Note: Descriptions are shown in the official language in which they were submitted.
13 4 1 5 2 3
1
Cloned DNA sequences, hybridizable with crenomic RNA of
lvmphadenopathv-associated virus (LAV)
The invention relates to cloned DNA sequences
hybridizable to genomic RNA and DNA of lymphadenopathy-
associated virus (LAV), a process for their preparation
and their uses. It relates more particularly to stable
probes including a DNA sequence which can be used for the
detection of the LAV virus or related viruses or DNA pro-
viruses in any medium, particularly biological, samples
containing of any them.
Lymphadenopathy-associated virus (LAV) is a human
15' retrovirus first isolated from the lymph node of a homo-
sexual patient with lymphadenopathy syndrome, frequently a
prodrome or a benign form of acquired immune deficiency
syndrome (AIDS) (cf.1). Subsequently other LAV isolates
have been recovered from patients with AIDS or pre-AIDS
(cf. 2-5). All available data are consistent with the
virus being the causative agent of AIDS (cf. 11).
The virus is propagated on activated T lymphocytes
and has a tropism for the T-cell subset OKT4 (cf. 2-6), in
which it induces a cytopathic effect. However, it has
been adapted for growth in some Epstein-Barr virus trans-
formed B-cell lines (cf. 7), as well as in the established
T-lymphoblastic cell line, CEM.
LAV-like viruses have more recently been indepen-
dently isolated from patients with AIDS and pre-AIDS.
These viruses called HTLV-III (Human T-cell Leukemia/
Lymphoma virus type III (cf. 12-15) and ARV (AIDS-
associated retrovirus) have many characteristics similar
to those of LAV and they represent independent isolates of
the LAV prototype. For the convenience of language they
will hereafter all be referred to as "LAV".
Detection methods so far available are based on
~ .~ 4 J
2
the recognition of viral proteins. Such a method is
disclosed in European application titled "Antig6nes, moyens
et methode pour le diagnostic de lymphadenopathie et du
syndrome d'immunodLpression acquise" filed on
September 14, 1984 under the priority of British
application Serial Nr. 83 24800 filed on
September 15, 1983. As a matter of fact, a high prevalence
of antibodies directed against the core protein having a
molecular weight of about 25000 (p25) of the LAV
retrovirus, has been found in the sera of AIDS and pre-AIDS
patients and to a lower but significant extent in the high-
risk groups for AIDS (cf. 8-10).
The present invention aims at providing new means
which should not only also be useful for the detection of
LAV or related viruses (hereafter more generally referred
to as "LAV viruses"), but also have more versatility,
particularly in detecting specific parts of the genomic DNA
of said viruses whose expression products are not always
detectable by immunological methods.
25
~
~3 4 1 5 2~
2a
The present invention relates to a DNA
molecule which contains a cDNA sequence of a LAV
virus sequence shown in Figures 4 to 11 or a cDNA
sequence that is complemetary to the sequence shown
in Figures 4 to 11 or a fragment of cDNA sequence,
wherein the fragment is characterized by at least
one of the following restriction sites:
Restriction Site Nucleotide position
Hind III 0
Sac I 50
Bam HI 460
Hind III 520
Bam HI 600
Pst I 800
Hind III 1,100
Bgl II 1,500
Kpn I 3,500
Kpn I 3,900
Eco RI 4,100
Eco RI 5,300
Sal I 5,500
Kpn I 6,100
Bgl II 6,500
Bgl II 7,600
Hind III 7,850
Barrm HI 8,150
Xho I 8,600
Kpn I 8,700
Bgl I 8,750
Bgl II 9,150
Sac I 9,200
Hind III 9,250.
-~rol
13 415 2~
2b
The present invention also relates to a
probe for the in vitro detection of LAV which
consists of a nucleotide sequence that hybridizes
to the sequence shown in Figures 4 to 11 under
conditions of high stringency or a nucleotide
sequence that hybridizes to a fragment under
conditions of high stringency, wherein the fragment
is characterized by at least one of the following
restriction sites :
Restriction Site Nucleotide position
Hind III 0
Sac I 50
Bam HI 460
Hind III 520
Bam HI 600
Pst I 800
Hind III 1,100
Bgl II 1,500
Kpn I 3,500
Kpn I 3,900
Eco RI 4,100
Eco RI 5,300
Sal I 5,500
Kpn I 6,100
Bgl II 6,500
Bgl II 7,600
Hind III 7,850
Bam HI 8,150
Xho I 8,600
Kpn I 8,700
Bgl I 8,750
Bgl II 9,150
,~
13 ~~~215~
2c
Sac I 9,200
Hind III 9,250.
