexuals, drug 
addicts and people from Haiti and Central America and individuals who may 
be receiving blood transfusions. It is also anticipated that temporary 
immunity for infants may be provided by immunization of mothers during 
pregnancy. 
EXAMPLE 7 
Diagnostic Test for AIDS 
It is clear that the env gene proteins of the instant invention may be used 
as diagnostic reagents for the detection of AIDS-associated antibodies. It 
is also apparent to one of ordinary skill that a diagnostic assay for AIDS 
using polyclonal or monoclonal antibodies to the AIDS env protein of the 
instant invention may be used to detect the presence of the AIDS virus in 
human blood. In one embodiment a competition immunoassay is used where the 
antigenic substance, in this case the AIDS virus, in a blood sample 
competes with a known quantity of labelled antigen, in this case labelled 
AIDS env protein, for a limited quantity of antibody binding sites. Thus, 
the amount of labelled antigen bound to the antibody is inversely 
proportional to the amount of antigen in the sample. In another 
embodiment, an immunometric assay may be used wherein a labelled AIDS-env 
antibody is used. In such an assay, the amount of labelled antibody which 
complexes with the antigen-bound antibody is directly proportional to the 
amount of antigen (AIDS virus) in the blood sample. In a simple yes/no 
assay to determine whether the AIDS virus is present in blood, the solid 
support is tested to detect the presence of labelled antibody. In another 
embodiment, monoclonal antibodies to AIDS env protein may be used in an 
immunometric assay. Such monoclonal antibodies may be obtained by methods 
well known in the art, particularly the process of Milstein and Kohler 
reported in Nature 256, 495-497 (1975). 
The immunometric assay method is as follows: Duplicate samples are run in 
which 100 .mu.l of a suspension of antibody immobilized on agarose 
particles is mixed with 100 .mu.l of serum and 100 .mu.l of soluble 
.sup.125 I-labelled antibody. This mixture is for specified times ranging 
from one quarter hour to twenty four hours. Following the incubation 
periods the agarose particles are washed by addition of buffer and then 
centrifuged. After removal of the washing liquid by aspiration, the 
resulting pellet of agarose particles is then counted for bound .sup.125 
I-labelled antibody. The counts obtained for each of the complexes can 
then be compared to controls. 
While the invention has been described in terms of certain preferred 
embodiments, modifications obvious to one with ordinary skill in the art 
may be made without departing from the scope of the invention. For 
example, it is understood that the env AIDS DNAs described herein 
represent only the precise structure of two naturally occurring gene 
segments. It is expected that slightly modified alleles will be found 
encoding for similarly functioning proteins, and such gene segments and 
proteins are considered to be equivalents for the purpose of this 
invention. It is also suspected that other variants in addition to those 
described herein will be found and that the envelope protein of said 
variants will differ slightly. These variant envelope proteins are 
likewise considered within the scope of the invention. DNA having 
equivalent codons is considered within the scope of the invention, as are 
synthetic gene segments that encode homologous proteins of the viral 
envelope. 
Various features of the invention are set forth in the following claims.