Human lymphoid cells expressing human immunodeficiency virus envelope protein gp160

The present invention provides mammalian cells modified to stably express at least the entire human immunodeficiency virus-1 envelope protein gp160. The invention provides a vaccine comprising the cells of the invention. The invention also provides methods for screening compounds for their ability to inhibit formation of syncytia between cells that express HIV-1 gp160 and cells that express CD4 comprising mixing cells of invention, cells that express CD4 on their surfaces, and a test compound for a length of time sufficient for syncytia to form; and then determining the amount of syncytia formation.

FIELD OF THE INVENTION 
The present invention relates to the field of cells transfected by 
recombinant DNA techniques to express heterologous proteins. More 
particularly, the present invention relates to cells transfected by 
recombinant DNA techniques to express viral proteins, and their use as 
vaccines for prevention of disease, and in assay systems in the drug 
discovery process. 
BACKGROUND OF THE INVENTION 
HIV-1 is the etiological agent of acquired human immune deficiency syndrome 
(AIDS) and related disorders. HIV-1 is an RNA virus of the Retroviridae 
family and exhibits the same 5'LTR-gag-pol-env-LTR3' organization as all 
retroviruses. In addition, it comprises a handful of genes with regulatory 
or unknown function, in particular the tat and rev genes. The env gene 
encodes the viral envelope glycoprotein that is first translated as a 
160-kilodalton (kDa) precursor (gp160), which is subsequently cleaved by a 
cellular protease to yield the external 120-kDa envelope glycoprotein 
(gp120) and the transmembrane 41-kDa envelope glycoprotein (gp41). Gp120 
and gp41 remain associated and are displayed on the viral particles as 
well as on the surface of HIV-infected cells. Gp120 is directly 
responsible for binding to the CD4 receptor present on the surface of 
helper T-lymphocytes, macrophages and other target cells. After gp120 
binds to CD4, gp41 mediates the fusion event responsible for virus entry. 
Infection begins as gp120 on the viral particle binds tightly to the CD4 
receptor on the surface of T4 lymphocytes or other target cells. The virus 
then merges with the target cell and reverse transcribes its RNA genome 
into double-stranded DNA. The viral DNA becomes incorporated into the 
genetic material in the cell's nucleus and directs the production of new 
viral RNA and viral proteins, which combine to form new virus particles. 
These particles bud from the target cell membrane and infect other cells. 
Destruction of T4 lymphocytes, which are critical to immune defense, is the 
major cause of the progressive immune dysfunction that is the hallmark of 
HIV infection. The loss of target cells seriously impairs the body's 
ability to fight most invaders, but it has a particularly severe impact on 
the defenses against viruses, fungi, parasites and certain bacteria, 
including mycobacteria. 
HIV-1 is known to kill the cells it infects by replicating, budding from 
them and damaging the cell membrane. HIV-1 might also kill target cells 
indirectly, by means of the viral protein, gp120, that is displayed on an 
infected cell's surface. The CD4 receptor on T cells has a strong affinity 
for gp120, and healthy T4 cells and cells expressing CD4 receptor can bind 
to gp120 and fuse with infected cells. In addition to the CD4-gp120 
interaction, other receptors may also play a role in the fusion process. 
The end result, called a syncytium, cannot survive, and all the once 
healthy cells it contains are destroyed along with the infected cell. 
HIV-1 can also elicit normal cellular immune defenses against infected 
cells. With or without the help of antibodies, cytotoxic defensive cells 
can destroy an infected cell that displays viral proteins on its surface. 
Finally, free gp120 may circulate in the blood of individuals infected 
with HIV-1. The free protein may bind to the CD4 receptor of uninfected 
cells, making them appear to be infected and evoking an immune response. 