The present invention also relates to a
method for the in vitro detection of a viral
infection due to LAV viruses which comprises
contacting a biological sample originating from a
person to be diagnosed for LAV infection and
containing DNA in a form suitable for hybridization
with a probe specific for LAV DNA under highly
stringent hybridizing conditions and detecting the
hybridized probe.
The present invention also relates to a
recombinant expression vector for the transformation
of prokaryotic or eukaryotic cells, which contains,
inserted in an expression vector, a DNA or a DNA
fragment as defined above.
The present invention also relates to a
microorganism, eukaryotic or prokaryotic cell, which
is transformed by a recombinant expression vector as
defined above and which expresses the polypeptide
encoded by the corresponding DNA fragment.
The present invention also relates to a
polypeptide encoded by the DNA as described above
and expressed by the microorganisms as described
above.
The present invention also relates to a
purified RNA of LAV viruses which has sizes from 9.1
to 9.2 kb and which hybridizes with the DNA molecule
as defined above under high stringency conditions.
The present invention also relates to a
RNA comprising LAV nucleotidic sequences and
obtainable by transcription of or back synthesis
13 415-2~
2d
from a cloned DNA which contains a DNA insertion
fragment being all or part of a cDNA of a LAV
retroviral genome contained in lambdaJl9 (CNCM I-
338) of LAV, wherein the DNA hybridizes under non
stringent conditions with the genomic RNA of the LAV
virus, but does not cross-hybridize with RNAs of
HTLV-I and HTLV-II retroviruses in a dot-blot
hybridization assay under low stringency conditions.
The present invention also relates to a
DNA in which a DNA insert comprises a sequence
extending from KpnI (6,100) to PvuII (8,500).
The present invention also relates to a
kit for detecting LAV comprising a biological sample
containing the RNA as described above.
The present invention also relates to a
kit for detecting LAV comprising an RNA as described
above.
The present invention also relates to a
DNA in which a DNA insert comprises a sequence
extending from KpnI (3,500) to PvuII (6,500).
The present invention also relates to a
DNA in which a DNA insert comprises a sequence
extending from PstI (800) to PvuII (3,800).
The present invention also relates to a
DNA which contains a DNA insert being a LAV
retroviral genome contained in kJ19 (C.N.C.M.
I-338) .
The present invention also relates to a
DNA hybridizing under stringent conditions to the
DNA insert as defined above.
,~
13 41523
2e
Reference is hereafter made to the drawings in
which:
- fig. 1 shows restriction maps of preferred cDNA inserts
contained in plasmid recombinants, wherein said cDNA
inserts consist of cDNA fragments corresponding to RNA
fragments of the LAV retroviral genome;
- fig. 2 shows restriction maps of cDNA fragments derived
from corresponding genomic retroviral fragments including
the full retroviral genome of LAV;
- fig. 3 is a restriction map of cDNA corresponding to a
complete LAV genome (clone IJ19);
- fig. 4 to 11 show the whole sequence of the LAV genoma,
the restriction map of which is shown in fig. 3.
The DNAs according to the invention contain DNA
fragments hybridizable with the genomic RNA of LAV.
Particularly any of said DNA fragments consist of a double-
stranded DNA resulting from the cloning of an initial
fragment obtained as a result of transcription of a
corresponding RNA fragment of the.LAV retroviral genome
into the first strand of said double-stranded DNA whose
second strand was transformed starting from the relevant
nucleotide in the presence of a polymerase. The DNA
according to the invention include recombinant DNAs
containing said cDNAs or cDNA fragments.
Preferred clones cDNA fragments respectively
contain the following restriction sites in the respective
orders which follow (from the 3' end to the 5' end):
1) HindIII, SacI, BglII (LAV75)
2) HindIII, SacI, BglII, BglIII, KpnI (LAV82)
~....~
13 41523
3
3) HindIII, Sacl, BglII, BglII, KpnI, XhoI, BamHI,
HindIII, Bg11I (LAV13).
