-
- Take a good look...read what your tax dollars have patented.
YOU own this.
- Imagine it being used on the American public or any people
on the planet
- as a weapon of mass illness and death. Imagine...
- ------------------------------------------------------------------------
- United States Patent 5,242,820
- Lo September 7, 1993
- ------------------------------------------------------------------------
- Pathogenic mycoplasma
- Abstract
-
- The invention relates to a novel pathogenic mycoplasma
isolated from
- patients with Acquired Immune Deficiency Syndrome (AIDS)
and its use in
- detecting antibodies in sera of AIDS patients, patients
with AIDS-related
- complex (ARC) or patients dying of diseases and symptoms
resembling AIDS
- diseases. The invention further relates to specific DNA
sequences,
- antibodies against the pathogenic mycoplasma, and their
use in detecting
- DNA or antigens of the pathogenic mycoplasma or other
genetically and
- serologically closely related mycoplasmas in infected
tissue of patients
- with AIDS or ARC or patients dying of symptoms resembling
AIDS diseases.
- The invention still further relates to a variety of different
forms of
- vaccine against mycoplasma infection in humans and/or
animals.
- ------------------------------------------------------------------------
- Inventors: Lo; Shyh-Ching (Potomac, MD)
- Assignee: American Registry of Pathology (Washington,
DC)
- Appl. No.: 710361
- Filed: June 6, 1991
-
- Current U.S. Class: 435/252.1; 435/5; 435/872
- Intern'l Class: C12N 005/00; C12N 005/02; C12N 001/00;
C12Q 001/70
- Field of Search: 435/870,5,872,240.2
- ------------------------------------------------------------------------
- References Cited [Referenced By]
-
- Other References
-
-
- Marquart et al (1985) Mycoplasma-Like Structures . .
. Eur J Clin Microbiol
- 4(1):73-74.
- Lo et al (1989) A Novel Virus-like Infectious Agent .
. . Am J Trop Med Hyg
- 40(2):213-226.
- Lo et al (1989) Identification of M Incognitus . . .
Am. J. Trop-Med. Hyg
- 41(5):601-616.
- Lo et al (1989) Association of the Virus-like Agent .
. . Am J Trop Med Hyg
- 41(3):364-376.
- Lo et al (1989) Fatal Infection of Silvered Leaf Monkeys
. . . Am. T Trop
- Med Hyg 40(4):399-409.
- Lo et al (1989) Virus-like Infectious Agent . . . Am
J Trop Med Hyg
- 41(5):586-600.
- Marquart et al (Feb. 1985) Abstract Only Eur J Clin Microbiol
4(1):73-74.
- Hu et al (1990) Gene 93:67-72.
- Primary Examiner: Nucker; Christine M.
- Assistant Examiner: Preston; D. R.
- Attorney, Agent or Firm: Venable, Baetjer, Howard &
Civiletti
- ------------------------------------------------------------------------
- Goverment Interests
- ------------------------------------------------------------------------
-
- The invention described herein was made in the course
of work under a grant
- or award from the United States Department of the Army.
- ------------------------------------------------------------------------
- Parent Case Text
- ------------------------------------------------------------------------
-
- CROSS-REFERENCE TO RELATED APPLICATIONS
- This is a continuation-in-part of U.S. patent application
Ser. No. 265,920,
- filed Nov. 2, 1988, now abandoned, which is a continuation-in-part
of U.S.
- patent application Ser. No. 875,535, filed Jun. 18, 1986,
now abandoned.
- ------------------------------------------------------------------------
- Claims
- ------------------------------------------------------------------------
-
- What is claimed is:
- 1. A biologically pure mycoplasma isolated from tissues
of patients with
- AIDS comprising the mycoplasma produced by the cell line
ATCC No. CRL 9127.
- 2. A biologically pure mycoplasma having the identifying
characteristics of
- M. fermentans incognitus, ATCC 53949.
- ------------------------------------------------------------------------
- Description
- ------------------------------------------------------------------------
-
- BACKGROUND OF THE INVENTION
-
- 1. Field of the Invention
-
- The present invention relates to a novel strain of mycoplasma
isolated from
- a patient with AIDS. The mycoplasma is closely related
to a species of
- human mycoplasma, M. fermentans. Upon characterization
of this mycoplasma,
- it may be classified as a unique strain within the species
M. fermentans
- incognitus.
-
- This novel strain of nycoplasma is referred to hereinafter
as the
- incognitus strain or M. fermentans incognitus.
-
- The invention also relates to use of the mycoplasma M.
fermentans
- incognitus as well as all strains of M. fermentans in
detecting specific
- antibodies in sera of patients with AIDS or an acute
fulminant systemic
- disease and/or animals and its use as a vaccine against
infection by the
- mycoplasma. The invention further relates to incognitus
strain-specific
- antibodies and cross-reactive which later break up into
individual cells
- that are capable of passing through membrane filters
of pore size 0.45
- .mu.m or even 0.22 .mu.m.
-
- A trilaminar cytoplasmic membrane contains sterols, phospholipid
and
- proteins. Therefore, the cells are generally susceptible
to polyene
- antibiotics and to lysis by digitonin.
-
- Replication of the Mycoplasma genome may precede cytoplasmic
division
- resulting in multinucleate filaments before individual
cells are delimited
- by constriction. Budding can also occur.
- Most Mycoplasma species are facultatively anaerobic,
and all known species
- are chemoorganotrophic. The fermentative species of Mycoplasma
utilize
- sugars such as glucose, while non-fermentative species
can utilize
- arginine.
-
- Known mycoplasmas may be grown on complex media, such
as Hayflick medium,
- while fastidious mycoplasmas may be grown on diphasic
SP-4 medium. The
- colonies are usually of the "fried egg" type,
i.e., an opaque, granular
- central region, embedded in the agar, surrounded by non-granular
surface
- growth. The optimal growth temperature of mammalian strains
is
- 36.degree.-37.degree. C.
- Many species of Mycoplasma produce weak or clear haemolysis
which appears
- to be due to the secretion of H.sub.2 O.sub.2. This H.sub.2
O.sub.2
- secretion is believed to be responsible for some aspects
of the
- mycoplasmas' pathogenicity. Known mycoplasmas are commonly
sensitive to
- chloramphenicol and
- tetracyclines.
