Non-infectious, replication-impaired, immunogenic human immunodeficiency virus type 1 retrovirus-like particles with multiple genetic deficiencies

An immunogenic retrovirus-like particle which is non-infectious and non-replicating and which is useful as a candidate vaccine component against retroviral infections, including AIDS and ATLL, is produced by genetic engineering. A DNA molecule comprising a retroviral genome devoid of long terminal repeats is incorporated into an expression vector, which is introduced into mammalian cells for expression of the retrovirus-like particle.

FIELD OF INVENTION 
The present invention relates to the preparation of human retrovirus-like 
particles, specifically HIV-like particles, which are immunogenic and 
non-infectious. These preparations will serve as candidates for 
whole-virus-like vaccines for human retrovirus diseases and should not be 
subject to the ethical concerns regarding the production of classical 
whole-virus vaccines from infectious virus preparations. 
BACKGROUND TO THE INVENTION 
Among all diseases caused by retroviruses in humans and animals, the 
acquired immunodeficiency syndrome (AIDS) and the adult T-cell 
leukemia-lymphoma (ATLL) represent the most dramatic human diseases due to 
HIV and HTLV-1 retroviruses, respectively. The etiologic agent of the 
acquired immune deficiency syndrome (AIDS) is a human retrovirus termed 
human immunodeficiency virus (HIV) of which there are presently two major 
subgroups, HIV-1 and HIV-2. These viruses are responsible for an ever 
widening world-wide epidemic of immune deficiency and central nervous 
system (CNS) disorders characterized by a slow, yet progressive, 
degeneration of immune and CNS functions. HIV-1 affects mainly North 
America, Western Europe, Haiti and Central Africa while HIV-2 is found 
predominantly in West African countries. The earliest symptoms of HIV 
infection include an acute influenza-like syndrome which persists for 2 to 
3 weeks. Several weeks to many months or years following infection, 
lymphadenopathy and/or progressive depletion in CD4.sup.+ T-helper 
lymphocytes becomes apparent and disease evolves to the point where immune 
deficiency becomes manifest. The diagnosis of HIV infection is confirmed 
by laboratory tests which include the detection of HIV-specific antibodies 
and/or HIV antigens in patient sera, and the isolation of infectious virus 
from patients body fluids or cells. A similar disease is observed in 
rhesus macaques infected with the simian acquired immunodeficiency virus 
(SIV). 
Immune deficiency in HIV infection is characterized by opportunistic 
infections with microbial agents which are not normally associated with 
disease in otherwise healthy individuals. The severity of these infections 
is exacerbated by the loss of helper T-cell function, which, when combined 
with other symptoms, such as diarrhoea and weight loss, leads to a general 
wasting syndrome. Death usually results from one or more opportunistic 
infections. As mentioned above, CNS involvement is another manifestation 
of AIDS and can be the result of direct HIV-induced neurological disease 
as well as that of opportunistic infections. 
The predominant host cells for HIV in infected individuals are the 
CD4.sup.+ T-helper cell and the monocyte/macrophage. However, more and 
more evidence points to the fact that HIV can infect a wide variety of 
cell types, CD4.sup.+ and CD4.sup.-, both in vivo and in vitro. These 
cell types include those of the haematopoietic system, the central nervous 
system, the gastrointestinal tract, and skin. This wide host cell tropism 
most likely accounts for the plethora of symptoms and the severity of 
disease associated with HIV infection. 
HIV-1 and 2 have been the subject of massive and unprecedented research 
efforts in recent years in a number of areas including vaccine strategies. 
The development of an efficacious vaccine for prevention of HIV infection, 
is of considerable importance as it can be easily recognized that 
prevention of infection is the best way to combat any infectious disease. 
Various strategies are currently being used in attempts to develop an 
effective vaccine against AIDS. Some of these strategies are briefly 
outlined along with their respective advantages and disadvantages. 
A subunit HIV vaccine consists of one or more purified HIV immunogens, 
either obtained from disrupted whole virus or produced in genetically 
engineered eukaryotic or bacterial expression systems. An important 
advantage of this type of vaccine is the relative ease with which these 
products can be produced. However, this advantage can be countered by the 
fact that subunit vaccines only contain a subset of HIV antigenic 
determinants, which in some cases can lead to a less than optimal immune 
response. Moreover, viral protein subunits may adopt different spatial 
conformations when extracted from the context of the whole-virus particle. 
This may affect the structure of important conformational epitopes and 
result in inefficient immune responses. 
Live recombinant virus vaccines consist of a non-pathogenic virus, such as 
vaccinia or adenovirus, which has one or more non-essential genes replaced 
by a nucleotide sequence encoding one or more HIV antigens. Live 
recombinant viruses can often induce efficient immune responses to single 
subunits of a particular pathogenic virus. However, as with subunit 
vaccines, recombinant virus vaccines express only a fraction of the total 
antigens of a given virus which can be disadvantageous when highly 
efficient immune responses are required. 
Future vaccines may consist of synthetic peptides containing multiple 
epitopes of a given pathogen. These peptides, coupled to a carrier protein 
and combined with an appropriate adjuvant, are potentially capable of 
eliciting good and lasting humoral and cellular immune responses against 
multiple components of a pathogen. The development of an efficacious 
synthetic peptide vaccine for AIDS is likely to require the full 
identification of all the functionally important immunological 
determinants of HIV-1 and HIV-2, a task which may not be completed in the 
very near future. An important disadvantage of peptide vaccines is the 
difficulty to produce synthetic molecules mimicking conformational 
epitopes (immunological determinants which are formed by distant amino 
acid residues brought together in space by protein folding). If 
conformational epitopes are important for protection against a particular 
infectious agent, it is unlikely that traditional peptide vaccine designs 
will prove successful. 
Inactivated, whole-virus vaccines consist of a purified preparation of 
intact particles from a given viral pathogen which has been rendered 
non-infectious by chemical or physical means. The inherent advantages of 
these vaccines are their relative ease of production and the fact that all 
or most of the important immunological epitopes of the virus are present. 
However, a major disadvantage of these vaccines is that infectious virus 
must be propagated on a large scale, thereby exposing production workers 
to significant risks, depending on the nature of the pathogen. Equally 
important is the fact that the virus must be rendered completely 
non-infectious. This poses ethical problems since it is extremely 
difficult to demonstrate that all infectious genetic material has been 
removed. Moreover, extensive inactivation regimes to kill all infectious 
viruses are likely to destroy or alter various immunological epitopes, 
thereby compromising the immunogenicity of the vaccine. 