The LAV75, LAV82 and LAV13 designations correspond
to the designations of the recombinant plasmids designated
as pLAV 75, pLAV 82 and pLAV 13 respectively, in which
they were first cloned. In other words LAV 75, LAV 82 and
LAV 13 respectively present as inserts in said recombinant
plasmids. For convenience the designations LAV 75, LAV 82
and LAV 13 will be further used throughout this specific-
ation to designate the cDNA fragments, whether the latter
are in isolated form or in a plasmid forms, whereby the
other DNA parts of said last mentioned recombinants are
identical to or different of the corresponding parts of
pLAV 75, pLAV 82 and pLAV 13 respectively.
Preferred cDNAs also (like LAV 75, LAV 82 and LAV
13) contain a region corresponding to the R and U 3 re-
gions of the LTR (Long Terminal Repeat) as well as the 3'
end of the coding region of the retroviral DNA. Particu-
larly if it is assumed that the retroviral structure of
LAV is in general agreement with the retroviral genomic
structures to date.
LAV 13 which has a size of about 2.5 Kbp has been
found of particular advantage. It is highly specific of
LAV or LAV related viruses and does also recognizes more
of the LAV retroviral genomes than do LAV75 or LAV82.
Particularly LAV 13 enabled the identification of the RU 5
junction of the retroviral genomes within the LTR and,
subsequently, the sizes of the LAV genomes, which average
from about 9.1 to about 9.2 kb.
LAV 13 is free of restriction sites for the
following enzymes Eco RI, Nru I, Pvu I, Sal I, Sma I, Sph
I, Stu I and Xba I.
LAV 13 further appears to contain at least part of
the DNA sequences corresponding to those which, in
retroviral genomes, code for the envelope protein.
The invention further relates to any of the
13 41523
4
fragments contained in the cDNA which seems to correspond
to part of the whole of the LAV retroviral genome, which
is characterized by a series of restriction sites in the
order hereafter (from the 5' end to the 3' end).
The coordinates of the successive sites of a
preferred whole LAV genome (restriction map) are indicated
hereafter too, with respect to the Hind III site (selected
as of coordinate 1) which is located in the R region. The
coordinates are estimated to within + 200 bp. Some coor-
dinates are better established than others.
Hind III 0
Sac I 50
Bam HI 460
Hind III 520
Bam HI 600
Pst I 800
Hind III 1 100
Bgl II 1 500
Kpn I 3 500
Kpn I 3 900
Eco RI 4 100
Eco RI 5 300
Sal. I 5 500
Kpn I 6 100
Bgl II 6 500
Bgl II 7 600
Hind III 7 850
Bam HI 8 150
Xho I 8 600
Kpn I 8 700
Bgl II 8 750
Bgl II 9 150
Sac I 9 200
Hind III 9 250
The abovesaid DNA according to the invention
optionally contains an additional Hind III approximately
4 1 b 2~
at the 5 550 coordinate.
More particularly the invention pertains to a DNA
having an approximate_ size of 9.1 kb and comprising the
series of restriction sites referred to in fig. 3.
5 More generally the invention relates more parti-
cularly to cDNA variants of the latter, which variants may
possess the same series of restriction sites in the order
hereafter (from the 5' end to the 3' end) yet with some of
them being deleted or added owing to mutatins either
induced willingly or not.
The invention further relates to other preferred
DNA fragments corresponding substantially to those which
in relation to the abovesaid restriction map extend res-
pectively
- from approximately Kpn I(6 100) to approximately Bgl II
(9150) said fragment being thought to correspond at least
in part to the gene coding for the proteins of the
envelope ; in particular a protein p110 of about 110,000
Daltons is encoded by this region ;
- from approximately Kpn I(3 500) to approximately Bgl II
(6500), said fragment being thought to correspond at least
in part to the pol gene, coding for the virus polymerase
- from approximately Pst (800) to approximately Kpn I
(3500), said fragment being thought to correspond at least
in part to the gag gene, which codes for the core anti-
gens, including the p25, the p18, and the p13 proteins.
The invention also relates to additional DNA frag-
ments, hybridizable with the genomic RNA of LAV as they
will be disclosed hereafter, as well as with additional
cDNA variants corresponding to the whole genomes of LAV
viruses. It further relates to DNA recombinants containing
said DNAs or cDNA fragments.
More particularly the invention relates to any
fragment corresponding to the above ones, having subs-
tantially the same sites at substantially same distances
from one another, all of these fragments having in common
13415 23
6
the capability of hybridizing with the LAV retroviral
genomes. It is of course understood that fragments which
would include some deletions or mutations which would not
substantially alter their capability of also hybridizing
with the LAV retroviral genomes are to be considered as
forming obvious equivalents of the DNA fragments more
specifically referred to hereabove.
