U.S. patent number RE38,824 [Application Number 10/087,882] was granted by the patent office on 2005-10-11 for antibodies against human herpes virus-6(hhv-6) and method of use.
This patent grant is currently assigned to The United States of America as represented by the Secretary of the Department of Health and Human Services, The United States of America as represented by the Secretary of the Department of Health and Human Services. Invention is credited to Dharam V. Ablashi, Robert C. Gallo, Steven F. Josephs, Syed Z. Salahuddin, Carl W. Saxinger, Flossie Wong-Staal.
United States Patent |
RE38,824 |
Salahuddin , et al. |
October 11, 2005 |
Antibodies against human herpes virus-6(HHV-6) and method of
use
Abstract
A new human B lymphotropic virus, also designated human
herpesvirus-6, has been isolated. DNA, molecular clones, antigenic
viral proteins and antibodies having specificity to the new virus
have been prepared. Various utilities of the new virus and products
derived therefrom have been described.
Inventors: |
Salahuddin; Syed Z. (Ventura,
CA), Ablashi; Dharam V. (Lewes, DE), Josephs; Steven
F. (San Diego, CA), Saxinger; Carl W. (Bethesda, MD),
Wong-Staal; Flossie (San Diego, CA), Gallo; Robert C.
(Bethesda, MD) |
Assignee: |
The United States of America as
represented by the Secretary of the Department of Health and Human
Services (Washington, DC)
|
Family
ID: |
27578715 |
Appl.
No.: |
10/087,882 |
Filed: |
March 1, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
392674 |
Feb 22, 1995 |
5604093 |
|
|
|
754220 |
Aug 27, 1991 |
|
|
|
|
255712 |
Oct 11, 1988 |
|
|
|
|
228550 |
Aug 4, 1988 |
|
|
|
|
901602 |
Aug 29, 1986 |
|
|
|
|
892423 |
Aug 4, 1986 |
|
|
|
|
895857 |
Aug 12, 1986 |
|
|
|
|
895463 |
Aug 11, 1986 |
|
|
|
Reissue of: |
774118 |
Dec 23, 1996 |
06054283 |
Apr 25, 2000 |
|
|
Current U.S.
Class: |
435/7.1;
424/130.1; 435/345; 435/7.94 |
Current CPC
Class: |
C07K
14/005 (20130101); C12N 7/00 (20130101); C12N
2710/16521 (20130101); C12N 2710/16522 (20130101); C12N
2710/16551 (20130101) |
Current International
Class: |
C07K
14/005 (20060101); C07K 14/03 (20060101); C12N
7/00 (20060101); C12N 7/02 (20060101); G01N
033/53 (); C12N 005/06 () |
Field of
Search: |
;435/5,7.44,7.1,345,7.2
;434/130.1,387.1,388.3 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Middeldrop et al , Journal of Clinical Microbiology, Oct. 1984, pp.
763-771. .
Rodgers et al , Journal of general Virology, 1985, vol. 66, pp.
2045-2049. .
Lawrence et al , Journal of Virology, 1990, pp. 287-299. .
Albashi et al., "HBLV (or HHV-6) in human cell lines)", Nature vol.
329, No. 6136 (Sep. 1987) p. 1723. .
Lusso et al., "In vitro Cellular Tropism of Human B-Lymphotropic
Virus . . . ", J Exp Med. vol. 167 (May 1988) pp. 1659-1670. .
Rodriquez et al., Recombinant DNA Techniques: An Introduction,
(published 1983), pp. 74-76. .
Josephs et al., "Genomic analysis of the human B-Lymphotropic
virus", Science, vol. 24, pp. 601-603 (1986). .
Lawrence et al., "Human Herpesvirus 6 is Closely Related to Human
Cytomegalovirus," J. Virol. 64(1):287-299, 1990. .
Frankel-Conrat et al., Virology, Prentice-Hall, Englewood, N.J.,
1982, pp. 207-211. .
Barnes, "Mystery Disease At Lake Tahoe Challenges Virologists and
Clinicians", Science, 234-542, 1986. .
Salahuddin et al., "Isolation of a New Virus, HBLV, in Patients
with Lymphoproliferative Disorders," Science, 234:596-601, 1986.
.
Robert et al., "Detection of Antibodies to Human Herpesvirus-6
using immunofluoresence Assay," Res. Virol., 141:545-555,
1990..
|
Primary Examiner: Salimi; Ali R.
Attorney, Agent or Firm: Townsend and Townsend and Crew
LLP
Parent Case Text
This is a .Iadd.reissue of application Ser. No. 08/774,118 filed
Dec. 23, 1996 (now U.S. Pat. No. 6,054,283) .Iaddend..Iadd.which
was a .Iaddend.Division of application Ser. No. 08/392,674, filed
Feb. 22, 1995 (now U.S. Pat. No. 5,604,093), which was a
continuation of Ser. No. 07/754,220, filed Aug. 27, 1991 (now
abandoned), which was a continuation of Ser. No. 07/255,712, filed
Oct. 11, 1988 (now abandoned), which was a CIP of Ser. No.
07/228,550, filed Aug. 4, 1988 (now abandoned), which was a CIP of
Ser. No. 06/901,602, filed Aug. 29, 1986 (now abandoned), which was
a CIP of Ser. No. 06/892,423, filed Aug. 4, 1986 (now abandoned),
which was a CIP of Ser. No. 06/895,857 filed Aug. 12, 1986, (now
abandoned), which was a CIP of Ser. No. 06/895,463, filed Aug. 11,
1986, the disclosure of which is incorporated by reference.
Claims
What is claimed is:
1. An isolated antibody which specifically binds to an antigenic
molecule from an isolated human herpes virus .[.having.].
.Iadd.wherein said isolated human herpes virus has .Iaddend.the
morphology of a human herpes virus and a double-stranded DNA genome
of about 170 Kb, wherein genomic DNA from said isolated human
herpes virus .[.hybridizes under stringent conditions with.].
.Iadd.comprises a .Iaddend.nucleic acid .Iadd.sequence .Iaddend.of
molecular clone ZVH14 (ATCC Accession No. 40,247); and further
wherein said .[.first nucleic acid.]. .Iadd.antibody .Iaddend.does
not .[.hybridize under said stringent conditions with the nucleic
acid of.]. .Iadd.specifically bind to an antigenic molecule from
.Iaddend. (a) Epstein-Barr virus; (b) human cytomegalovirus (CMV);
(c) Herpes Simplex virus (HSV); (d) Varicella-Zoster virus (VZV);
or (e) Herpes virus saimiri.
2. A method of detecting .[.HHV-6.]. .Iadd.human
herpesvirus-6(HHV-6) .Iaddend.in a biological sample comprising the
steps of: (a) contacting the biological sample with the antibody of
claim 1, under conditions such that the antibody will specifically
bind to a human herpes virus antigenic molecule present in said
biological sample whereby a complex is formed of antibody and
antigenic molecule; and (b) detecting for the presence or absence
of the complex. .[.
3. The method of claim 2, wherein said method comprises a western
blot..]..Iadd.
4. A method of detecting in a biological sample an antibody that
specifically binds an antigen from an isolated human herpes virus,
said method comprising the steps of: (a) contacting the biological
sample with said human herpes virus antigen, under conditions such
that the antibody will specifically bind to the human herpes virus
antigen, whereby a complex is formed of antibody and human herpes
virus antigen; and (b) detecting the presence or the absence of the
complex, wherein said isolated human herpes virus has the
morphology of a human herpes virus and a double-stranded DNA genome
of about 170 Kb, wherein genomic DNA from said isolated human
herpes virus comprises a nucleic acid sequence of molecular clone
ZVH14(ATCC Accession No. 40,247); and further wherein said antibody
does not specifically bind to an antigenic molecule from (i)
Epstein-Barr virus; (ii) human cytomegalovirus (CMV); (iii) Herpes
Simplex virus (HSV); (iv) Varicella-Zoster virus (VSV); or (v)
Herpes virus saimiri. .Iaddend..Iadd.