This invention describes a method to produce non-infectious, 
retrovirus-like particles as the basis for a candidate human vaccine 
against AIDS. This invention is also applicable to the production of 
similar particles serving as a candidate vaccine to other retrovirus 
diseases which include, but are not limited to, the simian acquired immune 
deficiency syndrome caused by the simian immunodeficiency virus (SIV), and 
certain forms of human T-cell leukemia-lymphoma caused by the human T-cell 
leukemia virus I (HTLV-1). 
A non-infectious retrovirus-like particle of this nature does not contain 
any infectious RNA and has the advantage of including all or part of the 
major viral antigens in their native configuration. The production of such 
a particle does not obligatorily require physical or chemical 
inactivation, thus avoiding the possible destruction of important 
immunological determinants. The ability of this particle to elicit potent 
immune responses to native viral proteins without any risk of inducing 
infection and disease makes it an important new candidate vaccine for 
evaluation in animal models and humans. 
SUMMARY OF INVENTION 
This invention describes a general method for the production of human 
retrovirus-like particles, specifically HIV-like particles, which are 
immunogenic and non-infectious. This preparation of genetically engineered 
HIV-like particles will serve as a candidate "whole-virus" vaccine for 
AIDS and should not be subject to the specific ethical concerns regarding 
the production of classical whole-virus vaccines from infectious virus 
preparations. It should be noted that the methodologies developed here for 
the AIDS virus are directly applicable to all human and non-human 
retroviruses and can be used to produce any retroviral candidate vaccine, 
including, but not limited to, the AIDS virus, and not limited to human 
pathogens. Furthermore, the same methodologies can be used to produce 
non-infectious retroviral particles to serve as antigens in diagnostic 
immunoassays for retroviral diseases. For the purpose of clarity, the 
discussion here pertains to actual examples employing the AIDS virus, but 
it is to be assumed that this invention covers all retroviruses. 
The present invention describes the engineering of cultured cells to 
produce retroviral proteins which self-assemble into virus-like particles 
in the absence of the production of an infectious retrovirus genomic RNA 
molecule. A virus-like particle can be defined here as a defective virion 
which is incapable of infecting a host cell due to the presence of one or 
more genetic modifications of viral genes or other genetic elements which 
are functionally critical at some stage of the virus life cycle. 
Virus-like particles may or may not contain all of the viral proteins 
normally found in infectious virions and may or may not contain RNA. If 
RNA is contained within the particle, it will be incapable of infecting a 
host cell. 
In the present examples pertaining to HIV, the production of non-infectious 
virus-like particles required the isolation of a DNA fragment from the HIV 
provirus containing relevant protein coding information and deficient in 
genomic elements required for replication and transcription of the 
retrovirus genome. This DNA fragment was linked to a heterologous promoter 
and transfected into a cultured cell line to allow for the expression of 
HIV-1 proteins and their assembly into virus-like particles. The unique 
features of this invention are described in detail below but centre on the 
fact that the present invention consists of a preparation of virus-like 
particles, which will not require chemical inactivation, and which can 
serve as a candidate vaccine for retroviral pathogens. Moreover, the 
method allows for the production of non-infectious virus-like particles 
containing modifications in one or more viral proteins so as to enhance 
the immunogenicity of the particles. Thus, the resultant candidate vaccine 
will represent a safe preparation of virus-like particles containing an 
optimized set of immunological epitopes necessary for stimulating a potent 
immune response. 
The safety of genetically engineered, non-infectious HIV-like particles can 
be guaranteed by the genetic engineering steps employed to produce these 
particles. Viral genetic elements required for replication are eliminated 
and a variety of genetic modifications can be introduced in the viral DNA 
sequences to be inserted into the producing cell line. These mutations can 
affect gene functions, gene products or genetic elements required for 
viral infectivity but not involved in the synthesis of the major viral 
proteins required for particle assembly or immunogenicity. With this 
strategy in hand, it becomes apparent to those skilled in the art that 
expression vectors employed to produce such non-infectious particles will 
not yield infectious viruses. 
The techniques for producing a preparation of safe, non-infectious 
virus-like particles stem from knowledge which is available to those 
skilled in the art, but which has been applied here in a unique fashion to 
produce whole virus-like particles devoid of infectious HIV viral RNA or 
RNA which can be replicated into double-stranded DNA in a recipient cell. 
We have developed an expression system for HIV antigens which results in 
the production of the major HIV-1 antigens in an engineered cell in the 
absence of the production of an infectious genomic RNA molecule. We have 
demonstrated co-expression of the envelope and core antigens and provide 
evidence that these products are assembled into retrovirus-like particles 
and can be purified by means employed to purify normal, infectious 
preparations of HIV particles. In addition, these purified virus-like 
particles induce efficient HIV-1 specific antibody responses in immunized 
mice and cross-reactivity with other HIV strains, including HIV-2. 
It is important to note that HIV-like particles and the cell lines used to 
produce these particles described here are significantly different from 
prior art retroviral studies involving the insertion of retrovirus genomes 
into cultured cell lines. Prior art experiments involved the construction 
of cell lines expressing murine retroviral proteins capable of packaging 
defective retroviral genomes carrying foreign genes. The packaging of 
defective retroviral genomic RNA molecules carrying foreign genes into 
virus particles allows for the efficient introduction of these RNAs into 
recipient cells. Once transduced into recipient cells, these recombinant 
viral RNAs become reverse transcribed into double-stranded DNA and 
integrated into the chromosomes of the cell, thereby genetically 
transforming the recipient cell. In the invention described here, the use 
of HIV-1 expression vectors devoid of long terminal repeat (LTR) elements 
results in the production of non-infectious virus-like particles which are 
intended for use as a vaccine and not for the transduction of recombinant 
retroviral RNA molecules into recipient cells for the purpose of genetic 
transformation. Indeed, in the present invention, RNA packaging into virus 
particles can be minimized and any RNA which is packaged cannot undergo 
the process of reverse transcription. Therefore, the present invention 
differs from prior art studies of retrovirus protein expression in the 
intended uses, methods employed, and the nature and characteristics of the 
resultant products. 