Additional features of the invention will appear
in the course of the disclosure of additional features of
preferred DNAs of the invention, including restriction
maps and nucleotide sequences, the preparation conditions
and the properties of which will be illustrated hereafter
in a non limitative manner.
1. Construction of a cDNA library
1.1 Virus purification
Virions were purified from FR8, an immortalized,
permanent =LAV producing B-Lymphocyte line (cf. 7)
(deposited at the "Collection Nationale de Cultures de
Micro-organismes" of the INSTITUT PASTEUR of Paris, under
Nr. 1-303 on May 9, 1984). The purification protocol was
described (cf. 1). The main steps were
polyethylene-glycol treatment of culture supernatant,
pelleting through 20 : sucrose cushion, banding on 20-60 :
sucrose gradient and pelleting of the virus-containing
fractions.
1.2 First-strand cDNA synthesis
The virus associated detergent activated endoge-
nous reaction is a technique bringing into play the
reverse transcriptase of the virus, after purification
thereof and lysis of its envelope.
For each reaction, purified virus corresponding to
250-300 ml of FR8 supernatant was used. Final reaction
volume was 1 ml. Incubation was at 37'C for 45 mn. Protein
concentration was about 250 microg/ml. Buffer was : NaCl
25 mM ; Tris HC1 pH 7.8 50 mM, dithiothreitol 10 mM, MgC12
6 mM, each of dATP, dGTP, dTTP at 0.1 mM, Triton X-100
13 4 1 5 2 3
7 _
0.02 t ; oligo dT primer 50 microg/ml. The cDNA-RNA was
labelled 15 mn with alpha 32p-dCTP 400 Ci/mmole to 0.6
microM plus cold dCTP to 4 microM. Afterwards, cold dCTP
was added to 25 microM to ensure optimal elongation of the
first strand.
The reaction was stopped 30 mn after the dCTP
chase by adding EDTA to 20 mM, SDS to 0.5%, digesting one
hour with proteinase K at 100 microg/ml and phenol-chloro-
form extraction.
cDNA-RNA was then purified on G-50 Sephadex
(Pharmacia) and ethanol precipitated.
1.3 2nd strand synthesis and cloning
Purified cDNA-RNA hybrids were treated with DNA
polymerase I and RNase H, according to GUBLER and HOFFMAN
(cf. 17). Double-stranded cDNA was dC-tailed with terminal
transferase and annealed to dG-tailed Pst-digested pBR 327
(cf. 34) a derivative of pBR 322.
A cDNA library was obtained by transfection of
coli C 600 recBC strain.
2. Detection of LAV-svecific clones
2.1 Screening of the library
500 recombinant clones were grown on nitrocellu-
lose filtres and in situ colony hybridization (cf. 35) was
performed with another batch of cDNA made in endogenous
virus-associated reaction as described (cf. 1.2) and
labelled with 32P. About 10 % of the clones could be
detected.
A major family was obtained by small-scale
amplification of these clones and cross-hybridization of
their inserts. Among these clones a major family of hybri-
dizing recombinants was identified. Three of these cDNA
clones, named pLAV 13, 75 and 82, carrying inserts of 2.5,
0.6 and 0.8 kb respectively were further characterized
(fig. 1).
All three inserts have a common restriction
pattern at one end, indicating a common priming site. The
1341 523
8
50 bp long common Hind III-Pst I fragment was sequenced
(fig. 1) and shown to contain a polyA stretch preceeding
the cloning dC tail. The clones are thus copies of the 3'
end of a polyA-RNA.
The LAV 13 specificity was shown by different
assays.
The specificity of pLAV 13 was determined in a
series of filter hybridization experiments using nick-
translated pLAV 13 as a probe. Firstly the probe hybri-
dized to purified LAV genomic RNA by dot and Northern
blotting (data not shown). pLAV 13 also hybridizes to the
genomic RNA of virus concentrated from culture supernatant
directly immobilized on filters (dot blot technique). LAV
RNA from different sources : normal T-cells, FR8 and other
B-cell LAV producing lines, CEM cells and, although less
strongly, LAV from the bone marrow culture from a haemo-
philiac with AIDS (cf. 3) were detected in a similar
manner. Uninfected cultures proved negative. This rapid
dot blot technique can be adapted with minor modifications
to the detection of LAV in serum or other body fluids.