5. The method of claim 4, wherein the biological sample is serum.
.Iaddend..Iadd.
6. The method of claim 4, wherein the biological sample is from a
patient. .Iaddend..Iadd.
7. The method of claim 4, wherein said method comprises an
immunofluorescence assay. .Iaddend..Iadd.
8. The method of claim 4, wherein said method comprises an ELISA.
.Iaddend..Iadd.
9. The method of claim 4, wherein the antigen is immobilized on a
solid surface before the step of contacting. .Iaddend..Iadd.
10. The method of claim 9, wherein the antigen is immobilized onto
nitrocellulose. .Iaddend..Iadd.
11. The method of claim 10, wherein said method comprises a Western
blot. .Iaddend..Iadd.
12. The method of claim 4, wherein the human herpes virus antigen
is present on an intact herpes virion. .Iaddend.
Description
The present invention is related generally to the isolation and
characterization of a new virus. More particularly, the present
invention is related to providing a biologically pure, isolated
human B lymphotropic virus, molecular clones, nucleic acids,
distinctive antigenic proteins and a method for detecting
antibodies to the new virus. A virus of the type as described
herein has not heretofore been known or characterized. The nature,
properties, importance and various utilities of the new virus are
now presented.
A virus, designated as human B-lymphotropic virus (HBLV or HHV-6
for human herpesvirus-6), was isolated from the peripheral blood
lymphocytes of six individuals: one HTLV-III(HIV-1) seropositive
patient with AIDS-related syndrome, 1 HTLV-III seropositive patient
with angio-immunoblastic lymphadenopathy, 1 patient with
dermatopathic lymphadenopathy, a patient with Mycosis fungoides, a
patient with immunoblastic lymophoma, and 1 patient(GS) with acute
lymphoblastoid leukemia (Table 1). All six isolates were closely
related by antigenic and molecular analysis, and sera from all 6
virus positive patients reacted immunologically with each virus
isolate (Table 1). In contrast, only 4 sera from more than 200
randomly selected healthy donors were seropositive. Subsequent
tests showed a high number of normal blood donors had titers to
HHV-6 (59.5%). It was found that HBLV contains a large
double-stranded DNA genome, and is morphologically similar to some
members of the human herpesvirus group. A detailed morphological
analysis of HBLV is given below.
It selectively infects freshly isolated human umbilical cord blood
lymphocytes, B-cells and T cells, where it induces the appearance
of characteristic large, retractile mononucleated or binucleated
cells containing nuclear and cytoplasmic inclusion. bodies. HBLV is
distinguishable from all known human and sub-human primate
herpesviruses by host range, biological effect on infected cells,
and by a lack of immunologic, antigenic and genomic relatedness
(Tables 2 and 3).
Despite morphological similarities, the host range of HBLV was
different from all other members of the human herpesvirus group.
For example, initial attempts to transmit the virus to a number of
T and B lymphoblastoid cell lines, and to a variety of other cell
types, were unsuccessful, but later tests showed that B- and
T-cells, megakaryocytes and neural cells could be infected with
HBLV. In contrast, Epstein-Barr virus (EBV) infects most B cells
and some epithelial cells. Furthermore, other herpesviruses [e.g.,
cytomegalovirus (CMV), Herpes Simplex I and II (HSV) and
Varicella-Zoster virus (VZV), infect a variety of cell types, often
inducing cytopathic effects. Immunological comparisons with EBV
further emphasized these differences. For example, no EBV nuclear
antigens were detected in HBLV-infected cord blood mononuclear
cells.
The virus of the present invention has been designated human
B-lymphotropic virus (HBLV) because the virus was initially
cultured from B-cells (the cells had cytoplasmic immunoglobulins)
because the virus initially infects B-cells in vitro in cord blood
cultures and because HBLV DNA sequences were found in only 3
lymphomas and all 3 were of B-cell origin. Comparative
morphological features which distinguish HBLV from other human
herpesviruses are listed in Table 4.
For the identification and isolation of HBLV, fresh peripheral
blood mononuclear cells from AIDS patients with associated
lymphoproliferative disorders were established in cell culture
(Table 1). In the cultures of eight patients, primary cell cultures
contained a small number of large, refractile mononucleated or
binucleated cells which survive for short periods of time. These
cells frequently contained intranuclear and/or intracytoplasmic
inclusion bodies. Electron microscope examination revealed that
these cells were infected by a DNA virus, 200 nm in diameter (FIG.
3). These large cells were also the only ones in culture expressing
viral antigens, as measured by fixed and unfixed cell indirect
immunofluorescence assays (IFA) (FIG. 2) and by in situ
hybridization (FIG. 1). All three virus-positive patients were
homosexual males (2 white and 1 black, between the ages of 35 and
44), who were seropositive for HTLV-III with AIDS-pneumocystic
pneumonia, with Kaposi's sarcoma, and with undifferentiated B-cell
lymphoma.
The presence of the unique large, refractile cells suggested the
need for further examination of patients demonstrating
morphologically similar cells in fresh tissues or culture.
HBLV from all six patients could be transmitted to freshly isolated
human leukocytes from umbilical cord blood, adult peripheral blood,
bone marrow, and spleen (previously stimulated with PHA-P
phytohemagglutinin-purified)). After in vitro infection the large
refractile cells, noted in primary cultures, appeared within 2-4
days post infection. These cells eventually became the predominant
cells in the culture, surviving for an additional 8-12 days. During
this time the other cells in the culture rapidly died. As in
primary cell cultures, these large cells expressed viral nucleic
acids as shown by in situ hybridization (FIG. 1), and viral
antigens as detected by IFA (immunofluorescent antibodies), (FIG.
2). Virus production was confirmed by electron microscopy (FIG. 3).
HBLV-infected cells were typed for surface markers defined by
specific monoclonal antibodies.
Molecular probes which were derived from HSV-1 (cross reactive with
HSV-2), CMV, EBV and VSV were used for comparisons with HBLV. While
each individual viral probe hybridized to its homologous nucleic
acids, HBLV was clearly distinct from these human herpesviruses
(FIG. 10). Furthermore, the size of the HBLV genome was shown to
contain a minimum complexity of 110 kb-pair as determined by
analysis of sucrose gradient purified viral DNA. Finer analysis
indicates the genomic size to be about 170 kb. This genome size, as
well as other features (such as morphology), also distinguished
HBLV from DNA viruses of the adenovirus, polyomavirus, papovavirus,
and papillomavirus groups.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the results of in situ hybridization of HBLV-infected
human cord blood cells using pZVH14 HBLV probe.
FIG. 2A is an immunofluorescent analysis of HBLV-infected acetone
fixed cells; FIG. 2B shows HBLV-infected live cells expressing
membrane fluorescence using HBLV antibody; and FIG. 2C shows
immunofluorescence of HBLV-infected cells with serum lacking HBLV
antibody.
FIG. 3 is an electron micrograph of HBLV showing extracellular
enveloped particles, the insert represents a virus particl showing
envelope, spikes, core, capsid and tegument.
FIGS. 4A, B, and C are electron micrograph of HBLV (negatively
stained).
FIGS. 5A and B are Southern blot analysis of HBLV genomic DNA,
lanes 1 and 2 are positive for HBLV and lane 3 is negative FIG. 5A:
Hind III digested HBLV genomic DNA. FIG. 5B: EcoRI digested HBLV
genomic DNA.
FIG. 6 shows HBLV proteins after radioimmunoprecipitation using a
positive patient (GS) serum and two dimensional (2D) gel
electrophoresis. HBLV specific proteins are indicated by arrows
according to apparent molecular size in KDa.