-
- The Mycoplasma genus currently consists of more than
60 known species which
- are differentiated on the basis of various tests, including
utilization of
- glucose and mannose, arginine hydrolysis, phosphatase
production, the "film
- and spots" reaction and haemadsorption. M. fermentans
antibodies (i.e.
- antibodies to homologous antigenic determinants), including
monoclonal
- antibodies of each, which are useful in detecting incognitus
strain
- antigens in infected tissue of patients or animals. The
invention also
- relates to incognitus strain-specific DNA probes which
are useful in
- detecting incognitus strain genetic materials in infected
tissues of
- patients or animals. Incognitus strain genetic materials
may also be
- detected in infected humans or animals by using specific
incognitus strain
- DNA sequences a homologous M. fermentans DNA sequences
and the polymerase
- chain reaction ("PCR") (U.S. Pat. No. 4,683,202
incorporated herein by
- reference).
-
- The ability to monitor AIDS or other acute fulminant
systemic disease
- status can be of great value. In addition to improving
- prognostication,knowledge of the disease status allows
the attending
- physician to select the most appropriate therapy for
the individual
- patient, e.g. highly aggressive or less aggressive therapy
regimens.
- Because of patient distress caused by more aggressive
therapy regimens, it
- is desirable to distinguish those patients requiring
such therapies. It has
- been found that M. fermentans incognitus is more directly
associated and
- functional deficits of the infected organ systems and
is capable of
- distinguishing such patients.
-
- Mycoplasma is a genus of cell wall-less sterol-requiring,
catalase-negative
- pathogens commonly found in the respiratory and urogenital
tracts of man
- and other animals. The cells of Mycoplasma are typically
non-motile and
- pleomorphic, ranging from spherical, ovoid or pear-shaped
to branched
- filamentous forms.
-
- Filaments are the typical forms in young cultures under
optimal conditions,
- which subsequently transform into chains of coccoid cells
-
- Mycoplasmas are the smallest and simplest free-living
organisms known.
- Mycoplasmas are not obligatory intracellular microorganisms
and are usually
- found extracellularly, but are often found intracellularly
in the infected
- tissues (Mycoplasma, Eds. Wolfgang, J. J., Willette,
H. P., Amos, D. B.,
- Wilfert, C. M., Zinsser Microbiology 19th Ed. 1988, Appleton
and Lange,
- 617-623). The term mycoplasma apparently was first used
by B. Frank in 1889
- (Frank B., Dent. Bot. Ges., 7, 332 (1889) and Krass,
C. J. et al., Int. J.
- Syst. Bacteriol. 23, 62 (1973)). Frank, after careful
microscopic
- observation, began writing about invasion of plants (legume)
by these
- microorganisms and stated: "the changed character
of the protoplasm in the
- cortical cells arising from infection, I will designate
as mycoplasma".
- Later, he had more explicitly defined mycoplasma as a
mixture of small
- fungus-like microorganisms and cell protoplasm (Frank,
B., Landwirt. Jahrb.
- 19, 523 (1890)). The description reflected the difficulty
of
- differentiating this unique microorganism from the infected
host cells
- morphologically.
-
- Even today with electron microscopy, it is still often
difficult to
- differentiate the mycoplasmas from the cellular protoplasmic
processes or
- the subcellular organelles of the infected host, because
ultrastructurally,
- these microorganisms have protoplasm-like internal structures
and are
- bounded by only an outer limited membrane (unit membrane)
without a cell
- wall. Thus, there have been few electron microscopic
studies of mycoplasmas
- identified directly in the infected tissues of animals
or humans.
-
- It has been reported that ultrastructural examination
of infected tissues
- has failed to localize the microbe, even in tissues where
very high titers
- (>10.sup.9 /gm) of microorganisms were recovered in
culture (Elizan, T. S.
- et al., Pro. Soc. Exp. Biol. Med. 139, 52 (1972) and
Schwartz, J. et al.,
- Pro. Soc. Exp. Biol. Med. 139, 56 (1972)). Therefore,
morphologically, the
- microbe might be mimicking certain normal cellular or
subcellular
- structures in the infected host tissues and preventing
direct
- identification.
-
- In addition to the natural difficulty of morphological
differentiation
- between the microorganisms and the protoplasm of infected
cells, the often
- poorly preserved formalin-fixed clinical materials present
further
- limitations to any attempt to directly visualize mycoplasma
organisms in
- the tissues.
-
-
- DESCRIPTION OF THE BACKGROUND ART
-
- Acquired Immune Deficiency Syndrome (AIDS) is a devastating
disease that
- has afflicted over 70,000 people worldwide (AIDS Weekly
Surveillance
- Report--United States, Centers for Disease Control, Aug.
29, 1988). The
- disease is clinically characterized by a set of typical
syndromes which
- manifests itself by the development of opportunistic
infections such as
- pneumocystic carinii pneumonia (PCP), toxoplasmosis,
atypical
- mycobacteriosis and cytomegalovirus (CMV). Further characteristics
of the
- AIDS associated syndromes are the clinical manifestation
of
- neuropsychiatric abnormalities, of AIDS encephalopathy
(Naura, B. A., et
- at., Ann.Neuro 19, 517 (1986)), kidney failure of AIDS
nephropathy, heart
- failure of AIDS cardiomyopathy infections and certain
uncommon malignancies
- such as Kaposi's sarcoma or B-cell lymphoma (Durack,
D. T., N.Eng.J.Med.
- 305, 1465 (1981); Reichert, C. M., et al., Am.J.Path.
112, 357 (1983);
- Ziegler, J. L., et al., N.Eng.J.Med. 311, 565 (1984)).
-
- Through co-cultivation of AIDS patients' peripheral blood
cells with
- mitogen-stimulated normal human lymphocytes or permanent
human T-cell
- lines, a number of laboratories have isolated T-cell-tropic
human
- retroviruses (HTLV-III/LAV), Barre-Sinoussi, F., et al.,
Science 220, 868
- (1983); Gallo, R. C., et al., Science 224, 500 (1984).