Secondly the probe detected DNA in the Southern
blots of LAV-infected T-lymphocytes and in the LAV-
producing CEM cell line. No hybridization was detected in
the DNA of uninfected lymphocytes nor in the DNA from
normal liver (data not shown) under the same hybridization
conditions.
A third characteristic resulted from the possibi-
lity of using LAV 13 to identify the whole retroviral
genome of the LAV viruses as disclosed hereafter. Parti-
cularly characteristic 1.45 kb Hind III fragment which co-
migrates with an internal viral fragment in Hind III
cleaved pLAV 13 was detected. Bands at 2.3 and 6.7 kb were
also detected. As the probe was only 2.5 kb long and as no
junction fragments could be detected, it is probable that
these extra-bands represent internal fragments arising
from a Hind III polymorphism of the LAV genome.
13 4 1 5 2 3
9
Together these data show that pLAV 13 DNA is exo-
genous to the human genome and detects both RNA and inte-
grated DNA forms derived from LAV infected cells. Thus
pLAV 13 is LAV specific. Being oligo-dt primed, pLAV 13
must contain the R and U3 regions of the LTR as well as
the 3' end of the coding region, assuming a conventional
retroviral genome structure.
Cloning of LAV Qenomic DNA
Having found a HindIII site within the R region of
the LTR, it was decided to clone the LAV genome by making
a partial Hind III digest of proviral DNA from LAV
infected cells. It was found that :(a) partial digestion
increased the chance of isolating complete clones and (b)
Hind III fragments were easily cloned in lambda replace-
ment vectors. The DNA isolated from T-cells of a healthy
donor after i} vitro infection with LAV was partially
digested with Hind III and fractionated. A 9*- 1.5 kb DNA
containing fraction was precipitated and ligated into the
Hind III arms of lambda-L47.1 (cf. 18).
The cloning of LAV genomic DNA was carried out
more particularly as follows :
cDNAs was prepared from LAV infected T cells as
described above, then partially digested with Hind III and
fractionated on a 5-40 % sucrose gradient in 10 mM Tris.Cl
pH 8, 10 mM EDTA, 1 M NaC1 (SW41 rotor, 16 hours at 40 000
rpm). A single fraction (9 0.5 kb) was precipitated with
20 microg/ml Dextran T40 as carrier and taken up in TE-
buffer (10 mM Tris.Cl pH 8, 1 mM EDTA). Lambda-L47.1 Hind
III arms were prepared by frist ligating the cos sites
followed by Hind III digestion and fractionation through a
5-40 % sucrose gradient. Fractions containing only the
lambda-Hind III arms were pooled, precipitated and taken
up in TE-buffer. Ligation of arms to DNA was made at
approximately 200 microg DNA/ml using a 3:1 molar excess
of arms and 300 units of T4 DNA ligase (Biolabs). ~}r vitro
packaging lysates were made according to (38). After i}
13 4 1 5 2 3
vitro packaging the phage lysate was plated out on NM538
on a C600 recBC strain. Approximately two million plaques
were screened by in situ hybridization (cf. 39) using ni-
trocellulose filters. Hybridization was performed at 68'C
5 in 1 x Denhardt solution, 0.5 % SDS, 2 x SSC, 2 mM EDTA.
Probe : 32P nick-translated LAV insert of pLAV 13 at
>108 cpm/microg Filters were washed 2 x 30 minutes in
0-1 SSC, 0.1 % SDS at 68'C, and exposed to Kodak XAR-5
film for 29-40 hours. Seven positive clones were identi-
10 fied and plaque purified on a C 600 rec BC strain. Liquid
cultures were grown and the recombinant phages banded in
CsCl. Plage DNA was extracted and digested under the
appropriate conditions.
Seven independent clones were so derived from
approximatively two million phage plaques after screening
in situ with a nick-translated pLAV 13 insert as a probe.
Restriction maps of lambda-J19 as well as of a Hind III
polymorph lambda-J81 are shown in fig. 2. Other recom-
binants lambda-J27, lambda-J31 and lambda-J57 had the same
Hind III map as lambda-J19. The map of lambda-J81 is
identical but for an additional Hind III site at
coordinate of approximately 5 550.
The restriction maps of fig. 2 were oriented by
hybridizing blots with respect to pLAV 13 DNA.