FIGS. 7A and B show the one dimensional (1D) gel electrophoresis
patterns of proteins recognized by human and rabbit anti-HBLV serum
by radioimmunoprecipitation FIG. 7A: 3 hrs .sup.35 S Cysteine
labeled HSB-2 infected cells. FIG. 7B: Identification of 120 kd
protein using HBLV positive serum.
FIG. 8 shows restriction enzyme hap of HBLV clone pZVH14.
FIGS. 9A and B show Western blot analyses of HBLV proteins FIG. 9A:
Concentrated HBLV from HSB 2 cells. FIG. 9B: HSB 2-Cell
Lysates.
FIG. 10 shows dot blot analysis of various herpesviruses, showing
specificity for the probes to their genomic DNA.
FIGS. 11A, B, and C show Southern blots using pZVH14 probe for
detecting HBLV in three human B-cell tumors FIG. 11A: HBLV
sequences in a follicular large cell lymphoma. FIG. 11B: Detection
of HBLV sequences in an African Burkitt tumor. FIG. 11C: Detection
of HBLV sequences in Multicentric Tumors arising in a Sjogren's
Syndrome patient.
FIG. 12 shows restriction enzyme bands generated using Eco R1 and
BamH1 as visualized on a 0.8% agarose gel using ethidium bromide
staining.
FIGS. 13A and B show restriction endonuclease comparison of a HBLV
isolate (HBLV Z29) obtained from the Center for Disease Control and
the prototype isolated HBLV (GS). Arrows show the restriction
enzyme differences in the EcoR1 restriction patterns between the
two isolates FIG. 13A Ethidium Bromide straining. FIG. 13B:
Hybridization of HHV6Z29 and HHV6 HBLV to HBLV probe pzVH14.
FIG. 14 shows the silver stained gel with enriched HBLV
proteins.
FIG. 15 is a Western blot of the gel run in parallel with gel of
FIG. 14. Clearly 120 and 72 KDa proteins are detected.
FIG. 16 is a map of HBLV clone pZVB70.
FIG. 17 shows HBLV infected human umbilical cord blood lymphocytes.
Large refractile infected cells are prominent.
Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
All publications mentioned hereunder are incorporated herein by
reference. Unless mentioned otherwise, the techniques employed
herein are standard methodologies well known to one of ordinary
skill in the art.
The term "substantially pure" as used herein means that the product
is as pure as can be obtained by standard methodology conventional
in the art.
Despite morphological and other properties similar to some of the
herpesviruses, human B lymphotropic virus (HBLV) is a new human
herpesvirus. It is distinguishable from other viruses by biological
properties and by the lack of immunological and genomic homology.
HBLV is highly lytic in vitro, as are CMV, HSV, HVS (Herpesvirus
Simia), and HVA (Herpesvirus ateles), but has a different host
range than these viruses. It is possible that HBLV could indirectly
cause abnormalities in B-cells leading to malignancy in vivo.
Even though in certain instances HBLV was associated with human
T-lymphotropic virus-III/lymphadenopathy-associated virus
(HTLV-III/LAV) seropositive donors, other evidence indicates that
it is not exclusively an AIDS-associated agent. Not only did all
HTLV-III seropositive patients have complicating
lymphoproliferative disorders, but HBLV was also isolated from a
HTLV-III seronegative ALL (acute lymphocytic leukemia) patient.
Furthermore, some seroepidemiological analyses have shown a
reactivity clearly dissociated from HTLV-III antibody positive
individuals.
Serological comparisons demonstrate the uniqueness of HBLV.
Immunofluorescence assay was developed following techniques
originally described for herpesviruses, and was used to analyze
patients and healthy control sera, and to monitor infected cells.
Sera from all six HBLV positive patients demonstrated an IgG
antibody titer to viral capsid antigens (>1:20). In contrast,
only 4 of the more than 200 sera from randomly selected healthy
donors were positive. Subsequent serological surveys indicate the
prevalence of HBLV antibodies in normal population to range from
about 9% to about 47% with regional differences. The pattern of
immunofluorescent staining in fixed, infected cells varied from
punctate nuclear staining to diffuse staining of the entire cell
(FIG. 2A). In live cells, the staining was confined to the cell
membrane either as a partial ring or in a capped form (FIG. 2B and
C). Uninfected cord blood mononuclear cells were negative when
tested with Sera from the 6 HBLV positive patients. Sera from these
positive patients also contained antibody to EBV and CMV. A careful
comparison of the titers of antibody to EBV, CMV, and HBLV yielded
a distinct titer for HBLV as compared to that for EBV and CMV.
Furthermore, the reactivity to EBV, CMV, HSV-1 and 2 and VSV was
completely removed by adsorption with disrupted, EBV-infected cells
or with purified viruses, without significantly affecting the
antibody titer to HBLV.
Sucrose gradient purification of HBLV. Heparinized peripheral blood
leukocytes or human umbilical cord blood mononuclear cells are
banded in Ficoll-Hypaque and established in cell culture at
36.degree. C. following PHA-P (5 ug/ml) stimulation for 48 hours.
The cells are then grown in RPMI-1640 medium supplemented with 10%
fetal bovine serum (heat inactivated, 56.degree. C. for 30 min.)
and 5 ug/ml hydrocortisone. Frozen supernatants obtained from the
infected cells are thawed, collected in 250 ml tubes and spun at
3500 rpm in a Sorvall GSA rotor at 5.degree. C. for 10 min. the
clarified supernatants are transferred to SW28 tubes and spun and
pelleted at 17,000 rpm for 90 min. at 5.degree. C. Pellets obtained
are resuspended in 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 1 mM EDTA
(TNE) to a volume of 300 microliters and layered onto a 15-60%
sucrose gradient and spun in an SW41 rotor (Beckman) at 20,000 rpm
for 30 min. at 5.degree. C. Fractions of 1 ml are collected from
the top of the gradient. Each fraction is diluted to 10 ml, spun,
and pelleted in an SW41 rotor at 17,000 rpm for 90 min. Pellets are
resuspended in 300 microliters of TNE and aliquots assayed (by
ELISA and Western Blot) for the presence of virus and for virus
infectivity. Human B Lymphotropic Virus is easily detected in
fractions 4-9 with a peak in fractions 5-7 by both assays.
Extraction of nucleic acids from each fraction shows the presence
of double stranded DNA in fractions 5-9 with a peak in fraction 7.
Virus is also detected by electron microscopy in the SW41 gradient
pellet as well. Virus purified from fresh unfrozen supernatants
according to this procedure is used for detailed electron
microscopy.
Aliquots of the sucrose gradient fractions can be definitively
assayed for the presence of HBLV by DNA dot blot analysis using the
pZVH14 9 kb insert (FIG. 8) as a probe. The pZVH14 molecular clone
is obtainable from the American Type Culture Collection under
Accession No. 40247.
The immunofluorescence, Western blot and radioimmuno-precipitation
assays are also employed for detecting HBLV infection and HBLV
antibodies in a variety of hematropoietic malignancies, including
B-cell lymphomas of both AIDS and non-AIDS origin. The presence of
HBLV antibodies is elevated in the following disease groups, but
the invention is not intended to be limited to these specific
diseases:
Roseola (Exanthum subitum)
Burkitt's lymphoma
Hodgkin's disease
Mononucleosis-like syndromes
Sarcoidoisis
Sjogren's Syndrome
A newly described infectious disease syndrome similar to that seen
in Lake Tahoe characterized as an "acute mononucleosis-like
syndrome" in adults, commonly known as chronic fatigue syndrome
(CFS).
ALL (acute lymphocytic leukemia) as diagnosed in children of
Japanese, Caribbean and African origin.
HIV-1 antibody positive AIDS, ARC and PGL (persistent generalized
lymphadenopathy) patients.