Epidemiologically,
- the newly isolated retroviruses have been shown to be
highly associated
- with patients of AIDS and/or AIDS-related complex (ARC).
Schupback, J., et
- al., Science 224, 503 (1984); Sarngadharan, M. G., et
al., Science 224, 506
- (1984). In vitro studies with HTLV-III/LAV have demonstrated
T-cell tropism
- and cytopathic changes. Barre-Sinoussi, F., et al., supra;
Popovic, M., et
- al., Science 224, 497 (1984). HTLV-III/LAV is believed
to be the causative
- agent of AIDS.
-
- However, the establishment of an animal model of AIDS
by HTLV-III-LAV
- injection has not been successful. Gajdusek, D.C., et
al., Lancet I, 1415
- (1984). The chimpanzee is the only primate other than
man found to be
- susceptible to infection by HTLV-III/LAV. However, overt
AIDS manifested by
- the development of opportunistic infections and/or unusual
malignancies has
- not yet been seen, despite evidence for persistent infection
and/or viremia
- in experiments on this species. Gajdusek, D.C., et al.
Lancet I, 55 (1985).
- Thus, the human retroviruses have not fulfilled Koch's
postulates, i.e.,
- producing transmissible AIDS-like diseases in experimental
animals.
- HTLV-III/LAV is not associated with the unusual malignancies
such as B-cell
- lymphoma and Kaposi's sarcoma, commonly found in patients
with AIDS. Shaw,
- G. M., et al., Science 226: 1165-1171, 1984; Delli Bovi,
P. et al., Cancer
- Research, 46: 6333-6338, 1986; Groopman, J. E., et al.,
Blood 67: 612-615,
- 1986. Furthermore, HIV infected patients often show a
wide variation in
- times of disease incubation and speed of disease progression.
It is not
- known whether any specific infectious agent other than
HIV can be
- responsible for the complex pathogenesis often seen in
this disease. One
- such candidate, initially identified as a virus or virus-like
infectious
- agent in parent application Ser. No. 265,920 has now
been discovered to be
- the mycoplasma M. fermentans (incognitus strain).
-
- Although a viral etiology of developing these malignancies
has long been
- suggested, conventional approaches for isolating infectious
viral agents
- have not been fruitful. The presence of a transforming
gene or transforming
- genes (oncogenes) has been associated with Kaposi's sarcoma
(Lo. S., et
- al., Am. J. Path., 118, 7 (1985)). A transformant carrying
the transforming
- gene can cause tumors in mice.
-
- However, there is no further characterization of this
transforming gene
- except for the presence of human repetitive DNA sequences.
The transforming
- gene has not been shown to be associated with any viral
or virus-like
- agent. An ongonege of AIDS Kaposi Sarcoma was similarly
identified
- following DNA transfection into NIH/3T3 cells and was
later characterized
- in detail (Delli Bovi O. et al., Proc Natl Acad Sci 84,
5660 (1987) and
- Delli Bovi P. et al., 50, 729 (1987). The oncogene was
found to be a
- rearranged human protooncogene of the fibroblast growth
factor (FGF)
- family.
-
-
- SUMMARY OF INVENTION
-
- The present invention relates to a novel strain of the
mycoplasma M.
- fermentans which has been isolated from Kaposi's sarcoma
of a patient with
- AIDS. This novel strain of mycoplasama has been designated
the incognitus
- strain of M. fermentans or M. fermentans incognitus.
The invention further
- relates to the use of this incognitus strain of M. fermentans
as well as
- other strains of M. fermentans with homologous antigenic
determinants for
- the detection of specific antibodies in sera of human
patients and animals,
- and for vaccines against mycoplasmas. The invention also
relates to
- antibodies, including monoclonal antibodies, to M. fermentans
incognitus
- and to homologous antigenic determinants of M. fermentans
and their use in
- detecting M. fermentans incognitus antigens in the infected
tissue of human
- patients and animals. The invention further relates to
sequencing the DNA
- of the M. fermentans incognitus and the manufacture of
DNA probes based on
- such sequencing and homologous sequences of M. fermentans
for use in the
- direct detection of the unique DNA sequences in the tissues
of human
- patients and animals.
-
- The present invention further relates to the detection
of the presence of
- M. fermentans incognitus in patients which are HIV-positive
or have other
- acute fulminant systemic disease as an indication of
the prognosis of the
- disease, which can be used to determine the appropriate
therapy regimen.
- The presence of M. fermentans incognitus is determined
as described above.
-
- The M. fermentans incognitus DNA is detected in the spleen,
liver, brain,
- lymph nodes, kidney, placenta, lungs, adrenal glands,
heart and peripheral
- blood mononuclear cells of patients with AIDS, or from
Kaposi's sarcoma
- tissue from patients with AIDS. The M. fermentans incognitus
DNA is capable
- of transfecting and transforming NIH/3T3 cells. M. fermentans
incognitus is
- a transmissible virus-like infectious agent in cell cultures,
experimental
- animals and humans. The DNA of the transformants does
not contain human
- repetitive DNA sequences. Two transformants are identified
as Sb51 and
- Kb43. These transformants are persistently infected by
the M. fermentans
- incognitus. M. fermentans incognitus is then isolated
from the
- transformants.
-
- The majority of M. fermentans incognitus cells have a
size of about 140 nm
- to about 280 nm, with an overall range of 100-900 nm.
Introduction of M.
- fermentans incognitus into nude mice and immunocompetent
mice (Balb/c)
- results in a significant morbidity and mortality of the
infected animals
- and the manifestation of many symptoms such as B-cell
tumor, spindle cell
- tumor or immunodeficiency.
-
- Similar diseases are transmitted from animal to animal
by introduction of
- infected tissues.
- M. fermentans incognitus was also found to infect non-human
primates
- (monkeys). M. fermentans incognitus antigens were identified
in the
- infected monkey's sera, and M. fermentans incognitus
DNA was found in DNA
- isolated from tissues of the infected monkeys.