The restriction map of the LAV 13 cDNA clone is
also shown in fig. 2. The restriction sites of lambda-J19
are . B-Bam HI, Bg-Bgl II, H-Hind III, K-Kpn I, P-Pst I,
R-Eco RI, S-Sac I, Sa-Sal-I and X-Xho I. Underneath the
scale is a schema for the general structure of the retro-
viruses showing the LTR elements U3, R and U5. Only the
R/US boundary has been defined and other boundaries are
only drawn figuratively.
There may be other Bam HI sites in the 5' 0.52 kb
Hind III fragment of lambda-J19. They generate fragments
3.5 that are too small to be detected.
Fig. 2 also shows those Hind III fragments of
1341 523
11
lambda-J19 and lambda-J81 which are detected by pLAV 13
(marked (+)), those which are not detected (-).
More particularly lambda-J19 shows four Hind III
bands of 6.7, 1.45, 0.6 and 0.52 kb the first two of which
correspond to bands in the genomic blot of Hind III res-
tricted DNA. The smallest bands of 0,6 and 0,52 kb were
not seen in the genomic blot but the fact that they appear
in all the independently derived clones analyzed indicates
that they represent internal and not junction fragments,
assuming a random integration of LAV proviral DNA. Indeed,
the 0,5 kb band hybridizes with pLAV 13 DNA (fig. 2)
through the small Hind III-Pst I fragment of pLAV 13. Thus
the 0,5 kb Hind III fragment of lambda-J19 contains the
R-U5 jur.ction within the LTR.
It appears that lambda-J81 is a restriction site
polymorph of lambda-J19. Lambda-J81 shows five Hind III
bands of 4.3, 2.3, 1.45, 0.6 and 0.52 kb. The 2.3 kb band
is readily detected in the genomic blot by a pLAJ 13
probe, but not the 4.3 kb fragment. That lambda-J81 is a
Hind III polymorph and not a recombinant virus is shown by
the fact that nick-translated lambda-J19 DNA hybridizes to
all five Hind III bands of lambda-J81 under stringent hy-
bridization and washing conditions. Also other restric-
tions sites in lambda-J81 are identical to those of lam-
bda-J19.
Sequencing of the LAV-derived cDNA.
The sequencing and determination of sites of par-
ticular interest was carried out on phage recombinant.
The whole recombinant phage DNA of clone XJ19 was
sonicated according to the protocol of DEININGER (1983),
Analytical Biochem. 129, 216. The DNA was repaired by a
Klenow reaction for 12 hours at 16'C. The DNA was
electrophoresed through 0.8 % agarose gel and DNA in the
size range of 300-600 bp was cut out and electroeluted and
precipitated. Resuspended DNA (in 10 mM Tris, pH 8 ; 0,1
mM EDTA) was ligated into M13mp8 RF DNA (cut by the
134152~
12
restriction enzyme SmaI and subsequently alkaline
phosphated), using T4 DNA- and RNA-ligases (Maniatis T et
al (1982) - Molecular cloning - Cold Spring Harbor
Laboratory). An JE. co i strain designated as TG1 was used
for further study. This strain has the following genotype:
Alac pro, supE, thi.F'traD36, proAB, IacIq, 2AM15,r
This E. c.oli TGI strain has the peculiarity of
enabling recombinants to be recognized easily. The blue
colour of the cells transfected with plasmids which did
not recombine with a fragment of LAV DNA is not modified.
To the contrary cells transfected by a recombinant plasmid
containing a LAV DNA fragment yield white colonies. The
technique which was used is disclosed in Gene (1983), 26,
101.
This strain was transformed with the ligation mix
using the Hanahan method (Hanahan D (1983) J. Mol. Biol.
166,557). Cells were plated out on tryptone-agarose plate
with isopropyl-B-D-thiogalactopyranoside (IPTG) and
5-bromo-4-chloro-3-indolyl-B-D-galactopyranoside(X-gal) in
soft agarose. White plaques were either picked and
screened or screened directly in situ using nitrocellulose
filters. Their DNAs were hybridized with nick-translated
DNA inserts of pUC18 Hind III subclones of I which are
identified in the table hereafter. In relation to this
table, it should also be noted that the designation of each
plasmid is followed by the deposition number of a cell
culture of JE. coli TGI containing the corresponding plasmid
at the "Collection Nationale des Cultures de Micro-
organismes" (C.N.C.M.) of the Pasteur Institute in Paris,
France. A non-transformed TGI cell line was also deposited
at the C.N.C.M. under Nr. 1-364. All these deposits took
place on November 15, 1984. The sizes of the corresponding
inserts derived from the LAV genome have also been
indicated.