As indicated above, the final products of the present invention are 
preparations of virus-like particles which are non-infectious due to the 
absence of an infectious retrovirus genome within the particles. Moreover, 
the present invention allows for genetic manipulations to optimize the 
immunogenicity of the particles and vaccine efficiency in immunized 
recipients. It is envisioned that significant alterations can be made to 
certain viral protein components of the genetically engineered particles 
through alterations in the DNA sequences encoding these components. These 
alterations are likely to involve the insertion of extra copies of various 
important immunological epitopes into virus protein regions, which are not 
critical for particle assembly, to generate candidate multivalent chimeric 
vaccines. Additional alterations might involve the deletion of certain 
protein regions which may be immunosuppressive or lead to the production 
of autoimmune disorders or enhancing antibodies. The alterations would be 
designed so as to avoid interference with particle assembly and create 
modified particles capable of eliciting an optimized immune response. The 
invention also allows for the production of hybrid viral particles 
containing antigens from multiple strains of a given infectious virus, or 
even from different viruses altogether. 
In summary, an invention is described which entails the expression of 
normal or modified retroviral proteins in a cultured cell line for the 
intended purpose of producing non-infectious, assembled virus-like 
particles as a candidate vaccine. The invention describes the use of 
stably engineered cell lines using inducible and constitutive promoters 
and an example of one method to produce such particles. It is not to be 
assumed that the genetic engineering examples described here are the only 
means of expressing non-infectious retrovirus-like particles for vaccine 
use.

GENERAL DESCRIPTION OF THE INVENTION 
FIG. 1 shows a functional map of the HIV-1 provirus indicating the location 
of various genes as well as the long terminal repeat (LTR) elements which 
are required for retroviral gene expression and genome replication. In the 
present invention, we have demonstrated that it was possible to produce 
HIV-1-like particles in transiently transfected monkey COS cells by 
inserting an 8.3 Kb SacI-XhoI DNA fragment from the provirus of 
LAV-1.sub.BRU into a simple eukaryotic expression vector employing the 
SV40 virus early promoter (FIG. 2). The proviral fragment employed 
contained the viral protein coding information found between the LTR 
elements and, therefore, lacked the genetic elements necessary for the 
reverse transcription of any RNA molecule transcribed from this fragment. 
Upon transfection of this vector into COS cells, HIV-1 protein expression 
was confirmed by metabolic labelling and immune precipitation, by Western 
blot analysis of pelleted material using a specific anti-HIV antiserum and 
detection of reverse transcriptase (RT) activity in the supernatant of 
transfected cells. Evidence for the formation of HIV-like particles was 
obtained by sucrose density gradient centrifugation of the high molecular 
weight material released in the supernatant of transfected cells. In this 
experiment, RT activity was shown to band at a density similar to that of 
intact retrovirus particles. It was further established that SIV-like 
particles could be engineered using the same method. 
To establish stably engineered cell lines for more efficient production of 
non-infectious HIV-1-like particles, HIV-1 protein coding information was 
inserted into an expression vector employing the adenovirus major late 
promoter and transfected into COS cell along with a plasmid specifying 
resistance to the antibiotic G418 (FIG. 3). After examination of a number 
of G418 resistant cell clones, several were identified which 
constitutively produced high molecular weight material containing RT 
activity in association with major HIV-1 antigens. That actual virus-like 
particles were being produced was demonstrated by sucrose density gradient 
centrifugation and electron microscopic analysis. The latter revealed the 
presence of virus-like particles budding from the plasma membrane of 
transfected cells. 
To demonstrate the potential for non-infectious HIV-1-like particles to 
function as a candidate vaccine, mice were immunized with a preparation of 
non-infectious HIV-1-like particles produced by stably transfected COS 
cells. In these experiments, particles were purified by sedimentation and 
sucrose banding and used as immunogens. After two injections, an efficient 
antibody response against various HIV-1 proteins and peptides was observed 
and virus neutralizing activity was detected for two HIV-1 strains. After 
a third injection, HIV-1 specific antibodies showed cross-reactivity with 
the envelope glycoprotein of HIV-2. 
To improve the levels of non-infectious HIV-1 particle production in 
engineered cell lines, an expression vector containing the human 
metallothionein IIa promoter and the HIV-1 protein coding DNA fragment was 
developed to allow for the inducible expression of virus-like particles in 
stably engineered cell lines (FIG. 10). After co-transfecting this vector 
into Vero cells along with the G418 resistance marker, numerous clones 
were identified which produced high levels of particulate RT activity. 
HIV-like particle production was verified by electron microscopy and p24 
antigen assays indicated that levels of particle production were higher 
than normally observed in an infection of human leukaemic T-cells with 
infectious HIV-1. Moreover, particle production in the stably transfected 
Vero cells was maintained for at least eight consecutive days after 
reaching confluence when the cells were given fresh medium and cadmium 
chloride every twenty four hours. Inducible and long-term expression was 
not limited to monkey Vero cells. Metal-responsive expression of 
substantial amounts of particles was also observed in a human colon 
adenocarcinoma cell line. These data indicate that a wide variety of cell 
lines are suitable for the large-scale production of non-infectious 
virus-like particles. 
Although one major advantage of this method is the possibility to produce 
virus-like particles which may potentially contain all of the viral 
proteins in their native configuration, it is nevertheless possible to 
produce modified virus-like particles containing viral antigens with 
altered structures. It may be determined that the introduction of epitopes 
from alternative viral strains into certain regions of the HIV-1 envelope 
glycoprotein may enhance its immunogenicity and is of net benefit in spite 
of potential negative effects associated with the possible alteration of 
native conformational epitopes. In addition, it may be possible to improve 
the immunogenicity of HIV-like particles by deleting certain enhancing or 
immunosuppressive regions of the envelope or other viral proteins. To 
demonstrate the feasibility of producing virus-like particles containing 
modified envelope proteins, we first mutated the proteolytic processing 
site of the gp160 envelope glycoprotein precursor to block processing of 
the gp160 molecule into the mature gp120 envelope glycoprotein and the 
gp41 transmembrane glycoprotein. This experiment was performed to 
determine if unprocessed gp160 could be targeted to the membrane of 
virus-like particles. Since modified gp120 molecules may be easily shed 
from virus-like particles as a result of a reduced affinity for the gp41 
transmembrane glycoprotein, it may be necessary to block proteolytic 
processing to efficiently retain modified envelope molecules on the 
particles. Sucrose density gradient analysis of particles produced from an 
expression construct containing a mutation in the region coding for the 
processing site revealed the presence of uncleaved gp160 tightly 
associated with RT activity in heavy molecular weight material released 
from transfected cells. Furthermore, additional expression constructs 
encoding envelope glycoproteins with epitope insertions at defined sites 
demonstrated the feasibility of producing particles with modified gp120 
envelope glycoproteins containing specific aminoacid insertions. 