-
- M. fermentans incognitus and other strains of M. fermentans
having
- homologous antigens are capable of detecting antibodies
in sera of patients
- with AIDS, ARC or non-AIDS patients with this mycoplasma
infection. Any
- method for detecting an antigen-antibody reaction may
be utilized,
- including enzyme-linked immunosorbent assay (ELISA),
immunoradiometric
- assay, direct and indirect immunofluorescent assay, Western
blot technique,
- and the like. In addition, M. fermentans incognitus-specific
antibodies (as
- well as antibodies to homologous antigens of other M.
fermentans strains)
- are raised in experimental animals or developed in monoclonal
antibodies
- which are capable of detecting M. fermentans incognitus-
related antigens
- in infected tissues. Furthermore, the probes having M.
fermentans
- incognitus-specific or homologous M. fermentans DNA sequences
can be used
- in the direct detection of M. fermentans incognitus DNA
in infected
- tissues, or specific M. fermentans incognitus or homologous
M. fermentans
- DNA sequences can be used in the polymerase chain reaction
("PCR") to
- identify M. fermentans incognitus DNA in infected tissues.
Since antibodies
- or antisera are successfully raised against M. fermentans
incognitus, the
- M. fermentans incognitus or homologous antigens of M.
fermentans antigens
- can be utilized to prepare vaccines which may be used
to protect animals,
- including humans, against infection by M. fermentans
incognitus or other
- mycoplasmas.
-
-
- BRIEF DESCRIPTION OF THE DRAWINGS
-
- FIG. 1A shows an electron photomicrograph of M. fermentans
incognitus.
- FIG. 1B shows an electron photomicrograph of M. fermentans
prototype strain
- (PG18).
- FIG. 1C shows the colony morphology of M. fermentans
incognitus.
- FIG. 1D shows the colony morphology of the prototype
strain (PG18) of M.
- fermentans.
- FIG. 2A shows antigenic comparison of M. fermentans incognitus,
M.
- fermentans and other human mycoplasmas in immunoblots
immunostained with
- rabbit antiserum raised specifically against M. fermentans
incognitus.
- FIG. 2B shows mycoplasmas in immunoblots immunostained
with mule antiserum
- raised specifically against M. fermentans.
- FIG. 3 shows a comparison of DNA homology and restriction
patterns between
- M. fermentans incognitus and other human mycoplasmas.
The samples were
- probed with A) pst-8.6, B) psb-2.2, C) RS48, D) MI-H
3.3, E) cDNA clone of
- E. coli rRNA.
- FIG. 4A shows direct immunofluorescence staining of M.
fermentans
- incognitus using FITC conjugated monoclonal antibody
D81E7 (X900).
- FIG. 4B shows direct immunofluorescence staining of M.
fermentans using
- FITC conjugated monoclonal antibody D81E7 (X900).
- FIG. 5A shows the genetic map of a repetitive segment
of a 2.2 Kb Eco RI
- fragment of M. fermentans incognitus.
- FIG. 5B shows the nucleotide sequence of a repetitive
segment of a 2.2 Kb
- Eco RI fragment of M. fermentans incognitus.
- FIG. 5C shows the genetic map of a repetitive segment
of a 2.2 Kb Eco RI
- fragment of M. fermentans incognitus.
- FIG. 6 shows the analysis of repetitive elements following
probing with A)
- psb-2.2 and B-K of FIG. 5A.
- FIG. 7A shows detection of M. fermentans from urine specimens
following PCR
- stained with ethidium bromide.
- FIG. 7B shows detection of M. fermentans from urine specimens
following PCR
- stained with Probe RU006.
- FIG. 8A shows detection of M. fermentans incognitus from
urine specimens
- following PCR stained with ethidium bromide.
- FIG. 8B shows detection of M. fermentans incognitus from
urine specimens
- following PCR stained with Probe RU006.
- FIG. 9 shows analysis of genomic DNA from various strains
or isolates of M.
- fermentans.
- FIG. 10A shows an electron micrograph of thin sections
of M. fermentans
- incognitus cells in the cytoplasm of degenerating Sb51
cells.
- FIG. 10B shows an electron micrograph of membrane bound
M. fermentans
- incognitus.
- FIG. 10C shows an electron micrograph of a partially
disrupted M.
- fermentans incognitus at high magnification.
- FIG. 11 shows a graph of body weight of monkeys over
time, after
- innoculation with M. fermentans incognitus.
- FIG. 12A shows immunocytochemical staining of Sb51 cells
with non-AIDS serum.
- FIG. 12B shows immunocytochemical staining of NIH/3T3
cells with AIDS serum.
- FIG. 12C shows immunocytochemical staining of Sb51 cells
with AIDS serum.
- FIG. 13 shows the immunocytochemical staining of the
subcapsular cortical
- sinus of a lymph node from a patient with AIDS.
- FIG. 14 shows the immunohistochemistry of the midbrain
of the brain stem of
- a patient with AIDS.
- FIG. 15A shows blotted filters of DNA from Sb51 cells
and control NIH/3T3
- cells probed with psb-8.6.
- FIG. 15B shows blotted filters of DNA from Sb51 cells
and control NIH/3T3
- cells probed with psb-2.2.
- FIG. 16 shows blotted filters of digested DNA from Sb51
cells, control
- NIH/3T3, cells, cell-free M. fermentans incognitus transmission
in NIH/3T3
- cells and DNA of partially purified M. fermentans incognitus
probed with
- psb-8.6.
- FIG. 17A shows a sucrose gradient banding of M. fermentans
incognitus.
- FIG. 17B shows DNA and antigen dot blot analysis of sucrose
gradient-banded
- M. fermentans incognitus in which the blot was probed
with .sup.32 P in a
- labeled insert fragment of psb-8.6.
- FIG. 18A shows DNA and antigen dot blot analysis of sucrose
gradient-banded
- M. fermentans incognitus in which immunochemical staining
using
- pre-immunized rabbit serum was performed.
- FIG. 18B shows DNA and antigen dot blot analysis of sucrose
gradient-banded
- M. fermentans incognitus in which immunochemical staining
using post-M.
- fermentans incognitus immunization rabbit antisera was
performed.
- FIG. 19A shows Southern blot hybridizations to compare
M. fermentans
- incognitus DNA to DNA from known human herpes viruses,
vaccinia virus, MCMV
- and HVS. The samples were probed with A) HSV-1 pHSV-106.