HBLV Virus Propagation. Infection of human umbilical cord blood or
peripheral blood mononuclear cells is conducted by cell-free
transmission as follows:
1) Fresh blood samples are diluted 1:1 with RPMI-1640 and spun (and
banded) on a Ficoll gradient.
2) The banded mononuclear cells are washed and put into culture in
the presence of PHA-P (5 ug/ml) and hydrocortisone (HC) (5 ug/ml)
in 20% fetal calf serum (FCS) and RPMI-1640.
3) After 24 hours, polybrene (2 ug/ml) is added to the culture and
after 6-24 hours, the cells are pelleted.
4) A one ml aliquot of freshly harvested or frozen infected culture
supernatant is added to the pellet and incubated at 37.degree. C.
for 1-2 hours, with frequent agitation.
5) Fresh medium [10% FCS and HC (5 ug/ml) in RPMI-1640] is then
added to the suspension, cultured, and incubated at 36.degree.
C.
6) Within 2-10 days post infection, the characteristic enlarged
refractile cells become visible. Supernatant is harvested at the
peak of infection as measured by immunofluorescence and by visual
observation of the culture for further transmission.
Cells infected by HBLV were also used to directly compare
immunological cross-reactivities with other human and nonhuman
primate herpesviruses using specific monoclonal antibodies,
hyperimmune sera, or sera from antibody positive control donors. As
summarized in Tables 2 and 3, monoclonal antibodies to EBV, CMV,
HSV, and hyperimmune sera to Rhesus CMV and African Green CMV, did
not react with HBLV-infected cells. Human sera possessing
anti-bodies to EBV, CMV, HSV, and VSV also did not react with
HBLV-infected cells. Furthermore, sera from several Old World and
New World primates, many of which had antibodies to nonhuman
primate herpesvirus (including EBV-like viruses and CMV), did not
show any cross-reactivity with HBLV-infected cells (Table 2).
Immunofluorescent Analysis of HBLV-infected cells. A modification
of the indirect immunofluorescence assay developed by Henle et al.
(J. Bacteriol. 91:1248-1256) for EBV was used for the detection of
antibody to HBLV capsid antigens. For this assay, HBLV-infected
cord blood mononuclear cells were isolated by Ficoll gradients to
remove dead cells. Uninfected human cord blood mononuclear cells
were used as controls. Uninfected and infected cells were washed 3
times for 10 minutes with PBS without Mg++Ca++, resuspended in PBS
containing Mg++Ca++, deposited on TEFLON coated slides, air dried,
and fixed in cold acetone for 10 minutes. Patient's sera (heat
inactivated at 56.degree. C. for 30 minutes and clarified by
centrifugation) were added to the acetone fixed cells, incubated in
a humidity chamber at 37.degree. for 40 minutes, washed with PBS,
air dried, and stained with affinity purified FITC conjugated
anti-human IgG (H and L) for 40 minutes. The cells were
counterstained with Evans blue (1:500 dilution to PBS) for 5 min to
further reduce background due to autofluorescence. The cells were
again washed as above, air dried, and mounted with IFA
immunofluorescence assay). mounting solution. Large cells with
greenish to yellow granular immunofluorescent and cytoplasmic
staining were scored as positive cells for HBLV. The example of
assays carried out 5 days post infection are shown in FIG. 2a.
Small cells in the background did not react with patient serum
(FIG. 2b with arrows).
As is shown in FIG. 2, detection of viral membrane antigen HBLV
infected as well as uninfected live cells (non-fixed) were washed 3
times in serum-free RPMI1640 medium and treated with patient's
serum for 30 minutes at 4.degree. C. The cells were again washed,
treated with affinity purified FITC anti-human IgG for another 30
minutes, washed in medium again and examined for membrane
fluorescence. HBLV infected cells showed surface markers when
tested with patient serum using the immunufluorescence technique
(FIG. 2b).
Southern blot analysis of HBLV genomic DNA. Supernatant fluid from
HBLV infected umbilical cord blood cells was layered onto 20%
glycerol cushions and pelleted by centrifuging at 25,000 rpm for 3
hr. in a Beckman SW41 rotor at 4.degree. C. The pellets were
suspended in TNE buffer (10 nM, Tris-HCl, pH 9; 100 mM, NaCl; 1 mM
EDTA), and extracted with PCI9 (Phenol:Chloroform:Isoamy alcohol;
50 mM Tris-HCl, pH9; 100:100:1:10::v:v:v:v) followed by
Chloroform:isoamy alcohol (24:1::v:v). Substantially enriched viral
DNA was precipitated by adding 2 volumes of 95% ethanol. DNA was
digested with Hind III and cloned into the Bluescribe vector
(commercially available from Vector Cloning Systems, Calif.).
Several clones obtained were prepared as radiolabeled probes and
screened for specificity of hybridization by Southern blotting to
HBLV infected human umbilical cord blood cell DNA and by in situ
hybridization to such infected cells. Results of hybridization of
HBLV clone pHV14 to DNA from pelleted virus digested with Hind III
and EcoR1 are shown in FIG. 5. Extracellular virus is shown in lane
1, virus infected human umbilical cord blood cells in lane 2 and
negative control DNA isolated from the skin of an AIDS patient in
lane 3. Clone pZVH14 scored positive in these assays and did not
hybridize to uninfected controls. The infected cell DNA shown in
lane 2 is isolated in substantially pure form after several rounds
of cell free virus transmission in human umbilical cord blood
cells.
In addition to the procedures described above, the following
specific methods and materials may also be employed.
Rather than using cord blood cells, HBLV can also be propagated by
infecting other suitable host cells such as HSB2 cells obtainable
from ATCC (CC1 1.20.1). HBLV(GS) strain was collected from 15 liter
cultures of infected HSB2 cells by continuous flow centrifugation
onto 10% to 60% sucrose gradients. Bands collected between 1.135
and 1.210 g/ml were pelleted at 20,000 rpm and resuspended in PBS
containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and 10 mM
MgCl.sub.2. The suspended virions were subjected to six strokes in
a Dounce homogenizer and 23 units per ml of RNAse free DNAse
(Boehringer-Mannheim) and incubated for 10 min. at 37.degree. C.
The total volume (2 ml) was layered onto 36 ml 5-30% dextran T10
gradient (w/w) in 0.5 mM phosphate buffer, pH7, and centrifuged in
a Beckman SW 27 rotor for 1 hr. at 20,000 rpm at 4.degree. C.
(Dolyniuk et al, J. Virol. 17:935, 1976). Fractions of 4 ml were
collected and a visible band was collected in fractions 7-9.
Examination of fraction 10 under the electron microscope revealed
highly enriched virions with very little cellular debris. Electron
microscope examination of virions filtered through 0.2%
polyvinylpyrollidone (PVP) treated 0.45 um Nalgene fitters also
gave excellent results and protein gel analyses showed a
purification indistinguishable from fractions 7-9 above by electron
microscopy (FIG. 3).
Purification of HBLV Genomic DNA
Infection and banding of the virions by continuous flow
centrifugation was as described herein supra. The sucrose-banded
virus was pelleted at 20,000 rpm in a Beckman SW27 rotor for 90
min. The virus was resuspended in 400 ul of TE buffer (20 mM
Tris-HCl, 1 mM EDTA) and 130 ul of 10% sodium lauryl sarkosinate
added. The viral lysate was incubated at .sub.60.degree. C. for 20
min. and then layered onto a 54% CsCl, 0.1 mg/ml ethidium bromide
solution and centrifuged in a Beckman SW50 at 45,000 rpm for 20 hr.
at 20.degree. C. The Viral DNA band (1/3 from the top of the
gradient) was visualized under UV illumination and removed by side
puncture with a needle and syringe. The HBLV DNA-CsCl aliquot was
extracted 5 times with equal volume of n-butanol and then dialyzed
against 2 changes of 1000 ml of TE buffer at 4.degree. C. Dialysis
membrane was placed over an Eppendorf tube and held in place with
an Eppendorf cap into which hole had been bored. The tube was
inverted and floated on the buffer for dialysis. DNA prepared in
this way was substantially pure to visualize the ethidium-stained
restriction digests on agarose gels and for the creation of plasmid
vector libraries. The DNA yield is usually greater than 30 ug per
25 liters of cell free supernatant depending on the extent of the
infection.