Conversely, deletion of DNA sequences coding for the major part of gp120 
resulted in the production of virus-like particles lacking the 
extracellular domain of the envelope protein. 
Genetically engineered, non-infectious HIV-like particles are designed to 
represent a completely safe candidate vaccine for use in humans. This is 
due to the fact that cell lines producing the virus-like particles do not 
contain the genetic elements required for reverse transcription. However, 
with the elimination of only the LTR elements, it may be argued that 
recombination of the protein coding sequences with heterologous LTR 
elements in either the vaccine-producing cell or in the vaccine recipient 
may result in the regeneration of an infectious retrovirus. This 
possibility can be completely eliminated by making a number of additional 
genetic modifications in regions of the HIV nucleotide sequences which are 
necessary for infectivity but dispensable for particle production and 
immunogenicity. Such regions include the RNA packaging signal, the region 
coding for the C-terminus of the gag p15 product, and the vif, integrase, 
reverse transcriptase, and tat genes. An expression construct containing 
mutations of this nature in addition to the deletion of the LTR elements 
could not give rise to infectious retrovirus, even if recombined with 
functional LTR elements. In the present invention, we have demonstrated 
that it was possible to delete 26 nucleotides between the first splice 
donor and the initiation codon for gag which encompasses part or all of 
the HIV-1 RNA packaging signal. In these experiments, virus-like particles 
expressed by this construct in stably transfected COS cells were 
indistinguishable from particles expressed from earlier constructs 
indicating that RNA packaging is not a critical function in the expression 
of non-infectious HIV-1 virus-like particles. Furthermore, we were able to 
show that the deletion in both the Integrase and the Vif genes did not 
affect particle formation. 
EXAMPLES 
Example 1 
This Example illustrates the expression of the major HIV protein antigens 
in cells transfected with an HIV expression construct. 
FIG. 2 shows a diagram of the expression construct termed pHIV-SV which 
contains a single DNA fragment containing HIV-1 protein coding information 
from the LAV-1.sub.BRU strain starting at nucleotide position 678 and 
ending at nucleotide position 8944. This 8.3 kb fragment was made 
blunt-ended using the Klenow fragment of E. coli DNA polymerase I and 
inserted into a blunt HindIII site of a Bluescript-based expression vector 
containing the SV40 virus early promoter and late polyadenylation site. 
The SV40 promoter and polyadenylation sites were obtained from 
commercially available cloning vectors. The pHIV-SV vector is suitable for 
transient expression in monkey COS cells due to the presence of the SV40 
virus origin of DNA replication. 
Fifteen micrograms of the plasmid pHIV-SV was transfected into COS-7 cells 
in a 25 cm.sup.2 flask using standard calcium phosphate transfection 
conditions. COS cells were maintained in Dulbecco's modified Eagle's 
medium containing 10% fetal bovine serum. HIV protein expression was 
analyzed by .sup.3 H-leucine metabolic labelling and immune precipitation 
of intracellular and extracellular antigens. Twenty four hours 
post-transfection, the cells were labelled with .sup.3 H-leucine for 15 
hours after which the medium was harvested and kept at 0.degree. C. Cells 
were lysed by the addition of 1 mL of NP-40 lysis buffer (20 mM Tris-HCl, 
150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM 
phenylmethylsulfonyl fluoride, pH 7.5) and cell lysates were held at 
0.degree. C. Supernatants and lysed cell samples were first reacted with 
normal human serum to clear any proteins non-specifically reacting with 
normal human immunoglobulins. These complexes were removed by binding to 
protein-A agarose. The cleared medium and cell lysate samples were 
subsequently reacted with a commercial human anti-HIV antiserum. 
HIV-specific immune complexes were isolated by binding to protein-A 
agarose and the bound material was subjected to SDS PAGE. Gel 
electrophoresis and fluorography demonstrated the expression of specific 
bands consistent with the p17, p24, gp41, p55, gp120 and gp160 products of 
HIV-1. These bands were not present in control samples from cells which 
were not transfected with pHIV-SV. 
Example 2 
This Example illustrates the sedimentation of high molecular weight 
material associated with reverse transcriptase activity from culture 
supernatants of transfected COS cells. 
Plasmid pHIV-CHO-SV (FIG. 3) is similar to pHIV-SV but employs the 
adenovirus major late promoter and two copies of the human cytomegalovirus 
immediate early enhancer instead of the SV40 virus early promoter. This 
plasmid, pHIV-CHO-SV, has been deposited with the American Type Culture 
Collection, Rockville, Md., on Jul. 14, 1994 under Accession No. 75828. 
This plasmid also contains the SV40 virus origin of replication for 
transient expression analysis. The two copies of the human cytomegalovirus 
immediate early enhancer span the region from nucleotide position -524 to 
-218 which was constructed from overlapping synthetic oligonucleotides. 
The enhancer fragments are located upstream from the adenovirus major late 
promoter which is contained within a 292 bp XhoI-PvuII fragment from 
adenovirus-2 genomic DNA encompassing the major late promoter and the 5' 
end of the first exon of the tripartite leader. This 292 bp fragment was 
ligated to a synthetic 140 bp fragment containing the 3' end of the first 
leader exon, all of the second leader exon, and two thirds of the third 
leader exon, in a pre-spliced configuration. 
Three 75 cm.sup.2 flasks of COS-7 cells were each transfected with 35 
micrograms of the plasmid pHIV-CHO-SV and 30 mL of the 75 mL medium 
supernatant was collected three days post-transfection. High molecular 
weight complexes were pelleted through a 20% glycerol cushion containing 
50 mM Tris-HCl and 0.1 mM KCl, pH 7.8 in an SW28 centrifuge tube. 
Centrifugation was performed at 100,000.times.g for 90 minutes at 
4.degree. C. Samples from mock-transfected COS cells were included as 
controls. The pellet was resuspended in 30 uL of Triton X-100 lysis buffer 
(50 mM Tris-HCl, 100 mM NaCl, 1 mM dithiothreitol, 0.1% Triton X-100, pH 
7.8) for subsequent reverse transcriptase activity analysis. One third of 
the resuspended sample was added to a 90 uL reaction mixture containing 40 
mM Tris-HCl, 4 mM dithiothreitol, 45 mM KCl, 10 mM MgCl.sub.2, 20 uCi 
.sup.3 H-dTTP (80 Ci/mmol), 50 ug poly rA, and 1 ug oligo dT at pH 7.8. 