Infection with HIV-1 is almost always fatal, and at present there are no 
cures for HIV-1 infection. Effective vaccines for prevention of HIV-1 
infection are not yet available. Because of the danger of reversion or 
infection, conventional live attenuated virus or killed whole virus cannot 
be used as vaccines. Also most subunit vaccine approaches have not been 
successful at preventing HIV infection to date. In addition, treatments 
for HIV-1 infection, while prolonging the life of infected persons to some 
extent, have serious side effects. There is thus a great need for 
effective treatments and vaccines to combat this lethal infection. 
Vaccination is an effective form of disease prevention and has proven 
successful against several types of viral infection. Determining ways to 
present HIV-1 antigens to the human immune system in order to evoke 
protective humoral and cellular immunity, is a difficult task. At the 
present time most attempts to generate an effective HIV vaccine have been 
unsuccessful. In AIDS patients, free virus is present in low levels only. 
Transmission of HIV-1 is enhanced by cell-to-cell interaction via fusion 
and syncytia formation. Hence, antibodies generated against free virus or 
viral subunits are generally ineffective in eliminating virus-infected 
cells. 
Vaccines exploit the body's ability to "remember" an antigen. After first 
encounters with a given antigen the immune system generates cells that 
retain an immunological memory of the antigen for an individual's 
lifetime. Consequently, subsequent exposure to the antigen results in 
elimination or inactivation of the pathogen. The immune system deals with 
pathogens in two ways: by humoral and by cell-mediated responses. In the 
humoral response lymphocytes generate specific antibodies that bind to the 
antigen thus inactivating the pathogen. The cell-mediated response 
involves cytotoxic lymphocytes that specifically attack and destroy 
infected cells. 
Vaccine development with HIV-1 virus presents problems because the virus 
infects some of the same cells the vaccine needs to activate in the immune 
system (i.e., T4 lymphocytes). Therefore it would be advantageous for the 
vaccine to inactivate the HIV before impairment of the immune system 
occurs. A particularly suitable type of HIV vaccine would generate an 
anti-HIV immune response which will recognize innumerable HIV variants and 
will extend its activity to HIV-positive individuals who are at the 
beginning of their infection. 
It is accordingly an object of the invention to provide vaccines for use in 
the prevention and treatment of HIV-1 infection. It is also an object of 
the invention to provide methods for screening compounds that inhibit the 
deleterious syncytia formation for use as treatments for HIV-1 infection. 
Sodroski, J. et al. (1986) Nature 322: 470-474 discloses T4+ Jurkat-tat-III 
cells (a T4+ Burkitt's lymphoma cell line that expresses the HIV-1 tat 
gene product) transfected with a plasmid designed to express both the rev 
and env gene products. These cells transiently expressed HIV-1 gp160, and 
were used in a syncytia formation assay in studies on the basis of the 
specific cytotoxicity of the AIDS virus. Although the cells expressed 
HIV-1 gp160, transient expression of the envelope protein is inconvenient 
and time-consuming for use in syncytium formation assays, since new cells 
would have to be transfected at frequent intervals. 
Palker, T. J. et al (1987) Proc. Natl. Acad. Sci. USA 84: 2479-2483 also 
discloses a syncytia formation assay. The assay was used for the study of 
antibodies specific for HIV-1 proteins. 
SUMMARY OF THE INVENTION 
The present invention provides mammalian cells modified to stably express a 
heterologous protein comprising at least the entire HIV-1 envelope protein 
gp160. The invention also provides vaccines for prevention of HIV-1 
infection comprising the cells of the invention. The invention further 
provides assays for the determination of syncytia formation for screening 
compounds for their ability to inhibit syncytia formation. This invention 
is more particularly pointed out in the appended claims and is described 
in its preferred embodiments in the following description.