Labeling of Cells
Media for 24 hr. labeling incubations was prepared by mixing 8 ml
of methionine Free DMEM (D-Met) (Gibco), 2 ml of 50% fetal calf
serum in RPMI 1640 and 0.1 ml gentamicin (100.times.concentrated, 5
mg/ml). Media for 2-3 hr. labeling incubations contained D-Met and
10% fetal calf serum. The amount of 5 mCi of [.sup.35 S] methionine
(or other radiolabeled amino acid) was lyophilized and
reconstituted with 400 ul of D-Met. Cells in the amount of
5.times.10 were pelleted at 1000 rpm for 5 min. in the Sorvall GLC
bench top centrifuge and resuspended in labeling media. For 24 hr.
labeling, the cells were split into two 0.8 ml aliquots in a 24
well microtiter plate and 50 ul of the recombinant [.sup.35 S]
methionine was added to each. For 2-3 hour labeling, 5.times.10
cells were resuspended in 1.0 ml of labeling medium and split into
two 0.5 ml aliquots and 50 ul of radiolabeled methionine added to
each. Cells were incubated at 37.degree. under 5% CO.sub.2 and 85%
humidity for the period of time necessary for labeling.
Radioimmunoprecipitatior
After metabolic labeling, (as described herein supra) the cells
were diluted in 10 ml of ice-cold phosphate buffered saline (PBS)
and pelleted for 5 min. at 1000 rpm in the Sorvall GLC bench top
centrifuge, resuspended in 10 ml of fresh ice-cold PBS and pelleted
a second time. The cells were resuspended in 1 ml of PBS and
transferred to an Eppendorf tube and centrifuged at half maximal
speed for 2 min. About 550 ul of lysis buffer [0.1% SDS (sodium
dodecyl sulfate), 1% TRITON X-100,
(T-octylphenoxypolyethoxyethanol, Sigma Chemical Company, St.
Louis, Mo.) X-100, 1% desoxycholate (free acid), 20 mM Tris-HCl, pH
8.0, 150 mM NaCl and 1 mM phenylmethylsulfonyl fluoride (PMSF,
Sigma)] was added. The lysate was vortexed at a setting of 5 for 15
sec., allowed to sit on ice for 0 min. and vortexed again. The
samples were then centrifuged at top speed in an Eppendorf
centrifuge for 3 min. A 50 ul stock aliquot was removed from each
tube and immediately frozen on dry ice. The remaining supernatant
was transferred to a clean Eppendorf tube and 20 ul of sera was
added. The tubes were placed on a rotor at 4.degree. C. and gently
inverted for 12 hr. The samples were then centrifuged at top speed
for 2 min. and all but 10 ul of the supernatant was removed to a
new Eppendorf tube. The amount of 100 ul of a 50% (v/v) slurry of
protein A SEPHAROSE, beaded agarose (Pharmacia) in lysis buffer was
added to each tube and the tubes gently inverted for 30 min. The
samples were centrifuged for 2 min. at top speed and the
supernatants discarded. The protein A SEPHAROSE, beaded agarose
pellet was washed 6 times by resuspension in lysis buffer and
centrifuged for 15 sec. at top speed. After removal of the
supernatant of the sixth wash, the pellet was frozen and sent to
Protein Data Bases, Inc. (a commercial analytical service
laboratory in Huntington Station, N.Y.) for the gel runs.
All radioimmunoprecipitations were performed using serum from
patient GS, the source of the prototype HHV-6 isolate. Specificity
of the antisera was demonstrated by adsorbing the sera against
virion preparations of human cytomegalovirus, Epstein-Barr virus,
Varicella Zoster virus, and Herpes Simplex type 1.
High Resolution 2 Dimensional Gels (HR2D) of HHV-6 Proteins
The viral proteins were prepared by SDS-BME (sodium dodecyl
sulfate-basic maintenance emulsion) lysis of gradient-banded
virions and RNA-DNAse treatment as described herein supra and then
frozen on dry ice according to the standard protocols of Protein
Data Bases Incorporated (PDI), Huntington Station, N.Y. The samples
were run at PDI on 12.5% broad range non-equilibrium and
equilibrium polyacrylamide gels and silverstained. The protein-A
Sepharose bound radiolabeled immunoprecipitates run on 12.5% broad
range non-equilibrium polyacrylamide gels were then exposed for
autoradiography at PDI.
It should be noted that in addition to radioimmunoprecipitation
(RIP), Western blot, indirect immunofluorescence assay (IFA) enzyme
linked immunosorbent assay (ELISA), and the like can also be
utilized to detect viral antigens or antibodies. These techniques
are well established and known to one of ordinary skill in the art
to which this invention belongs.
HR2D Western Blotting
Immunoblotting was performed after HR2D electrophoretic resolution
of fractions of HBLV prepared from sucrose gradients or filtered
virus as described herein supra. The nitrocellulose sheets were
stored at 20.degree. C. prior to use. Sheets were incubated for one
hour in a blotte solution of 4% normal goat serum, 4% fetal bovine
serum, 5% non-fat dry milk and 0.02% thimerosal for blocking.
Sheets were then incubated with serum from a known HBLV infected
patient, diluted 1:1000 in the blotto solution. After 3 successive
5 minute washes with PBS, they were reacted in sequence for 1 hour
with 1:500 dilution of affinity purified goat anti-human IgG
labeled with biotin and for one hour with 1:1000 of horseradish
peroxidase streptavidin (Kirkegaard and Perry Labs., Inc.,
Gaithersburg, Md.) at room temperature (about 21.degree.-25.degree.
C.) in 5% normal goat serum in PBS and 0.02% Thimersol. A stock
solution of 4-chloronaphthol (4CN stock) was prepared by dissolving
0.3 g of 4-chloronaphthol in 100 ml of methanol. Staining was
carried out in a solution containing 2 ml of 4CN stock, 8 ml of PBS
and 4 microliters of hydrogen peroxide. The reaction was stopped by
washing with distilled water.
In order to obtain a better size estimate, DNA was purified from
virus collected by continuous flow centrifugation from 15 liters of
HBLV infected HSB2 cell culture supernatant and pooled. The regions
of the 10% to 60% sucrose gradient pooled were from 1.14 to 1.17
g/ml, fraction A, and from 1.17 to 1.21, fraction B. The virions
were pelleted, lysed and the DNA purified by banding on cesium
chloride gradients and dialyzed. FIG. 12 shows the restriction
enzyme bands generated using EcoRI and BamHI as visualized on 0.8%
agarose gels by ethidium bromide staining. Over 26 bands were
generated by EcoRI digestion (A to Z, top to bottom) and at least
15 fragments with BamHI (A to O). The bands seen were of similar
intensity with a marked absence of submolar fragments, compared to
other herpesviruses. Possible exceptions were the EcoRI A' and the
BamHI F' and M' fragments which had intensities equivalent to 1/4
M. The reasons for the generation of these bands are not
understood; however, they are possibly due to genomic inversions
and were not counted for the genome size estimates. Table 5 shows
the results of restriction enzyme analyses of HBLV.
The construction of BamHI plasmid libraries from the DNA showed
that nearly 100% of the fragments cloned were HBLV thereby
providing further evidence that the bands visualized in FIG. 12 can
be used as a reliable estimate of the HBLV genome size. The
molecular weights of the fragments listed in Table 5 gave genome
size estimates of 168,000 and 172,000 for the EcoRI and BamHI
digests, respectively. By this estimate, the genome of HBLV is
approximately the size of the Epstein-Barr virus genome.