. :1
13 1341523
TABLE
Essential features of the recombinant plasmids
- pJ19 - 1 plasmid (1-365) 0.5 kb
Hind III - Sac I - Hind III
- pJ19 - 17 plasmid (1-367) 0.6 kb
Hind III - Pst 1- Hind III
- pJ19 - 6 plasmid (1-366) 1.5 kb
Hind III (5')
Bam HI
Xho I
Kpn I
Bgl II
Sac I (3')
Hind III
- pJ19-13 plasmid (1-368) 6.7 kb
Hind III (5')
Bgl II
Kpn I
Kpn I
Eco RI
Eco RI
Sal I
Kpn I
Bgl II
Bgl II
Hind III (3')
Positively hybridizing M13 phage plates were grown
up for 5 hours and the single-stranded DNAs were
14 ~3 4 1 5 2 3
extracted.
M13mp8 subclones of AJ19 DNAs were sequenced
according to the dideoxy method and technology devised by
Sanger et al (Sanger et al (1977), Proc. Natl. Acad. Sci.
USA, 74 , 5463 and M13 cloning and sequencing handbook,
AMERSHAM (1983). the 17-mer oligonucleotide primer
a-35SdATP (400Ci/mmol, AMERSHAM), and 0.5X-5X buffer
gradient gels (Biggen M.D. et al (1983, Proc. Natl. Acad.
Sci. USA, 50, 3963) were used. Gels were read and put into
the computer under the programs of Staden (Staden R.
(1982), Nucl. Acids Res. 10. 4731). All the appropriate
references and methods can be found in the AMERSHAM M13
cloning and sequencing handbook.
The complete sequence of AJ19 is shown in figs
5-12.
Relationship to other human retroviruses
HTLV-I and HTLV-II constitute a pair of C-type
transforming retroviruses with a tropism for the T-cell
subset, OKT4 (cf. 20). An isolate of HTLV-I has been
totally sequenced (cf. 21) and partial sequencing of an
HTLV-II has been reported (cf. 22-24). Both genomes (one
LTR) were approximately 8.3 kb in length, have a pX region
and show extensive sequence homology. They hybridize
between themselves under reasonably stringent conditions
(40 % formamide, 5 XSSC) and even at 60 % formamide the pX
regions hybridize (cf. 26). Thus a conserved pX region is
a hallmark of this class of virus.
We have compared cloned LAV DNA and cloned HTLV-II
DNA (pMO (cf. 27)) by blot-hybridization and found no
cross-hybridization under low stringency conditions of
hybridization and washing. For example, Hind III digested
lambda-J19, lambda-J27 and lambda-J81 were electropho-
resed, blotted and hybridized overnight with 32P
nick-translated pM0 (HTLV-II) DNA (having a specific
activity greater than 0.5 X 108 cpm/microg) in 20 %
formamide, 5 XSSC, 1 X Denhardts solution, 10 % Dextran
1341523
sulphate,at 37'C. Filters were washed at 37'C (tm.50)
tm.50 using a 53.1 % GC content derived from the HTLV-I
sequence (21). The washings were repeated at 50'C and 65'C
in 1 x SSX, 0.1 % SDS. Even when hybridized in 20 %
5 formamide, 8 X SSC (tm.50) and washed at 37'C in 2 X SSC
(tm.50) no hybridization was detected after two days
exposure at -70'C using an intensifying screen.
Thus there is no molecular evidence of a relation-
ship between LAV and the HTLV viruses. In addition, the
10 LAV genome is approximately 9 kb long in contrast to 8.3
kb for the HTLV viruses. Despite their comparable genome
sizes LAV and Visna (cf. 29) cloned viral genomes do not
cross-hybridize, nor does LAV with a number of human
endogenous viral genomes (cf.30) under non stringent
15 conditions (hybridization-20 -o formamide, 8 SSC, 37'C
washing - 2 SSC, 0.1 % SDS, 37 C.
The invention also relates more specifically to
cloned probes which can be made starting from any DNA
fragment according to the invention, thus to recombinant
DNAs containing such fragments, particularly any plasmids
amplifiable in procaryotic or eucaryotic cells and carry-
ing said fragments. As mentioned earlier a preferred DNA
fragment is LAV 13.