EXAMPLE 1 
Preparation of BJAB Cells Expressing HIV-1 gp160 Construction of CLDN-gp160 
plasmid 
The HIV-1 gp160 coding sequence was isolated on an MstII-XhoI 2945-base 
pair (bp) restriction endonuclease fragment from the BH10 HIV-1 viral 
isolate described by Shaw et al. (1984) Science 226: 1165-1171. This 
restriction endonuclease fragment carries the intact HIV-1 gp160 env gene 
as well as the two intact rev exons (coordinates 5535 to 8480, according 
to the numbering of Ratner et al (1985) Nature 313: 277), the disclosures 
of which are incorporated by reference as if fully set forth herein. The 
DNA sequence coding for HIV-1 gp160 used in the preferred embodiment is 
set out in FIG. 1 which shows the sequence of the MstII-XhoI restriction 
from BH10 carrying the full-length gp160 envelope gene and the two intact 
exons of the rev regulatory protein. The position of the gp120, gp41 and 
rev coding sequences is shown. The position of the XbaI linkers at each 
end of the restriction fragment is shown by an asterisk (*). The HIV-1 
restriction fragment sequence shown is taken from sequence of HIV-1 
disclosed in Rather et al. supra. 
After ligation to XbaI linkers and digestion with XbaI, this env-carrying 
fragment was inserted downstream of the cytomegalovirus (CMV) promoter in 
the CLDN expression vector. CLDN is a vector derived from pUC 19 which 
contains an SV40 origin of replication and three independent 
transcriptions cassettes for stable integration, high expression level, 
and amplification in a variety of mammalian cells. The first 
transcriptional cassette consists of the strong and constitutive 
cytomegalovirus (CMV) immediate early (IE) promoter isolated on an 
Spe1-Sac1 restriction endonuclease fragment from the CDM8 vector described 
by Seed (1987) Nature 329: 840-842, a unique polylinker region (including 
an XbaI site) for insertion of genes and the bovine growth hormone (BGH) 
polyadenylation region (Pfarr, D. et al. (1986) DNA 5: 115-122). 
Downstream of the expression cassette are two dominant selectable markers. 
First is the mouse dihydrofolate reductase (DHFR) gene cassette which is 
used for methotrexate MTX selection and amplification (Subramani, S. et 
al. (1981) Mol. and Cell. Biol. 1: 854-864). Second, downstream from the 
DHFR cassette, is the bacterial neomycin resistance (NEO) cassette which 
is used for neomycin G418 selection (Colbere-Garapin, F. et al. (1981) J. 
Mol. Bio. 150: 1-14). 
BJAB Cells 
BJAB cells are human cells (human Burkitt's lymphoma cell line) which are 
EBV (Epstein-Barr Virus) and EBNA (EBV nuclear antigen) negative. BJAB 
cells were obtained from Dr. Henle, Children's Hospital of Pennsylvania, 
Philadelphia, Pa. The cells are Epstein-Barr virus and Mycoplasma free. 
Cells grow in suspension, and have doubling time of approximately 15 
hours. The karyotype of BJAB cells is stable after transfection. BJAB 
cells were cultured in TY medium supplemented with 15% fetal calf serum 
(FCS) at 37 C in a 7% CO.sub.2 atmosphere. 
Transfection and Expression of gp160 
BJAB cells were transfected with CLDN-gp160 plasmid DNA by electroporation 
and clones selected for neomycin resistance. Selected clones were screened 
for expression of gp160 with a panel of antibodies (monoclonal and 
polyclonal) by immunocytochemistry and by their capability to form 
syncytia with human lymphoid cells of CD4+ phenotype. 
The following media and solution were used in the transfection and 
selection of clones expressing gp160. 
1) HY Medium--To 500 ml of DMEM-HB (Dulbecco's Modified Eagle--high glucose 
medium with 10% fetal calf serum (FCS) add: 5 ml of L-glutamine 
(100.times.)[200 mM], 5 ml of oxaloacetate, pyruvate, insulin 
(OPI)(100.times.) (to prepare 100 ml of 100.times. OPI combine: CIS-oxalic 
acid, 1500 mg, Sigma D-7753, insulin, 2000 U, SIGMA I-6634; pyruvic acid, 
500 mg. Sigma P-5280), 5 ml hypoxanthine, thymidine (HT) (100.times., 
Gibco 32D-1067 (to prepare 100 ml of 100.times. HT, combine: hypoxanthine 
10.sup.-2 M, thymidine 1.6.times.10.sup.-3 M), 0.5 ml gentamicin (10 
mg/ml, Gibco #600-5710), (HY-medium--Gibco). 