This mixture was incubated at 37.degree. C. for 30 minutes. Radioactive 
incorporation into trichloroacetic acid-precipitable nucleic acids 
indicated the presence of reverse transcriptase activity. The results 
presented in the following Table I show the radioactivity incorporated for 
each sample: 
TABLE I 
______________________________________ 
Sample CPM incorporated 
______________________________________ 
Blank 14,110 
Mock transfected 18,053 
Transfected 390,220 
Purified MuLV reverse transcriptase 5,416,920 
______________________________________ 
These data demonstrate that reverse transcriptase activity is present in a 
high molecular weight material released in the culture supernatants of COS 
cells transfected with an HIV expression plasmid. 
Example 3 
This Example illustrates the presence of multiple HIV-1 antigens associated 
with high molecular weight material released in the supernatants of 
transfected COS cells. 
Twenty five ug of the plasmid pHIV-SV was transfected into COS cells in a 
75 cm.sup.2 culture flask using a commercial Lipofectin transfection kit. 
Eight mL of the 10 mL medium supernatant was harvested three days 
post-transfection and particulate material was pelleted at 100,000.times.g 
through a 20% glycerol cushion for subsequent Western blot analysis. 
Pellets were suspended in 100 uL of TNE (150 mM NaCl, 50 mM Tris-HCL, 1 mM 
EDTA, pH 7.5) prior to the addition of SDS-PAGE sample buffer and 
electrophoresed on a 12.5% SDS-polyacrylamide gels using standard 
methodologies. Proteins were electrophoretically transferred to Immobilon 
membranes (Millipore) for subsequent reaction with a cocktail of four 
monoclonal antibodies specific for gp120 (DuPont, NEA-9384), p24 (Dupont, 
NEA-9306), p17 (DuPont, NEA-9282) and gp41 (DuPont, NEA-9303), 
respectively. The second antibody was a goat anti-mouse IgG antibody 
conjugated to alkaline phosphatase (Promega). Antibody reactions were 
performed in 5% Carnation instant milk in PBS and developed using an 
alkaline phosphatase substrate solution (Bethesda Research Laboratories). 
Bands corresponding to all four HIV-1 products were present in the 
transfected cell samples but not observed with a control sample from 
mock-transfected cells. The identity of the gp41 and gp120 products was 
confirmed using single monoclonal antibodies in subsequent experiments. 
Example 4 
This Example illustrates the buoyant density analysis of HIV-like particles 
produced from transfected COS cells. 
Seventy micrograms of pHIV-CHO-SV were transfected into each of five 150 
cm.sup.2 flasks of COS cells using the calcium phosphate method and 
culture supernatants were harvested three days post-transfection. HIV-like 
particles were precipitated from growth medium by the addition of NaCl to 
0.15 M and PEG-8000 to 9.3%, and layered on an 11 mL 20-60% sucrose 
density gradient in 100 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA, pH 7.4. The 
gradient was centrifuged for 15 hours at 35,000 rpm in an SW40 rotor at 
4.degree. C. The gradient was subsequently fractionated and aliquots from 
individual fractions were assayed for reverse transcriptase activity after 
the addition of Triton X-100 to 0.2% and KCl to 0.25 mM to disrupt 
virus-like particles. A single peak of reverse transcriptase activity 
banded at a buoyant density of approximately 1.16 g/mL consistent with the 
production of retrovirus-like particles (FIG. 4). The density of 
individual fractions was measured gravimetrically to determine the density 
of the peak fraction. 
Example 5 
This Example illustrates the ability to generate stably transfected COS 
cell clones expressing high molecular weight material containing reverse 
transcriptase activity. 
The HIV expression plasmid pHIV-Ad (FIG. 5) is similar to pHIV-CHO-SV but 
does not contain the SV40 virus origin of replication which would not be 
compatible with stable transfection in COS cells. Five ug of linear 
pHIV-Ad was co-transfected into COS cells in a 5 cm culture dish along 
with a linearised pLTRneo vector specifying resistance to the antibiotic 
G418. Two days following transfection, the cells were split 1:10 into 
medium containing 0.6 mg/mL G418 and resistant colonies were allowed to 
develop. Individual colonies were isolated and expanded and RT activity 
was measured in 8 mL supernatants from each clone as previously described. 
RT activity was detected in the supernatants of two of the first 44 
clones. These clones continued to produce high molecular weight material 
associated with RT activity after continuous passage in the presence of 
0.6 mg/mL of G418. 
Example 6 
This Example illustrates the production of HIV-like particles in stably 
transfected COS cells as demonstrated by Western blot analysis and sucrose 
density gradient centrifugation. 
For Western blot analysis, a culture supernatant from stably transfected 
COS cells was layered over a 20% glycerol cushion and centrifuged at 
100,000.times.g for 90 minutes to pellet virus-like particles. The pellet 
was resuspended in 1.5 uL of TNE for each mL of cell supernatant. Two 
twenty uL aliquots of the concentrated material were analyzed by Western 
blotting with either a commercially available human HIV-1 specific 
antiserum (BioRad) or a cocktail of four commercial monoclonal antibodies 
specific for HIV-1 gp120, gp41, p24 and p17 as described above. These 
results demonstrated the presence of several bands with molecular weights 
consistent with those of the following HIV-1 proteins: gp120, p55, gp41, 
p30, p23 and p17. 
For sucrose density gradient centrifugation, HIV-like particles were 
pelleted from a 500 mL supernatant from stably transfected COS cells by 
sedimentation through a 20% glycerol cushion. The pellets were resuspended 
in TNE and layered on a 20-60% sucrose density gradient and centrifuged as 
described above. After centrifugation, the gradient was fractionated and 
RT activity measured in each fraction. The results are shown in FIG. 6 and 
revealed the presence of a major peak of RT activity banding at a density 
consistent with the formation of HIV-like particles. 
Example 7 
This Example illustrates the detection of budding particles in stably 
transfected COS cells by electron microscopy. 
For thin section analysis, stably transfected COS cells were scraped from a 
culture flask, washed with growth medium, and pelleted by centrifugation. 