- FIG. 19B shows the Southern blot of FIG. 19A using B)
VZV pEco A.
- FIG. 19C shows the Southern blot of FIG. 19A using C)
EBV pBam W.
- FIG. 19D shows the Southern blot of FIG. 19A using D)
CMV pCMH-35.
- FIG. 19E shows the Southern blot of FIG. 19A using E)
HBLV pZVH-70.
- FIG. 19F shows the Southern blot of FIG. 19A using F)
Vaccinia pEH-1.
- FIG. 19G shows the Southern blot of FIG. 19A using G)
MCMV pAMB-25.
- FIG. 19H shows the Southern blot of FIG. 19A using H)
HVS pT 7.4.
- FIGS. 20A and 20B shows DNA amplification analysis of
various tissue DNA
- isolated from patients with AIDS and control subjects
without AIDS.
- FIG. 21A shows M. fermentans incognitus-induced histopathological
changes
- of fulminant necrosis in the spleen of a patient without
AIDS dying of an
- acute systemic disease.
- FIG. 21B shows the advancing margin of FIG. 21A.
- FIG. 21C shows M. fermentans incognitus-induced histopathological
changes
- of fulminant necrosis in the lymph node of a patient
without AIDS dying of
- an acute systemic disease.
- FIG. 21D shows M. fermentans incognitus-induced histopathological
changes
- of fulminant necrosis in the adrenal gland of a patient
without AIDS dying
- of an acute systemic disease.
- FIG. 22A shows the immunohistochemistry of M. fermentans
incognitus-induced
- necrotizing lesions in the spleen using M. fermentans
incognitus-specific
- rabbit antiserum.
- FIG. 22B shows the margin of microsis of FIG. 22A.
- FIG. 22C shows the immunohistochemistry of M. fermentans
incognitus-induced
- necrotizing lesions in the lymph node using M. fermentans
- incognitus-specific rabbit antiserum.
- FIG. 22D shows the peripheral zone of necrosis of FIG.
22C.
- FIG. 22E shows the immunohistochemistry of M. fermentans
incognitus-induced
- necrotizing lesions in the adrenal gland using M. fermentans
- incognitus-specific rabbit antiserum.
- FIG. 23A shows in situ hybridization for M. fermentans
incognitus nucleic
- acids in the necrotizing lesions of splenic tissue in
the peripheral zone
- around necrosis.
- FIG. 23B shows a higher magnification of FIG. 23A.
- FIG. 23C shows an area of differing necrosis in splenic
tissue.
- FIG. 23D shows an area of differing necrosis in splenic
tissue.
- FIG. 24A.sub.1 shows an electron micrograph of the margin
of necrosis of an
- adrenal gland highly positive for M. fermentans incognitus-specific
- antigens.
- FIG. 24A.sub.2 is a higher magnification of FIG. 24A.sub.1.
- FIG. 24B.sub.1 shows an electron photomicrograph of the
peripheral zone of
- necrosis in lymph node highly positive for M. fermentans
- incognitus-specific antigens.
- FIG. 24B.sub.2 shows an electron photomicrograph of the
peripheral zone of
- necrosis in lymph node highly positive for M. fermentans
- incognitus-specific antigens.
- FIG. 24B.sub.3 is higher magnification of FIG. 24B.sub.1.
- FIG. 25A shows analysis and comparison of DNA restriction
patterns of VLIA
- and M. fermentans incognitus probed with psb-8.6.
- FIG. 25B shows analysis and comparison of DNA restriction
patterns of VLIA
- and M. fermentans incognitus probed with psb-2.2.
- FIG. 26A shows the immunohistochemistry of thymic tissues
derived from
- patients with AIDS.
- FIG. 26B is a higher magnification of FIG. 26A.
- FIG. 26C is a higher magnification of FIG. 26B.
- FIG. 26D shows the immunohistochemistry of thymic tissues
derived from
- patients with AIDS.
- FIG. 26E is a higher magnification of FIG. 26D.
- FIG. 27A shows an electron micrograph of an AIDS thymus
immunostained
- positively for M. fermentans incognitus-specific antigens
showing
- mononuclear lymphohistiocytes.
- FIG. 27B shows an electron micrograph of an AIDS thymus
immunostained
- positively for M. fermentans incognitus-specific antigens
showing
- mononuclear lymphohistiocytes.
- FIG. 27C shows an electron micrograph of an AIDS thymus
immunostained
- positively for M. fermentans incognitus-specific antigens
showing
- mycoplasma-like particles.
- FIG. 27D shows an electron micrograph of an AIDS thymus
immunostained
- positively for M. fermentans incognitus-specific antigens
showing
- mycoplasma-like particles.
- FIG. 28A shows the immunohistochemistry of livers from
patients with AIDS
- using monoclonal antibody C42H10.
- FIG. 28B shows the immunohistochemistry of livers from
patients with AIDS
- using monoclonal antibody C42H10.
- FIG. 28C shows the immunohistochemistry of livers from
patients with AIDS
- using a non-specific monoclonal antibody.
- FIG. 28D shows the immunohistochemistry of livers from
patients with AIDS
- using monoclonal antibody C42H10.
- FIG. 29A shows an electron micrograph of AIDS liver immunostained
- positively for M. fermentans incognitus-specific antigens
at low
- magnification.
- FIG. 29B is a higher magnification of FIG. 29A.
- FIG. 29C is a higher magnification of FIG. 29B.
- FIG. 29D shows an electron micrograph of AIDS liver immunostained
- positively for M. fermentans incognitus-specific antigens
at low
- magnification.
- FIG. 29E is a higher magnification of FIG. 29D.
- FIG. 30A shows the immunohistochemistry of a brain derived
from a patient
- with AIDS using monoclonal antibody C42H10.
- FIG. 30B shows the immunohistochemistry of a brain derived
from a patient
- with AIDS using monoclonal antibody C42H10.
- FIG. 30C shows the immunohistochemistry of a brain derived
from a patient
- with AIDS using a non-specific monoclonal antibody.
- FIG. 30D shows the immunohistochemistry of a brain derived
from a patient
- with AIDS using monoclonal antibody C42H10.