Using the cloned provirus DNA as a molecular hy-
bridization probe - either by marking with radionucleo-
tides or with fluorescent reagents - LAV virion RNA may be
detected directly in the blood, body fluids and blood
products (e.g. of the antihemophylic factors such as
Factor VIII concentrates) and vaccines, i.e. hepatitis B
vaccineIt has alredy been shown that whole virus can be
detected in culture supernatants of LAV producing cells. A
suitable method for achieving that detection comprises
immobilizing virus onto said a support e.g. nitrocellulose
filters, etc., disrupting the virion and hybridizing with
labelled (radiolabelled or "cold" fluorescent- or
enzyme-labelled) probes. Such an approach has already been
13 41523
16
developed for Hepatitis B virus in peripheral blood
(according to SCOTTO J. et al. Hepatology (1983),
379-384).
Probes according to the invention can also be used
for rapid screening of genomic DNA derived from the tissue
of patients with LAV related symptoms, to see if the pro-
viral DNA or RNA is present in host tissue and other
tissues.
A method which can be used for such screening
comprise the following steps : extraction of DNA from tis-
sue,
restriction enzyme cleavage of said DNA, electrophoresis
of the fragments and S_outhern blotting of genomic DNA from
tissues, subsequent hybridization with labelled cloned LAV
provival DNA. Hybridization in situ can also be used.
Lymphatic fluids and tissues and other non-lympha-
tic tissues of humans, primates and other mammalian spe-
cies can also be screened to see if other evolutionnary
related retrovirus exist. The methods referred to here-
above can be used, although hybridization and washings
would be done under non stringent conditions.
The DNA according to the invention can be used
also for achieving the expression of LAV viral antigens
for diagnostic purposes as well as far the production of a
vaccine against LAV. Of particular advantage in that res-
pect are the DNA fragments coding core (gag region) and
for envelope proteins, particularly the DNA fragment
extending from Kpn I(6 100) to BglII(9 150).
The methods which can be used are multifold
a) DNA can be transfected into mammalian cells
with appropriate selection markers by a variety of tec-
hniques, calcium phosphate precipitation, polyethylene
glycol, protoplast-fusion, etc..
b) DNA fragments corresponding to genes can be
cloned into expression vectors for E. coli , yeast- or
mammalian cells and the resultant proteins purified.
13 4 1 5 2 3
17
c) The provival DNA can be "shot-gunned" (frag-
mented) into procaryotic expression vectors to generate
fusion polypeptides. Recombinant producing antigenically
competent fusion proteins can be identified by simply
screening the recombinants with antibodies against LAV
antigens .
d) The invention also relates to oligopeptides
deduced from the DNA sequence of LAV antigen-genes to
produce immunogens and antigens and which can be
synthethised chemically.
All of the above (a-d) can be used in diagnostics
as sources of immunogens or antigens free of viral par-
ticles, produced using non-permissive systems, and thus of
little or no biohazard risk.
The invention further relates to the hosts (proca-
ryotic or eucaryotic cells) which are transformed by the
above mentioned recombinants and which are capable of
expressing said DNA fragments.
-Fina,lly it also relates to vaccine compositions
whose active principle is to be constituted by any of the
expressed antigens, i.e. whole antigens, fusion polypep-
tides or oligopeptides.
The invention finally refers to the purified
genomic mRNA, which can either be extracted as such from
the LAV viruses or resynthesozed back from the cDNA,
particularly to a purified mRNA having a size appro-
ximating 9.1 to 9.2 kb, hybridizable to any of the DNA
fragments defined hereabove or to parts of said purified
mRNA. The invention also relates to parts of said RNA. The
nucleotidic structures of this purified RNA or of the
parts thereof can indeed be deduced from the nucleotidic
sequences of the related cDNAs.
It will finally be mentioned that lambda-J19 and
lambda-J81 have been deposited at the Collection Natio-
nale des Cultures de Micro-organismes (C.N.C.M.) of the
INSTITUT PASTEUR of Pasteur (France) under Nr. 1-338 and
18
1-339 respectively, on September 11, 1984.
The invention finally refers to the genomic DNA,
the DNA sequence of which can be determined and used to
predict the aminoacid sequences of the viral protein
(antigens) and to the RNA probes which can be derived from
the cDNA.
There follows the bibliography to which references
have been made throughout this specification by bracketted
numbers.
1341523
19
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