Resuspended cells were fixed in 2.5% buffered glutaraldehyde, followed by 
1% buffered osmium tetroxide, dehydrated through alcohol and propylene 
oxide and embedded in an epon-araldite epoxy resin mixture using standard 
techniques. Thin sections were stained with uranyl acetate and lead 
citrate and examined in a Philips EM3000 transmission electron microscope. 
The results demonstrated the presence of budding, immature particles 
consistent with the production of HIV-like particles. 
Example 8 
This Example illustrates the immunogenicity of HIV-like particles purified 
from stably transfected COS cells by sucrose density gradient 
centrifugation as described in Example 6. 
Sucrose gradient fractions containing RT activity were diluted with ten 
volumes of TNE and virus-like particles were concentrated by 
centrifugation at 100,000.times.g for 90 minutes at 4.degree. C. Purified 
virus-like particles were quantified as a function of their p24 content 
using commercial HIV-1 p24 assay kits (Abbott Laboratories and Coulter 
Immunology). 
Six to eight week-old female mice, (C57BL/6.times.C.sub.3 H)F1 (Charles 
River, Montreal) were immunized with HIV-1 virus-like particles and serum 
samples were assayed for HIV-1 specific antibodies. Each mouse received 
two subcutaneous injections equivalent to 6 ug of p24 antigen per 
injection at a three week interval. The primary and boosting immunizations 
were performed in complete Freund's adjuvant and incomplete Freund's 
adjuvant, respectively. Nine days after the second immunization, sera were 
collected and heat-inactivated at 56.degree. C. for 30 minutes. HIV-1 
specific antibodies were detected by enzyme-immunoassays (EIAs). EIA 
plates (Maxisorp, Nunc) were coated with recombinant (r) antigens: rgp120 
(American Biotech. Inc.), rgp160 (Repligen), rgp41 (DuPont), or rp24 
(DuPont) in phosphate buffered saline (PBS, pH 7.0) at 0.4 ug per well. 
Adsorption of the antigens was allowed to proceed overnight at 4.degree. 
C. Unbound antigen was aspirated and the plates were blocked with 300 uL 
per well of 2% skim milk powder (Carnation) in PBS for two hours at room 
temperature. The plates were then washed three times with PBS containing 
0.025% Tween 20 (BioRad). Serum samples were serially diluted in PBS and 
added to individual wells for one hour at room temperature. The plates 
were then washed three times with PBS/Tween 20 as described above. A goat 
anti-mouse-IgG antibody conjugated to horseradish peroxidase (Jackson 
Laboratories) was diluted 1 in 5000 with PBS and added for one hour at 
room temperature. After washing with PBS/Tween 20, 100 uL of a 
tetra-methylbenzidine substrate solution diluted 1 in 10 with peroxide 
reagent as described by the manufacturer (ADI Diagnostics) was added to 
each well for 10-15 minutes at room temperature. One hundred uL of 1N 
H.sub.2 SO.sub.4 was added to stop the reactions and the plates were read 
in an EIA plate reader at 450 nm. 
The results shown in FIG. 7 demonstrate significant IgG antibody responses 
against recombinant gp120, gp41, and p24 proteins. The presence of IgG 
antibodies against a major synthetic HIV-1 neutralization epitope 
encompassing residues 311 to 320 of gp120 and a synthetic gp41 B-cell 
epitope (Residues 727 to 751) was detected in peptide-specific EIA's using 
microtitre plates coated with synthetic peptides produced by solid-phase 
chemical synthesis on an ABI Model 430A peptide synthesizer. Murine 
antisera also demonstrated HIV-1 specific neutralization activity for 
HIV-1 strains LAV-1.sub.BRU and HTLV-III.sub.MN in a standard tissue 
culture syncytial inhibition assay at a serum dilution of 1/10. These 
results indicate that HIV-1-like particles have the capacity to induce 
cross-reactive immune responses against different viral isolates. Further 
evidence of antibody cross-reactivity was obtained with sera from animals 
boosted a second time with virus-like particles at 1.5 ug of p24 core 
antigen per dose. The IgG responses specific for various HIV recombinant 
antigens are shown in FIGS. 8 and 9 and demonstrate not only potent immune 
responses to HIV-1 gp120 and gp160, but also significant reactivity with 
HIV-2 gp120 as well. These data indicate that HIV-like particles may prove 
to be an efficient immunogen for the induction of cross-reactive immune 
responses. 
Example 9 
This Example illustrates the inducible expression of HIV-like particles 
following transfection of an inducible expression vector into Vero cells. 
FIG. 10 shows a map of the HIV expression vector pMT-HIV which is similar 
to previous expression vectors but contains the human metallothionein IIa 
promoter from nucleotide positions -742 to +59, placed directly upstream 
of the HIV-1 coding sequences which begin at nucleotide position 678. This 
plasmid has been deposited with the American Type Culture Collection on 
Oct. 12, 1990 (#40912). After co-tansfection of pMT-HIV into Vero cells 
with the G418 resistance marker, numerous G418 resistant colonies were 
isolated and tested for RT production in response to cadmium chloride 
(CdCl.sub.2) addition. Individual clones were grown to confluence in 9 cm 
culture dishes and treated with 5 uM CdCl.sub.2 for twenty four hours, 
after which a standard assay for RT activity was performed on high 
molecular weight material pelleted from 8 mL culture supernatants as 
previously described in Example 2. These results demonstrated the 
production of significant levels of RT activity in at least 30% of the 
clones. RT activity in the absence of induction was detectable in the 
supernatants of only a few Vero cell clones. The following Table II shows 
the levels of p24 antigen production detected in the culture supernatants 
of six clones in the presence and absence of CdCl.sub.2. p24 levels were 
measured using a commercial HIV-1 p24 assay kit and the results 
demonstrated a very high expression level for one clone (clone 11) and 
high induction ratios for all six clones. 
TABLE II 
______________________________________ 
p24 Production in Induced and Non-Induced Vero Cell Clones 
Clone Number 
Non-Induced (ug/L) 
Induced (ug/L) 
Ratio 
______________________________________ 
7 &lt;.01 16 &gt;1600 
10 &lt;.01 33 &gt;3300 
11 .41 600 1463 
30 .02 33 1650 
62 .02 112 5600 
76 &lt;.01 74 &gt;7400 
______________________________________ 
Example 10 
This Example illustrates the production of mature HIV-like particles in 
Vero cell clone 11 which was stably transfected with the inducible 
expression vector pMT-HIV. 