- FIG. 31A shows electron microscopy of CNS encephalopathy
AIDS brains which
- were histologically unremarkable but immunostained positively
for M.
- fermentans incognitus-specific antigens.
- FIG. 31B is a higher magnification of FIG. 31A.
- FIG. 31C is a higher magnification of FIG. 31B.
- FIG. 31D is a higher magnification of FIG. 31C.
- FIG. 32A shows the immunohistochemistry of a placenta
delivered by a
- patient with AIDS using monoclonal antibody C42H10.
- FIG. 32B is a higher magnification of FIG. 32A.
- FIG. 33A shows electron microscopy of an AIDS patient's
placenta
- immunostained positively for M. fermentans incognitus
specific antigens
- showing Hofbauer cell.
- FIG. 33B shows electron microscopy of an AIDS patient's
placenta
- immunostained positively for M. fermentans incognitus
specific antigens
- showing Hofbauer cell.
- FIG. 33C shows electron microscopy of an AIDS patient's
placenta
- immunostained positively for M. fermentans incognitus
specific antigens
- showing stronal connective tissue.
- FIG. 33D shows electron microscopy of an AIDS patient's
placenta
- immunostained positively for M. fermentans incognitus
specific antigens
- showing stronal connective tissue.
- FIG. 33E shows electron microscopy of an AIDS patient's
placenta
- immunostained positively for M. fermentans incognitus
specific antigens
- showing stronal connective tissue.
- FIG. 34A shows in situ hybridization for M. fermentans
incognitus nucleic
- acid in liver from patients with AIDS.
- FIG. 34B shows in situ hybridization for M. fermentans
incognitus nucleic
- acid in liver from patients with AIDS.
- FIG. 34C shows in situ hybridization for M. fermentans
incognitus nucleic
- acid in spleen from patients with AIDS.
- FIG. 34D shows in situ hybridization for M. fermentans
incognitus nucleic
- acid in spleen from patients with AIDS.
- FIG. 35 shows the inhibition of HIV-1-induced syncytium
formation by M.
- fermentans incognitus.
- FIG. 36A shows the augmentation of cytocidal effect and
inhibition of RT
- activity in HIV-1 infected A3.01 cells cultures by M.
fermentans
- incognitus.
- FIG. 36B shows the inhibition of RT activity in HIV-1
infected A3.01 cell
- cultures by M. fermentans incognitus.
- FIG. 37A shows continued viral production of HIV-1 and
M. fermentans
- incognitus in culture supernatant by ELISA.
- FIG. 37B shows continued viral production of HIV-1 and
M. fermentans
- incognitus in culture supernatant by electron micrograph.
-
-
- DETAILED DESCRIPTION OF THE INVENTION
-
- A. Definitions
-
- In order to provide a clear and consistent understanding
of the
- specification and the claims, including the scope given
to such terms, the
- following terms as used herein are defined below.
-
- The term "AIDS-like syndrome" is used to describe
a set of physiologic
- conditions or clinical presentations which are commonly
used to identify
- individuals who are suspected of having the disease AIDS,
but who have not
- had confirmation of the disease by blood test. The physiologic
conditions
- are those that are common to individuals with blood test-confirmed
AIDS,
- and include the development of opportunistic infections
such as
- pneumocystic carinii pneumonia (PCP), atypical mycobacterial
infection,
- toxoplasmosis and cytomegalovirus (CMV), the clinical
manifestation of
- progressive weight loss, persistent diarrhea, neuropsychiatric
- abnormalities of AIDS encephalopathy, kidney failure
of AIDS nepthropathy,
- heart failure of AIDS cardiomyopathy, respiratory distress
syndrome and
- infections and uncommon malgnancies such as Kaposi's
sarcoma or B-cell
- lymphoma.
-
- The term "substantial sequence homology" is
used to describe substantial
- functional and/or structural equivalence between sequences
of nucleotides
- or amino acids. Functional and/or structural differences
between sequences
- having substantial sequence homology will be de minimus.
-
-
- B. Previous Related Applications
-
- The present invention relates to a novel strain of infectious
mycoplasma
- (M. fermentans incognitus) isolated from patients with
AIDS. The
- recognition of this pathogen as a mycoplasma has been
a slowly evolving
- process as evidenced by the history of the present specification.
-
- The predecessor patent applications (Ser. No. 875,535,
filed Jun. 18, 1986
- and Ser. No. 265,920, filed Nov. 2, 1988) identified
the subject pathogen
- as a virus and a virus-like infectious agent (VLIA),
respectively. However,
- continuing study of the pathogen has resulted in the
present identification
- of the pathogen as an infectious mycoplasma. Ser. Nos.
265,920 and 875,535
- are incorporated herein by reference.
-
- The presently identified mycoplasma like many other mycoplasmas
has many of
- the characteristics of a virus, which resulted in its
identification as
- such in the original patent application (Ser. No. 875,535,
filed Jun. 18,
- 1986). Further research then showed characteristics which
were not typical
- of classic viruses, thus the characterization as a VLIA
in Ser. No.
- 265,920, filed Nov. 2, 1988. Additional research has
now revealed
- characteristic traits of a mycoplasma as fully explained
below.
-
-
- C. Deposits
-
- A mycoplasma (M. fermentans incognitus) according to
the invention, in
- persistently infected cells, is deposited with the American
Type Culture
- Collection under Deposit No. CRL 9127, deposited on Jun.
17, 1986. M.
- fermentans incognitus, itself is also deposited with
the American Type
- Culture Collection under Deposit No. 53949, deposited
on Sep. 29, 1989.
-
- Deposit is for the purpose of completeness but is not
intended to limit the
- scope of the present invention to the materials deposited
since the
- description as further illustrated by the Examples fully
enables the
- practice of the instant invention. Access to the cultures
will be available
- during the pendency of the patent application to those
determined by the
- Commissioner of Patents and Trademarks to be entitled
thereto. All
- restrictions on availability of said cultures to the
public will be removed
- irrevocably upon the grant of the instant application
and said cultures
- will remain available permanently during the term of
said patent 30 years
- or five years after last request, whichever is longer.
Should any culture
- become nonviable or be destroyed, it will be replaced.