2) TY Medium.sup.19 --To 500 ml of HY medium with 15% FCS add: 0.5 ml ITS 
[insulin, transferrin, selenium (CR-ITS Premix, Collaborative Research)], 
2 .mu.l 2-aminoethanol (Sigma ED135), 2 .mu.l 2-mercaptoethanol (Sigma 
Chemical Co., St. Louis, Mo.), 
Electroporation was done using a BTX Electro Cell Manipulator, Model 401-AM 
(BTX, Biotechnologies--Experimental Research, Inc., 3742 Lawell Street, 
San Diego, Calif., U.S.A.). The chambers used for electroporation were 
Flat chamber 481 (0.5 ml volume/1.0 mw gap). Electroporation was monitored 
using an optimizer (BTX Optimizor 50) with graphic pulse analyzer. For 
BJAB cells suspended in phosphate buffered saline (PBS), optimal 
conditions were an amplitude of 500-999 (2.4 kV/cm-4.2 kV/cm) and a pulse 
width of 99 .mu.sec. At an amplitude of 999 and pulse width of 99 .mu.sec, 
viability of human BJAB cells was 85% in PBS. 
BJAB cells were grown in HY medium with 15% FCS. The cells were split and 
refed the day before electroporation. Prior to electroporation, cells were 
washed twice with ice-cold PBS pH 7.0, and resuspended in ice-cold PBS at 
10.sup.7 cells/ml (this ensures 95% cell viability). Prior to use, plasmid 
DNA was ethanol precipitated and resuspended in sterile PBS. 10-50 .mu.g 
of plasmid DNA (supercoiled or linearized plasmid) per 5.times.10.sup.6 
cells is added to the cell suspension, mixed well, incubated for 10 
minutes on ice, and then this mixture is placed into the electroporation 
chamber. The electroporation chamber, previously stored in 70% ethanol was 
washed thoroughly with cold PBS, placed on ice and connected to the 
electrocell manipulator. 0.5 ml of the BJAB-DNA mixture was added to the 
chamber. The cells received single/multiple pulses under at the conditions 
described above. Under these conditions approximately 75% cells were 
viable after one hour at 37.degree. C. The electroporated cells were 
transferred into a tube and kept on ice for ten minutes. TY medium was 
added to the cells and the cells were transferred to culture dishes. 
Medium was replaced weekly. 
For selection of clones expressing gp160, a selection medium containing TY 
medium supplemented with 800 .mu.g/ml G418 (Geneticin, G418 sulphate, 
Gibco) was used, and clones were selected according to the method in 
Harlow et al., Antibodies, A Laboratory Manual, Cold Spring Harbor 
Laboratories, Cold Spring Harbor, N.Y. 
BJAB cells incubated with either plasmid DNA alone or calcium phosphate 
failed to be transfected with the plasmid DNA and did not form 
proliferating colonies. 
Clone TF228.1 showed the highest expression levels of gp160 per cell and 
uniformity of gp160 expression with respect to total cell population 
expressing the gene. To assure monoclonality, cells were cloned by limited 
dilution and were screened for function, expression and stability. 
Characterization of TF228.1 
Indirect immunofluorescence immunocytochemistry to test anti-gp120, 
anti-gp41 and soluble T4 (sT4) binding was performed using standard 
techniques such as those described in Harlow et al., Antibodies, A 
Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, 
N.Y. Pandex assay to test anti-gp120 and anti-gp41 binding was performed 
according to the method in "Whole-cell applications of PCFIA" in: 
Proceedings of the 1987 Pandex Symposium on Particle Concentration 
Fluorescence Immunoassay (Baxter Corporation, Chicago, Ill.). Western blot 
analysis was performed using standard techniques such as those described 
in Harlow et al., Antibodies, A Laboratory Manual, Cold Spring Harbor 
Laboratories, Cold Spring Harbor, N.Y. 