Restriction endonuclease comparison of another independent HBLV
isolate, HBLV(Z29), to the prototype HBLV (GS) strain is shown in
FIG. 13. The arrows indicate the areas where the EcoRI digests of
each stain differ as visualized by ethidium bromide staining.
Hybridization to one HBLV probe, ZVH14,revealed identical
restriction patterns between the two isolates; however, by probe
ZVB70, the HBLV(GS) BamHI B fragment, showed differences (not
shown). This indicates that restriction site heterogeneity can be
observed among different isolates of HBV-6. Another isolate, the
HBLV(DV), was identical by hybridization with both the ZVH14 and
ZVB70 probes to HBLV (GS).
Studies of the complexity of the enveloped HHV-6 proteins were
attempted by banding the virus collected by continuous flow
centrifugation on DEXTRAN T-10 gradients similar to methods used to
purify the proteins of the enveloped EBV (Dolyniuk et al, 1976,
supra). The virions obtained from continuous flow centrifugation
were pelleted, treated with DNAse 1 and then banded on 10% to 30%
DEXTRAN T-10 gradients. The various fractions collected from the
top were analyzed by electron microscopy and the virus was pelleted
from those which looked relatively free of cellular debris. A viral
band was seen toward the bottom of the gradient (fractions 7-9) and
the fraction immediately below (fraction 10) was considered to be
relatively free of cellular debris when compared to virus obtained
after a single banding. The virus was found in clusters with little
cellular material. Virus prepared by this method when inoculated
into rabbits resulted in the generation of HBLV specific antibodies
in 14 days which were readily detected by indirect
immunofluorescence assay on infected cells. Subsequent bleeds gave
some non-specific cellular background in IFA tests. Hence, the
animals should be bled about 14 days post inoculum. These
antibodies can be utilized for detection of HBLV by established
techniques.
The pure preparations of the virus as revealed by electron
micrographs, were used to determine the proteins by direct
visualization on high resolution two dimensional polyacrylamide
gels (HR2D). As mentioned herein supra by procedures developed at
Protein Data Bases, Inc., several 12.5% broad range
non-equilibrium, gels were run and a gel of the virions obtained
from the DEXTRAN T-10 fraction 10 was silverstained (FIG. 14). A
parallel gel was run and Western blotted using GS serum and
peroxidase conjugated goat anti-human antibody. Two major proteins
were detected at 120 and 72 kDa as shown in FIG. 15.
Radioimmunoprecipitation of HBLV-infected cell lysates with the GS
serum showed several additional proteins (FIG. 6). In addition to
the proteins detected by Western blots, radioimmunoprecipitations
performed on proteins from lysates of metabolically labeled cells
showed a protein at 120 kDa: however, additional major proteins at
200, 80 and 19 kDa, as well as some minor proteins at 60 kDa, 80
kDa and several in the 30 kDa range were also detected. Two forms
of the 19 kDa proteins were observed, a more acidic form, 19a, and
a more basic major form, 19b, possibly due to differences in
phosphorylation (FIG. 6). The antigen proteins can then be isolated
in substantially pure form following standard purification
techniques, such as column chromatography, HPLC, preparative gel
electrophoresis, and the like. These proteins can be identified,
for example by Western blot using HBLV antibody positive sera. A
pharmaceutical composition in accordance with the present invention
comprises an immunogenic amount of the antigenic protein in a
pharmaceutically acceptable carrier. Antigenic proteins or portions
thereof can also be obtained from gt11 expression libraries or the
like.
Antigenic proteins of the present invention also allow detection of
the presence of HBLV antibodies in a biological sample by reacting
said sample with the viral antigens, a positive, antigen-antibody
complex formation being indicative of HBLV infection.
Antigen-antibody reactions can be detected by any standard
immunological techniques well known to one of ordinary skill in the
art, such as radiommuno, Western blot, ELISA, immunofluorescence,
histoimmunological tests and the like.
EXAMPLES
Example 1
Fresh tissue sections from 3 patients were found to contain a low
number of HBLV-infected cells. One patient, a 40 year old Hispanic
with a history of IV drug use, was seropositive for both HTLV-I and
HTLV-III, and was diagnosed with AIDS-pneumocystic pneumonia with
associated dermatopathic lymphadenpathy. Another was a 61 year old
white male who received multiple blood transfusions in conjunction
with open heart surgery 4 years prior to death. This patient was
seropositive for HTLV-III and was diagnosed with immunoblastic
lymphadenopathy with some skin involvement. A third patient (GS)
was a 16 year old black male diagnosed with acute lymphocytic
leukemia of the T-cell type. Unlike the others, this patient was
seronegative for HTLV-III. Primary peripheral blood mononuclear
cell cultures from these patients also contained a small number of
the unique cells which, upon close examination, were also found to
be infected by HBLV.
Example 2
A direct comparison of molecularly cloned sequences of the HBLV
genome with the genomes of other herpesviruses was also conducted.
Several DNA clones obtained from nucleic acids extracted from
purified virus were examined for specificity and for comparison
with other DNA viruses. Two HBLV clones, designated, pZVH14 (FIG.
8) and pZVB70 (FIG. 16, ATCC No. 40473), were used in these
studies. Southern blot analysis (FIG. 5) showed the presence of
viral specific DNA in Hind III and EcoRI digests of DNA from both
purified virus and HBLV-infected human cord blood cells. In situ
hybridization experiments with the pzVH14 probe also confirmed that
these sequences were confined to the infected cells (FIG. 1).
Example 3
Monoclonal antibodies and hyperimmune sera prepared against human
and simian herpesviruses were tested for reactivity with HBLV
infected cells by indirect immunofluorescence procedures as
described herein above. Monoclonal antibodies to EBV and HCMV were
used at 1:40 dilution; HSV-I and II, VZV and HVS at a 1:10 dilution
and normal ascites fluid was used as 1:5 and 1:10 dilutions.
Hyperimmune sera to African green and Rhesus monkey CMV were heat
inactivated (50.degree. C. 30 min.), clarified at 10,000 rpm, and
then were used at 1:10 dilutions. In addition to the sera shown,
human sera containing antibodies to EBV, CMV, HSV-I and II, and VZV
also did not react with HBLV infected cells. African green monkey
and Rhesus sera containing antibody to CMV were also negative when
tested with HBLV. Monoclonal antibodies to EBV and HCMV, and
ascites fluid from normal mouse were gifts from Dr. Gary Pearson,
School of Medicine, Georgetown University, Washington, D.C.
Monoclonal antibodies to VZV and HVS were obtained from Dr. Nancy
Chang, Baylor College of Medicine, Houston, Tex., and Dr. John
Dahlberg, NCI, Bethesda, Maryland, respectively. HSV-I and II
monoclonal antibodies were purchased from Dupont, Boston, Mass.
Hyperimmune serum to purified African green and Rhesus CMV were
previously prepared in rabbits by Dr. Ablashi. The specificity of
the serum containing antibodies to HBLV was shown by adsorbing it
against the other human herpesviruses (either whole virus or
infected cells).
Abbreviations used: HBLV, Human B lymphotropic virus; EBV,
Epstein-Barr virus; HCMV, Human cytomegalovirus; HSV, Herpes
simplex virus; VSV, Varicella-Zoster virus; HVS, Herpes virus
saimiri, VCA (Viral capsid antigen); EA, early antigen; MA,
membrane antigen.
HBLV infected cord blood mononuclear cells were stained with an
HBLV negative serum resulting in a considerable number of large
cells with no immunofluorescence.
Example 4
Serum from Old World and New World primates were tested for
antibody to HBLV by indirect immunofluorescence as described.