Subconfluent cells grown in a 75 cm.sup.2 culture flask were treated with 5 
uM CdCl.sub.2 for twenty four hours, then scraped from the flask and 
processed for thin section electron microscopy as described in Example 7. 
Electron micrographs showed numerous mature and immature virus-like 
particles budding from the plasma membrane of the transfected cells. These 
levels of particle formation were consistent with the high levels of p24 
and RT production observed for this clone. 
Example 11 
This Example illustrates the continuous production of RT activity and p24 
core antigen in a single flask of Vero cell clone II over an eight day 
period of continuous induction with CdCl.sub.2. 
Clone 11 cells were grown to confluence in a 75 cm.sup.2 culture flask and 
were then treated with 5 uM CdCl.sub.2 for twenty four hours after which 
all of the medium was harvested and fresh medium containing CdCl.sub.2 
added. This process was repeated every twenty four hours for eight days 
and the levels of RT and p24 production in each twenty four hour culture 
supernatant are shown in FIG. 11. The levels of production of p24 and RT 
activity correlated extremely well and peaked five days after the cells 
reached confluence. 
Example 12 
This Example illustrates the feasibility of producing HIV-like particles 
with a genetically modified envelope glycoprotein. 
It is possible that structural alterations of gp120 may result in a 
reduction of its binding affinity for gp41, and therefore in its shedding 
from the surface of the particles. Mutations preventing the proteolytic 
processing of the gp160 envelope glycoprotein should circumvent this 
problem and result in the expression of uncleaved glycoprotein precursors 
stably associated with virus-like particles. Codon position 455 in the 
LAV-1.sub.BRU gp160 envelope glycoprotein gene encodes an arginine residue 
which is the site of proteolytic cleavage in the gp160 envelope 
glycoprotein. This codon was mutated to encode a threonine residue using a 
commercial M13 phage-based site-directed mutagenesis kit (Amersham) and an 
appropriate synthetic mutagenic oligonucleotide complementary to the 
regions flanking this codon. This mutation was created in a cloned 
subfragment of the envelope glycoprotein gene which was subsequently used 
to replace the corresponding region in the pHIV-SV particle expression 
plasmid. Transfection of this modified plasmid into COS cells resulted in 
the expression of high molecular weight virus-like material in association 
with the gp160 envelope glycoprotein precursor as detected by Western blot 
analysis. The fact that gp120 was not detected was consistent with the 
disruption of proteolytic processing. That the gp160 envelope protein 
remains associated with virus-like particles was demonstrated by banding 
particles produced in a transient transfection experiment by sucrose 
density gradient centrifugation. Western blot analysis demonstrated that 
the gp160 precursor protein was found only in gradient fractions 
containing RT activity and not in heavier or lighter fractions. 
The feasibility of modifying the structure of the gp120 envelope 
glycoprotein was further demonstrated by inserting two pairs of synthetic 
oligonucleotides into the Bg1II site at nucleotide position 7008 in the 
LAV-1.sub.BRU DNA sequence. Insertion of these oligonucleotides resulted 
in a modified coding sequence in which the inserted element encoded the 
HTLV-III.sub.MN V3 neutralization epitope encompassing amino acid residues 
306 to 329 of the viral envelope glycoprotein. The resultant sequence 
encoded the complete LAV-1.sub.BRU envelope glycoprotein with an MN strain 
V3 loop insertion at amino acid position 272. The resulting plasmid is 
termed pV3Bg and has been deposited with the American Type Culture 
Collection on Oct. 12, 1990 (#40910). The oligonucleotides used in the 
modification were as follows: 
5' GATCTCGGACCGCCTACAATAAAAGAAAAAGGATACATATAGGA 3' 
3' AGCCTGGCGGATGTTATTTTCTTTTTCCTATGTATATCCTGGTCCCTC 5' (SEQ ID NO:1) 
- 5' CCAGGGAGAGCATTTTATACAACAAAAAATATAATAGGAACGCGTA 3' 
3' TCGTAAAATATGTTGTTTTTTATATTATCCTTGCGCATCTAG 5' (SEQ ID NO:2) 
Western blot analysis of pellets obtained by centrifugation of transfected 
cell supernatants through a 20% glycerol cushion demonstrated the 
production of particulate material containing gp120 at a level similar to 
that observed with the unmodified expression vector. These data 
demonstrate that the insertion of 24 additional aminoacid residues at 
amino acid position 272 of the LAV-1 envelope did not disrupt its 
targeting to the viral membrane, the processing of the envelope 
glycoprotein precursor, or its non-covalent association with the gp41 
transmembrane glycoprotein. It is therefore likely that additional gp120 
alterations might be tolerated and, thus, allow for further modulation of 
the immunogenicity of the envelope glycoprotein. 
Example 13 
This Example illustrates the feasibility of creating a mutation in an HIV-1 
sequence element which is required for infectivity but dispensable for 
virus-like particle production. 
To demonstrate the feasibility of producing HIV-like particles using an 
expression construct containing an additional mutation in a genetic 
element required for infectivity, an expression plasmid containing a 
deletion of the RNA packaging signal was created. This signal has been 
shown to lie between the first splice donor at nucleotide position 744 and 
the initiation codon for Gag at nucleotide position 790 and to be critical 
for HIV RNA packaging and infectivity. This region was deleted from a 
clone containing an 8.9 kb SacI-SacI fragment of LAV-1.sub.BRU (nucleotide 
positions 678-9619) by removing a 114 bp SacI-HgaI restriction fragment 
encompassing nucleotide positions 678 to 791. The deleted fragment was 
replaced with a double-stranded synthetic fragment containing a deletion 
of 26 bp from nucleotide positions 753 to 777. The modified SacI-SacI 
fragment was then used as a source of DNA to isolate the SacI-XhoI 
fragment (nucleotide positions 678 to 8944) for construction of an HIV 
expression vector utilizing the adenovirus major late promoter similar to 
pHIV-Ad described in Example 5. The new plasmid, termed pHIV-Ad-d26 was 
transfected into COS cells as described in Example 5 and G418 resistant 
clones constitutively expressing RT activity were isolated. Virus-like 
particles were pelleted from 200 mL of culture supernatants from two COS 
cell clones stably transfected with the original and "RNA 
packaging-deleted" expression constructs, respectively. FIG. 12 shows a 
comparison of the sucrose density gradient centrifugation profiles for the 
virus-like particles derived from these expression constructs. Virus-like 
particles derived from the construct containing the packaging deletion 
were not significantly different from those derived from the original 
expression construct. 