As indicated in Table I, several assay systems showed that gp120 is 
expressed on the surface of BJAB cells. Several different antibodies 
indicated positive binding to gp120 and gp41 molecules. The binding of 
soluble CD4 (sT4) to gp160 indicated that the gp120 portion of the 
molecule has appropriate conformational structure to bind the CD4 
receptor. 
TABLE I 
______________________________________ 
CHARACTERIZATION OF TF228.1 CLONE (HIV-gp160) 
Assay System: TF228.1 
______________________________________ 
Immunocytochemistry 
Anti-gp120 binding 
+ 
Anti-gp41 binding + 
sT4 binding + 
Pandex assay 
Anti-gp120 binding 
+ 
Anti-gp41 binding + 
Western blot analyses 
+ 
______________________________________ 
EXAMPLE 2 
Syncytia Formation 
3.5.times.10.sup.4 five to six day old cultures of SupT1 cells a (CD4+ 
human T lymphocytic line) and 1.75.times.10.sup.4 five to six day old 
cultures of TF228.1.16 (gp160) cells in forty microliters of buffer per 
each cell type were placed in the same well of 96 well plates. No test 
compound was added to controls. Twenty microliters of test compound was 
added to the remaining wells. The cells were incubated for 16-18 hours at 
37 C, 7.5% CO.sub.2 to allow formation of syncytia. Syncytia were then 
counted under a microscope (Biostar) at 40.times. magnification. The 
number of syncytia formed in wells containing test compound is expressed 
as percentage reduction of syncytia formed in control wells. (i.e., the 
number of syncytia formed in wells containing test compound divided by the 
number of syncytia formed in control wells.) 
As indicated in Table II, several human T cell lines and primary human 
lymphocytes showed syncytia formation with the TF228.1 clone. When 
compounds are tested for their ability to inhibit syncytia formation, they 
may be tested with one or several combinations of cells. 
TABLE II 
______________________________________ 
SYNCYTIA FORMATION 
CD4 + Human T-Cells/Cell lines 
CD4 human 
HIV-gp160 cell line 
BJAB Primary human 
TF112B3.1 
Transfectant 
SUPT1 MOLT4 T lymphocytes 
(control) 
______________________________________ 
TF228.1 + + + - 
______________________________________ 
EXAMPLE 3 
Syncytia Formation Assay 
Soluble CD4 (sT4), a soluble recombinant form of the CD4 receptor found on 
human lymphocytes, and Leu-3a anti-CD4 monoclonal antibodies (Becton 
Dickinson Immunocytochemistry Systems, San Jose, Calif.) were tested using 
the syncytia formation assay described in Example 2. 20 .mu.l of Leu-3a or 
sT4 dilution were added to the cells after they were placed in the 
microtiter wells. Inhibition of syncytia formation was followed by 
counting the number of syncytia present and by [.sup.3 H]TdR uptake. 
Increasing concentrations of sT4 increased the inhibition of syncytia 
formation by sT4. At a concentration of 10 .mu.g/ml sT4 inhibited syncytia 
formation by 10 per cent when compared to controls. At a concentration of 
50 .mu.g/ml sT4 inhibited syncytia formation by about 25-35 per cent when 
compared to controls. Syncytia formation was inhibited by about 45-55 per 
cent when compared to controls when sT4 was present at a concentration of 
100 .mu.g/ml. At a concentration of 250 .mu.g/ml sT4 inhibited syncytia 
formation by about 85 to 100 per cent when compared to controls. When sT4 
was present in the medium at a concentration of 500 .mu.g/ml syncytia 
formation was inhibited by 100 per cent when compared to controls. 