Some sera from the Old World primates were gifts from Dr. P. Kanki,
Harvard School of Public Health, Boston, Mass. All sera were heat
inactivated at 50.degree. C. for 30 minutes, and clarified by
centrifugation before use. HBLV-infected cord blood leukocytes,
P3HR-1 (an established cell line expressing EBV-VCA), and Owl
monkey kidney cells infected by HSV-strain II were used for
comparisons. When infected cells showed cytopatic effects, the
cells were fixed in acetone and used for the IFA test.
Three owl monkeys and one cottontop marmoset were previously
inoculated with HVS. Sera from these animals possessed antibody to
HVS late antigen which cross-reacted with Herpesvirus ateles. The
results are presented in Table 2.
Example 5
In situ hybridization of HBLV-infected human cord blood cells.
Tests were performed utilizing .sup.35 S-labeled RNA probes as
described herein supra. Clone pZVH14 of the HBLV genome was used as
a template for radiolabeled RNA using T7 RNA polymerase, .sup.35
S-labeled GTP, and unlabeled ribotriphosphates. Less than one grain
per cell was observed in uninfected negative control cultures.
Large retractile cells characteristic of the infected cultures were
heavily labeled, indicating the expression of abundant viral
messages (FIG. 1).
Example 6
Two dimensional gel electrophoresis patterns of proteins recognized
by human sera against human B cell lymphotropic virus (HBLV) are
shown in FIG. 6. Human umbilical cord blood lymphocytes or
HSB.sub.2 cells were infected with HBLV and then labeled by
incubation with .sup.35 S-methionine for periods of either 3 hours
or 24 hours. H9 cells were used as negative controls. The labeled
cells were lysed and the proteins immunoprecipitated according to
established procedures (Protein Data Bases, Inc., New York). Spots
seen on the gels of the lysates from infected cells but not seen on
the control gels represent candidate virus proteins arrayed in
unique virus specific patterns. These patterns serve as a
fingerprint which can specifically identify HBLV. The proteins
detected are antigenic proteins, the coding sequence of which can
be cloned and expressed, and the purified proteins thus obtained
can be used as diagnostic reagents.
Preparation of the Clones
Of course, the availability of the biologically pure HBLV and its
DNA, allows the preparation of the clones of HBLV. A general method
of cloning the Human B Lymphotropic Virus (HBLV) genome involves
isolating viral DNA after infection of suitable host cells (such as
HSB.sub.2 and the like), primary cells or cord blood cells with the
HBLV virus. The unintegrated viral DNA is then cloned in a suitable
cloning vector such as a plasmid or a lambda phage to create
libraries which can be screened for the presence of viral specific
DNA fragments.
Infected cells and cultured peripheral cord blood cells produce
HBLV virus and serve as the principal source of the virus for
immunological assays and the like for detecting virus-specific
antigens and antibodies in human sera. Cultures of infected cells
are grown and the virus harvested from the supernatant and the high
molecular weight DNA extracted from the virus. This produces viral
DNA containing the HBLV genome of the present invention. This DNA
is then subcloned in a suitable plasmid to produce a clone. A
complete description of the procedures for preparing clones can be
found in such standard publications as Maniatis et al: "Molecular
Cloning," Cold Spring Harbor, N.Y.
Two elements of the above process are well known are a part of the
recombinant DNA procedures: the DNA library and the differential
screening of DNA inserts to infected and uninfected cells. The
library is formed by taking the total DNA from the enriched or
purified virus DNA, cutting the DNA into fragments with suitable
restriction enzyme(s), joining the fragments to plasmid vectors,
and then introducing the recombinant DNA into a suitable host. The
viral specific DNA fragments are distinguished by their
hybridization to infected cell DNA and/or by in situ hybridization
to infected cells but not to uninfected cells.
As shown herein infra, a molecular clone, pZVH14, of the HBLV
genome is useful as a template for radiolabeled RNA using T7 RNA
polymerase, .sup.35 S-labeled GTP, and unlabeled
ribotriphosphates.
In the preferred embodiment of the present invention, supernatant
fluid from HBLV infected cells is layered onto 20% glycercol
cushions and pelleted by centrifuging at 25,000 rpm for 3 hr. in a
Beckman SW41 motor at 4.degree. C. The pellets are suspended in TNE
buffer (10 mM, Tris-HCl, pH 9; 100 mM, NaCl; 1 mM EDTA), and
extracted with PCI9 (Phenol:Chloroform:isomayl alcohol; 50 mM
Tris-HCl, pH9; 100:100:1:10 v:v:v:v) followed by chloroform:isoamyl
alcohol (24:1::v:v). Enriched viral DNA is precipitated by adding 2
volumes of 95% ethanol DNA is digested with Hind III and cloned
into the Bluescribe vector (commercially available from Vector
Cloning Systems, CA). Several clones obtained after screening with
labeled, enriched, DNA were examined for specificity of
hybridization to the HBLV DNA and by in situ hybridization to HBLV
infected cells.
Clones pZVH14 and pzZVB70 which were thus produced, scored positive
when tested by hybridization techniques and did not hybridize to
uninfected controls. The infected cell DNA is isolated after
several rounds of cell free virus transmission in human umbilical
cord blood cells or HSB.sub.2 cells. Clone pzVB70 was obtained from
CsCl gradient banded DNA of sucrose banded virus. DNA was BamH1
digested as described herein supra.
It is noted that these probes, either alone or in combination, can
be employed for detecting the viral DNA or RNA and virus-infected
cells containing HBLV nucleic acids by any of several standard
techniques well known to one of ordinary skill in the art. Examples
of such well established techniques are Southern and dot-blot for
DNA analysis, Northern blot for RNA analysis and in situ
hybridization. Furthermore, a probe for in situ by hybridization
can be made by any of well established procedures such as
radiolabeling or covalent linkage of hapten or enzyme to DNA. A few
illustrative examples are now provided.
Example 7
Several DNA clones obtained from nucleic acids extracted from
purified virus obtained as described above, were examined for
specificity relative to other DNA viruses. HBLV clone designated
pZVH14, contained a 9.0 kb Hind III fragment. Souther blot analysis
showed the presence of viral specific DNA in Hind III and EcoRI
digests of DNA from both purified virus and HBLV-infected human
cord blood cells. In situ hybridization tests with the same probe
also confirmed that these sequences were confined to infected
cells.
Example 8
Human B Lymphotropic Virus clone pZVH14 has been restriction enzyme
mapped as shown in FIG. 8.
Example 9
Similarly, HBLV clone pZVB70 has been restriction enzyme mapped as
shown in FIG. 16.
It is noted that based on the sequence information, any number of
specific clones can be generated and used as probes. The techniques
are well established and known to one of ordinary skill in the art
to which this invention belongs.
Example 10
In situ hybridization HBLV-infected cells. Tests were conducted
utilizing .sup.35 S-labeled RNA probes as described herein supra.
Clone pZVH14 of the HBLV genome were used as a template for
radiolabeled RNA using T7 RNA polymerase, .sup.35 S-labeled GTP,
and unlabeled ribotriphosphates. Less than one grain per cell was
observed in uninfected negative control cultures. Large retractile
cells characteristic of the infected cultures were heavily labeled,
indicating the expression of abundant viral messages (FIG. 1).
Example 11
Based on the nucleotide sequence, polymerase chain reaction
technique (Saiki et al, 1985, BioTechnology, 3:1008; Science,
230:1350) was employed to obtain increased levels of nucleic acids
from specimens (tissue or cell culture) suspected of HBLV infection
from diseased and normal A (control) populations and the presence
of HBLV detected by Southern blotting of the amplified HBLV DNA or
other method of detecting the amplified DNA with radiolabeled or
nonradiolabeled probes as are well known to one of ordinary skill
in the art.