-
-
- D. Physical Characteristics of M. fermentans incognitus
-
- The M. fermentans incognitus cell is roughly spherical
and about 140-200 nm
- in diameter, has an outer limiting membrane (about 8
nm thick), and has a
- buoyant density of about 1.17 g/ml to about 1.20 g/ml
in a sucrose
- gradient. Although M. fermentans incognitus could be
identified in the
- nuclei, mature M. fermentans incognitus cells are usually
seen in the
- cytoplasm or associated with the plasma membrane of disrupted
cytolytic
- cells.
-
- Using Southern blot hybridization analysis, the M. fermentans
incognitus
- was distinct from all known members of human herpes virus.
M. fermentans
- incognitus was also distinct from vaccinia virus, monkey
herpesvirus
- saimiri (HVS) and mouse cytomegalovirus (MCMV). M. fermentans
incognitus
- can be transmitted from culture to culture by cell-free
filtrate, after
- 0.22 micron filtration.
-
- M. fermentans incognitus was also found to be distinct
from any other known
- strain of Mycoplasma. One unique feature of M. fermentans
incognitus is its
- ability to catabolize glucose both aerobically and anaerobically
and also
- to hydrolyze arginine. M. fermentans incognitus cannot
hydrolyze urea in a
- biochemical ssay. When grown in culture, M. fermentans
incognitus produces
- a prominent alkaline shift in pH after an initial brief
acidic shift. The
- only other human mycoplasma which is known to metabolize
both glucose and
- arginine is the rarely isolated M. fermentans.
-
- However, the incognitus strain differs from M. fermentans
in that it
- appears to be is more fastidious in its cultivation requirements
and has
- only been grown in a cell-free modified SP-4 medium.
M. fermentans also
- grows in modified SP-4 medium, but at a much faster rate
than M. fermentans
- incognitus.
-
- Furthermore, M. fermentans incognitus can be grown in
a variety of commonly
- used mycoplasma media, whereas M. fermentans incognitus
cannot.
-
- When grown in the modified SP-4 medium, M. fermentans
incognitus displays
- smaller spherical particle size than M. fermentans incognitus,
and
- occasional filamentous morphology which is not seen with
M. fermentans
- incognitus. Furthermore, M. fermentans incognitus forms
only irregular and
- very small colonies with diffuse edges when grown on
agar plates. The M.
- fermentans incognitus are cell wall-free and bound by
a single triple
- layered membrane. The average size of an M. fermentans
incognitus cell is
- about 180 nm, compared to an average size of about 460
nm for an M.
- fermentans cell.
-
- FIG. 1 shows electron photomicrographs and colony morphology
of M.
- fermentans incognitus and M. fermentans. Thin sections
of concentrated M.
- fermentans incognitus (A) and M. fermentans incognitus
(B) reveal
- pleophorphic microorganisms with trilaminar outer unit
membrane as
- designated by the arrows. The bars in 1A and 1B represent
100 nm. M.
- fermentans incognitus (C) and M. fermentans (D) formed
colonies of
- apparently different size and morphology after 14 days
and 10 days of
- incubation, respectively. In these figures, the bar represents
50 .mu.m and
- 20 .mu.m, respectively.
-
-
- E. Antigenic differentiation of M. fermentans incognitus
and M. fermentans
-
- Further differentiation of M. fermentans incognitus from
prototype strain
- of M. fermentans (PG18) was displayed by antigenic analysis
using both
- polyclonal and monoclonal antibodies, as well as DNA
analysis of sequence
- homology and restriction enzyme mapping. These analyses
showed that the
- incognitus strain is distinct from all other mycoplasmas,
but is most
- closely related to previously isolated M. fermentans
strains.
-
- M. fermentans incognitus was distinguished from M. fermentans
(PG18 strain)
- by comparing their specific antigenicity. Polyclonal
rabbit antiserum
- (raised originally against VLIA-sb51) was found to react
with both M.
- fermentans (PG18 strain) and M. fermentans incognitus,
but not with any of
- the other mycoplasmas tested. However, in the same assay
a larger amount of
- M. fermentans (PG18 strain) protein (>0.63 .mu.g)
was required to elicit a
- positive immunochemical response, and the positivity
of the reaction
- rapidly disappeared when the M. fermentans (PG18 strain)
protein was
- further diluted. In contrast, a 250-fold to 1000 fold
lower concentration
- of M. fermentans incognitus protein still carried a sufficient
amount of
- antigenic determinants to elicit positive reactions in
the assay.
-
- In a parallel assay, antiserum raised specifically against
M. fermentans
- (PG18 strain) also reacted intensely with M. fermentans
incognitus. The M.
- fermentans incognitus-specific antiserum reacted as effectively
with the
- antigens of M. fermentans incognitus as with the antigens
of M. fermentans
- (PG18 strain). There was approximately an equal amount
of antigens which
- could be recognized by the M. fermentans incognitus antiserum
in each unit
- of M. fermentans (PG18 strain) and M. fermentans incognitus
proteins. Both
- M. fermentans and M. fermentans incognitus proteins could
be diluted to 40
- ng per well and still elicit a positive reaction.
-
- However, when M. fermentans incognitus proteins and M.
fermentans (PG18
- strain) proteins were reacted with monoclonal antibodies
raisedspecifically
- against M. fermentans incognitus, only M. fermentans
incognitus proteins
- reacted positively. Six M. fermentans incognitus monoclonal
antibodies
- (many with different isotypes) reacted with only M. fermentas
incognitus,
- but not with M. fermentans. Therefore, M. fermentans
incognitus carries
- additional specific antigens which can not be identified
in the prototype
- of M. fermentans (PG18 strain).
-
- FIG. 2 shows antigenic comparison of M. fermentans incognitus,
M.
- fermentans and other human mycoplasmas in immunoblots.
Upper blot (2A) was
- immunostained with rabbit antiserum raised specifically
against M.
- fermentans incognitus. Lower blot (2B) was immunostained
with mule
- antiserum raised specifically against M. fermentans (PG18
strain). The
- concentration of mycoplasma protein was dot-blotted decrementally
(1:4
- dilution) from lane 1 (10 .mu.g) to lane 12 (2.5 pg).