Syncytia formation was also significantly inhibited when increasing 
concentrations of Leu-3a were added. When Leu-3a was present at a 
concentration of 2.4 ng/ml syncytia formation was inhibited by 20 per cent 
when compared to controls. Increasing Leu-3a concentrations up to 19.5 
ng/ml resulted in similar inhibition of syncytia formation (about 5-15 per 
cent inhibition with 4.8 ng/ml; about 5-24 per cent inhibition with 9.7 
ng/ml; and about 18-25 per cent inhibition with 19.5 ng/ml). At 39 ng/ml 
Leu-3a inhibition of syncytia formation increased dramatically to about 
50-65 per cent inhibition when compared to controls. And at 78 ng/ml 
Leu-3a inhibition of syncytia formation was 100 per cent when compared to 
controls. Similarly, inhibition of syncytia formation at 156 ng/ml was 
85-100 per cent. 
EXAMPLE 4 
In vitro Stimulation/Activation of Human Lymphocytes With TF228.1.16 Cells 
Peripheral blood mononuclear (PBM) cells were isolated from day old Red 
Cross blood on a ficoll-hypaque density gradient and cultured together 
with irradiated (3000R) TF228.1 cells at a ratio of 50:1 or 100:1 PBM to 
TF228.1 cells. The final cell density was 5.times.10.sup.6 cells/ml. The 
cell mixtures were incubated at 37.degree. C. in a 7% CO.sub.2 atmosphere 
in TY media containing 25% mixed lymphocyte culture (MLC) supernatant and 
5 U/ml interleukin-2 (IL2). (Boehringer-Mannheim Corporation, 
Indianapolis, Ind.) for either 6 or 10 days. (MLC supernatant was 
generated by mixing together the peripheral blood lymphocytes of two 
patients, incubating the cells for 8-10 days, and collecting the 
supernatant from the cells.) Culture supernatants containing human 
antibodies were obtained by low speed centrifugation at 1,500.times.g. 
Culture supernatants (1.times. final concentration) were added to the 
syncytia assay described in Example 2 with the modification that TF228.1 
cells were preincubated with the culture supernatants containing human 
antibodies thirty minutes prior to adding SupT-1 cells. Syncytia were 
counted after twenty hours of incubation. The results of the syncytia 
assay are shown in Table 3. Very small syncytia indicate partial 
inhibition of syncytia formation. 
Specificity of human antibodies in the culture supernatants to HIV-gp120 
was measured by ELISA using recombinant gp120 from a Drosophila cell line 
as antigen. Purified gp120 was bound to the wells of a 96-well microtiter 
plate by incubating at 4.degree. C. for twenty four hours. After blocking 
with 1% BSA in PBS containing 0.02% NaN.sub.3 for one hour, culture 
supernatants were added to the wells and incubated for two hours. Antibody 
to gp120 was detected using peroxidase labeled goat-anti-human IgG, IgM 
and o-phenylenediamine. Results are represented in Table 3 as optical 
density readings at 460 nm. 
As shown in Table 3, after six days of in vitro immunization, the number of 
antibodies specific for gp120 was not significant at either cell ratio 
when compared to controls which did not contain culture supernatant and 
the number of syncytia counted was not significantly less than the number 
found in the controls. After ten days of in vitro immunization, the number 
of antibodies specific for gp120 was significantly higher at both cell 
ratios than the number found after six days and no syncytia formation 
could be detected. Thus TF228.1 cells expressing HIV-1 gp160 are effective 
in provoking antibodies that prevent syncytia formation in vitro. 
TABLE 3 
______________________________________ 
In Vitro Immunization of human PBM with TF228.1 cells 
Days of In Vitro 
Ratio Number of ELISA (gp120 
Immunization 
PBM:TF228.1 
Syncytia Drosophila) 
______________________________________ 
Day 6 50:1 48.sup.1 0.117 
100:1 69.sup.1 0.191 
Day 10 50:1 0 0.540 
100:1 0 0.361 
Control 1 44.sup.2 0.131 
Control 2 50.sup.2 0.089 
______________________________________ 
.sup.1 small syncytia 
.sup.2 large syncytia