A deposit of the clones pZVH14 and pZVB70 have been made at the
ATCC, Rockville, Md. under the accession numbers 40,247 and 40,473,
respectively. The deposit shall be viably maintained, replacing if
it becomes non-viable, for a period of 30 years from the date of
the deposit, or for 5 years from the last date of request for a
sample of the deposit, whichever is longer, and made available to
the public without restriction in accordance with the provisions of
the law. The Commissioner of Patents and Trademarks, upon request,
shall have access to the deposit.
In summary, as demonstrated herein, high level production of HBLV
can now be obtained by the use of the HSB2 or other cell lines.
This allows purification of the enveloped virus and the viral
nucleic acids. Purification of the viral DNA has been demonstrated
by hybridization with specific cloned viral DNA such as clone
paVH14. Although the size estimate (170,000) of the HBLV genome is
similar to that of EBV, evidence by molecular hybridization shows
distant relationships to the human cytomegalovirus and to the
Marek's disease cirrus of chickens (data not shown).
Comparison of the Western blots from the HR2D gels to the
radio-immunoprecipitation revealed a major antigenic protein of 120
kDa and other antigenic proteins described herein supra which is
detectable by both (RIP and Western blot) methods. The 120 Kd
protein seems to be a major antigenic protein as demonstrated by
anti-HBLV patient sera. Increased resolution of the minor proteins
on 2D gels indicates that it would be easier to verify the presence
of characteristics viral proteins by this method than by 1D gels.
Very clean background seen in the two dimensional Western blot, in
which 120 Kd and 72 Kd proteins were detected, may be the method of
choice.
Although not necessary, because biologically pure virus can be
obtained by following the standard procedures described herein by
anyone of ordinary skill in the art, nevertheless a deposit of the
isolated virus has been made at the ATCC, Rockville, Md. under
accession number VR2225. A deposit of the anti-HBLV positive serum
has also been made at the ATCC under accession number 40476. The
deposits shall be viably maintained, replacing if it becomes
non-viable, for a period of 30 years from the date of the deposit,
or for 5 years from the last date of request for a sample of the
deposit, whichever is longer, and made available to the public
without restriction in accordance with the provisions of the law.
The Commissioner of Patents and Trademarks, upon request, shall
have access to the deposit.
A diagnostic kit in accordance with the present invention comprises
containers separately containing anti-HBLV antibodies, one or more
purified or cell associated antigenic viral protein(s) produced by
HBLV in any part of its replicative cycle (i.e., HBLV infected
cells or cells expressing specific HBLV proteins); HBLV specific
nucleic acid probes; positive and negative controls and
instructional material to perform diagnostic test employing said
antibodies, antigenic viral protein(s), probes and the like. Of
course, the present invention also allows the detection of HBLV
present in any biological sample. Any suitable method mentioned
herein can be utilized as deemed most appropriate by one of
ordinary skill in the art, depending on such factors as the
location, nature, amount of the sample available and the like.
It is understood that the examples and embodiments described herein
are for illustrative purposes only and that various modifications
or changes in light thereof will be suggested to persons skilled in
the art and are to be included within the spirit and purview of
this application and scope of the appended claims.
TABLE 1 ISOLATION OF HBLV FROM PERIPHERAL BLOOD LYMPHOCYTES OF
PATIENTS WITH LYMPHOMA AND LYMPHADENOPATHY Serology* HBLV Patient
Description HTLV HBLV Isolation** 1 RC 29 WM AIDS +III 1:80 + KS B
cell lymphoma 2 HA 57 WM OHS 1:40 + AILD 3 PD 40 WM Dermatopathic
+II 1:80 + lymphadeno- and pathy; IVDA +III T8* skin infiltrate 4
GS 17 BM T-cell ALL -- 1:160: + (T-4*) 5 RW 66 BM Mycones -- 1:80 +
Pungtides (T-4*) Outaneous T- cell Lymphoma 6 BD 35 BF
Immunoblastic -- 1:80 + Lymphoma *Serology was done by indirect
immunofluoracetate using as standard a reference virus isolated
from patient GS. **PBL from patients were cultured as the primary
source of virus. Virus particles were transmitted to fresh human
cord blood. Positive cultures were identified by morphology IF, and
EM. ***Definitions: KS - Kaposi's sarcoma, AILD -
angio-immunoblastic lymphadenopathy, IVDA - intravenous drug
abuses.
TABLE 2 Cross-Reactivity of Nonhuman Primate Sera Virus Used to
Infected Target Cells EBV HBLV No. Positive RSV No. Positive
(VCA)/No. No. Positive Serum No. Tested Tested No. Tested Sources
(Percent Positive) (Percent Positive) (Percent Positive) Old World
Primate Chimpanzee 0/5 (0) 5/5 (100%) 0/4 (0) Gorilla 0/3 (0) 2/3
(66.6%) 0/3 (0) Orangutan 0/2 (0) 1/2 (50%) 0/2 (0) Baboons 0/3 (0)
3/3 (100%) 0/3 (0) Stumptail 0/2 (0) 1/2 (50%) 0/2 (0) Rhesus 0/9
(0) 6/9 (66.6%) 0/7 (0) African 0/10 (0) 6/10 (60%) 0/10 (0) Green
New World Primates Squirrel 0/10 (0) 0/10 (0%) 8/10 (80) monkeys
Owl 0/6 (0) 0/6 (0%) 3/6 (50) monkeys Marmouets 0/6 (0) 0/6 (0) 0/6
(0) (common) Marmouet 0/3 (0) 0/3 (0) 1/3 (33.3) (cottontop) ZHV14
on ZVB70 probe, anti-HBLV antibodies or purified HBLV
TABLE 3 Immunological Cross Reactivities of HBLV to Other Human and
Nonhuman Primates Herpesviruses Antibody Viruses Used to Infect
Target Cells HSV- Af. 1 Gr. Rhesus Source HBLV EBV HCMV and II VZV
HVS CMV CMV ERV - + - - - - - - Mono- clonal Anti- body (VCA, EA,
MA) HCMV - - + - - - - - Mono- clonal Anti- body (VCA and EA) HSV I
- - - + - - - - and II Mono- clonal Anti- body (early and late
anti- gens) VZV - - - - + - - - Mono- clonal Anti- body (late anti-
gens) HVS - - - - - + - - Mono- clonal Anti- body (late anti- gens)
Af. - - - - - - + - Green Monkey CMV (hyper- immune serum) Rhesus -
- - - - - - + Monkey CMV (hyper- immune serum)
Morphologic comparison of HBLV with other herpes viruses Feature
HBLV HSV* HCMV (6) EBV (7) Diameter of 60-80 nm 50-70 nm 64.3 nm 48
nm nucleoid Diameter of 95-105 nm 95-110 nm 106.4 nm 80 nm capsid
Symmetry of Icosahedral Icosahedral Icosahedral Icosahedral capsid
No. of 162 162 162 162 capsomeres in capsid Thickness of Dense,
Often Dense, Variable, tagument prominent, indistinct, prominent,
20 nm 25-40 nm 20-40 nm 24.4 nm Diameter of 160-200 nm 150-200 nm
174 nm 120 nm enveloped vision *HSV used for this comparison was
prepared simultaneously and under identical conditions as HBLV.
TABLE 5 BcoR1 BarH1 Fragment MW (kb) Fragment MW (kb) A 20.0 A 40.0
B 17.0 B 30.0 C 16.0 C 23.1 D 10.5 D 13.5 E 8.0 E 11.8 F 7.7 F 10.9
G 7.4 G 8.5 H 6.6 H 6.5 I 6.3 I 6.2 J1, J2 5.9 J 5.95 K 5.4 K 5.6 L
5.0 L 3.4 M 4.5 M 2.6 N 4.36 N 2.05 O 3.85 O 1.95 P 3.75 172.05 Q
3.5 R 3.25 S 3.05 T 2.95 U 2.5 V 2.4 W 2.3 X 2.25 Y 2.1
* * * * *