Example 14 
This Example illustrates the production of non-infectious SIV-like 
particles. 
To demonstrate the applicability of this invention to the production of 
virus-like particles derived from a retrovirus other than HIV, an SIV 
expression vector containing a DNA fragment from the provirus of 
SIV.sub.mac239 was constructed. Although the nucleotide sequence of the 
SIV.sub.mac239 provirus has yet to be published, comparison of its 
restriction map with that of SIV.sub.mac142 which has been sequenced 
revealed a number of similarities. The NarI restriction site at nucleotide 
position 835 of SIV.sub.mac142 and the two SstI restriction sites at 
positions 5756 and 9236 are conserved in the SIV.sub.mac239 provirus. 
Therefore, two fragments from the SIV.sub.mac239 provirus consisting of 
the 4.9 kb NarI-SstI and the 3.4 kb SstI-SstI fragments were isolated from 
the cloned SIV.sub.mac239 provirus and inserted into an expression vector 
utilizing the SV40 virus early promoter as described in Example for 
LAV-1.sub.BRU. Transfection of this plasmid, termed pSIV-SV, into COS 
cells resulted in the transient expression of RT activity as described for 
LAV-1.sub.BRU' also in Example 2. The co-transfection of Vero cells with 
pSIV-SV and a G418 resistance marker resulted in the isolation of drug 
resistant clones constitutively expressing RT activity as described in 
Example 5 for the transfection of pHIV-Ad into COS cells. 
To provide further evidence for the production of SIV-like particles, 
culture supernatants were collected from Vero cells stably transfected 
with pSIV-SV and virus-like particles were pelleted by centrifugation at 
100,000.times.g. Pelleted material was analyzed by SDS-PAGE and Western 
blotting using a monkey SIV-specific antiserum. Analysis of the data 
revealed the presence of several bands with molecular weights consistent 
with the expression of the major SIV antigens. These bands were not 
detected in the culture fluid of non-transfected Vero cells. 
Example 15 
This Example illustrates the feasibility of expressing HIV-like particles 
using an expression vector containing a deletion of the provirus genome 
which encodes proteins required for viral infectivity but which are 
dispensable for particle assembly. 
The pMT-HIV expression vector described in Example 9 was modified to 
contain a deletion of the 26 nucleotides in the RNA packaging region as 
described in Example 13. A further deletion of HIV coding sequences which 
encode the Vif and Integrase genes was also performed. The Vif and 
Integrase genes are required for viral infectivity but are dispensable for 
particle assembly. This second deletion was accomplished by removing a 746 
bp fragment between the BspmI restriction sites at nucleotide positions 
4345 and 5091 of the LAV-1.sub.BRU genome. The resultant expression 
plasmid, termed pMT-HIV-dVI-d26, has been deposited with the American Type 
Culture Collection on Oct. 12, 1990 (#40911). Transfection of 
pMT-HIV-dVI-d26 into COS cells in a transient expression assay resulted in 
the expression of significant quantities of p24 antigen in the culture 
supernatant but no RT activity was detected. The absence of RT activity 
indicates that the neighbouring integrase mutations may have affected RT 
protein processing. Evidence for the assembly of virus-like particles was 
obtained from a Western blot analysis of material pelleted from the 
culture supernatant of transfected cells by high speed centrifugation 
through a 20% glycerol cushion which demonstrated the presence of gp120 in 
significant quantities. Production of p24 and gp120 was not detected in 
the culture supernatant of non-transfected cells. 
Example 16 
This Example illustrates the production of HIV-like particles which are 
deficient in the gp120 envelope glycoprotein due to a deletion of gp120 
coding sequences in a modified expression vector. 
The HIV expression vector pHIV-SV was modified so as to encode the 
production of virus-like particles lacking the gp120 envelope 
glycoprotein. This was accomplished by deleting a fragment between the 
KpnI site at nucleotide position 6379 and the BglII site at nucleotide 
position 7668. The remaining restriction ends were treated with Klenow DNA 
polymerase to remove the 3' overhang at the KpnI end and to fill in the 5' 
overhang at the Bgl lI end. The blunt ends were ligated to a BglII linker 
(Pharmacia, 5'-pd[CAGATCTG]-3') which was then cleaved with BglII and the 
sticky ends were ligated together. This ligation restored what remained of 
the gp120 reading frame to allow for the later insertion of heterologous 
coding sequences. This plasmid, termed pBL-HIV-dgp120-6, has been 
deposited with the American Type Culture Collection on Oct. 12, 1990 
(#40913). 
Transfection of pBL-HIV-gdp120-6 into COS cells in transient expression 
assays resulted in the expression of high molecular weight material 
containing RT activity. Western blot analysis of the pellet obtained by 
high speed centrifugation of the culture supernatant through a 20% 
glycerol cushion revealed the expression of the p24 and p17 gag proteins 
but not of gp120. In a control experiment where the original pHIV-SV 
expression plasmid was transfected, significant quantities of HIV-1 gp120 
were detected in the pelleted fractions. 
SUMMARY OF DISCLOSURE 
In summary of this disclosure, the present invention provides 
enetically-engineered non-infectious and immunogenic retrovirus-like 
particles which are candidates for vaccines against the respective 
retroviruses, such as AIDS and procedures for producing the same. 
Modification are possible within the scope of this invention. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- - - - (1) GENERAL INFORMATION: 
- - (iii) NUMBER OF SEQUENCES: 2 
- - - - (2) INFORMATION FOR SEQ ID NO:1: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 92 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:1: 
- - GATCTCGGAC CGCCTACAAT AAAAGAAAAA GGATACATAT AGGAAGCCTG GC - 
#GGATGTTA 60 
- - TTTTCTTTTT CCTATGTATA TCCTGGTCCC TC - # - # 
92 
- - - - (2) INFORMATION FOR SEQ ID NO:2: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 88 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:2: 
- - CCAGGGAGAG CATTTTATAC AACAAAAAAT ATAATAGGAA CGCGTATCGT AA - 
#AATATGTT 60 
- - GTTTTTTATA TTATCCTTGC GCATCTAG - # - # 
88 
__________________________________________________________________________