Row A (M. arginini),
- row B (A. laidlawii), row C (M. fermentans), row D (M.
hominis), row E (M.
- orale), row F (M. hyorhinis), row G (M. pneumonia), row
H (M. fermentans
- incognitus). In FIG. 2C row A, B, C, D and F were immunostained
with
- monoclonal antibodies D81E7, C69H3, F89H7, B109H8, F11C6
and C42H10,
- respectively. The concentration of mycoplasma protein
was dot-blotted
- decrementally (1:10 dilution) from lane 1 (10 .mu.g)
to lane 8 (1 pg). Row
- a (M. fermentans incognitus) and Row b (M. fermentans).
-
-
- F. DNA Homology
-
- DNA was isolated from M. fermentans incognitus and ten
other species of
- mycoplasmas (M. orale), M. hyorhinis, M. pneumonia, M.
arginini, M.
- hominis, M. fermentans, M. genitalium, M. salivarium,
U. urealyticum and A.
- laidlawii) and analyzed on Southern blots, being probed
with .sup.32
- P-labeled cloned M. fermentans incognitus DNA (psb-8.6,
psb 2.2) or
- synthetic oligonucleotide RS48 (SEQ ID NO:1) a M. fermentans
- incognitus-specific sequence. An additional molecular
clone, carrying a 3.3
- kilobase insert of M. fermentans incognitus DNA (MI-H
3.3) was also used as
- a probe.
-
- Although some homology with psb-2.2 was observed in the
M. orale genome, no
- homology with RS48 (the specific DNA sequences occurring
at one terminal
- end of psb-2.2) and no homology with psb-8.6 or MI-H
3.3 were identified in
- the M. orale genome. Although DNA homology with psb-8.6,
psb-2.2, RS48 and
- MI-H 3.3 were all found in the M. fermentans (PG18 strain)
genome, the
- restriction patterns revealed by these probes were different
between M.
- fermentans (PG18 strain) and M. fermentans incognitus.
-
- FIG. 3 shows a comparison of DNA homology and restriction
patterns between
- M. fermentans incognitus and other human mycoplasmas.
The blots were probed
- with .sup.32 P nick-translated psb-8.6 (3A) and psb-2.2
(3B), .sup.32 P
- end-labeled RS48 (3C), .sup.32 P labeled MI-H 3.3 (3D)
and .sup.32 P
- end-labeled cDNA probe of E. coli ribosomal RNA (3E).
Each lane contained
- 0.2 microgram of EcoRI enzyme pre-digested DNA from Acholeplasm
laidlawii
- (lane 1), M. arginini (lane 2), M. hominis (lane 3),
M. hyorhinis (lane 4),
- M. pneumoniae (lane 5), M. orale (lane 6), M. fermentans
(PG18 strain)
- (lane 7) and M. fermentans incognitus (lane 8). Arrows
indicate the
- positions of standard size marker 23, 9.4, 6.7, 4.4,
2.3, and 2.0 kb,
- respectively.
-
- Furthermore, there is significant homology between the
ribosomal RNA
- (r-RNA) genes of procaryotive mycoplasmas and those of
Escherichia coli
- bacterium. The same blot which was consecutively probed
with RS48 and MI-H
- 33 was reprobed with .sup.32 P-labeled cDNA of E. coli
or r-RNA, after
- removing the previously incorporated probe by boiling
the filter. The
- analysis of r-RNA genes revealed both a difference in
numbers and size of
- the hybridization bands with each different species of
mycoplasma tested.
- The EcoRI restriction pattern of the r-RNA genes for
M. fermentans
- incognitus and M. fermentans (PG18 strain) appeared to
be identical, but
- were different from any other mycoplasma tested.
-
-
- G. Immunofluorescence Staining
-
- Further support for the conclusion that M. fermentans
incognitus differs
- from any other mycoplasma came from a study of direct
immunofluorescence
- staining. An FITC probe was conjugated to the purified
M. fermentans
- incognitus monoclonal antibodies, and again revealed
positive staining only
- in M. fermentans incognitus, but not in M. fermentans
(PG18 strain) or six
- other species of human mycoplasmas. FIG. 4 shows direct
immunofluorescence
- straining of M. fermentans incognitus (A) and M. fermentans
(PG18 strain)
- (B) using FITC conjugated monoclonal antibody D81E7 (X900).
-
-
- H. M. fermentans incognitus Infection
-
- A high prevalence of M. fermentans incognitus infection
has been found in
- patients with AIDS by using the polymerase chain reaction.
The genetic
- material specific for M. fermentans incognitus has been
isolated from
- spleens, Kaposi's sarcoma, livers, lymph nodes, peripheral
blood
- mononuclear cells and brains of patients with AIDS.
-
- Furthermore, M. fermentans incognitus infection has been
found in
- previously healthy non-AIDS subjects with an acute fatal
disease. The M.
- fermentans incognitus infection in these patients was
directly identified
- in the necrotizing lesions in lymph nodes, spleens, livers,
adrenal glands,
- heart and brain.
- The pathogensis of M. fermentans incognitus infection
is unusual in that
- despite fulminant tissue necrosis, there is lymphocyte
depletion and an
- apparent lack of cellular immune response or inflammatory
reaction in the
- infected tissues. It is believed that infection of M.
fermentans incognitus
- either has concomittantly caused damage to key components
of the hosts'
- immune system, or this pathogen has special biological
properties which
- enable it to elude immunosurveillance of the infected
hosts.
-
- Coinfection with Mycoplasma fermentans (incognitus strain)
enhances the
- ability of human immunodeficiency virus type-1 (HIV-1)
to induce cytopathic
- effects on human T lymphocytes in vitro. Syncytium formation
of
- HIV-infected T cells was essentially eliminated in the
presence of M.
- fermentans (incognitus strain), despite prominent cell
death. However,
- replication and production of HIV-1 particles continued
during the
- coinfection. Furthermore, the supernatant from cultures
coinfected with
- HIV-1 and mycoplasma may be involved in the pathogenesis
of acquired
- immunodeficiency syndrome (AIDS).
-
- Abstract from Science 251, 1074 (1991). Since the presence
of M. fermentans
- incognitus is most often associated with AIDS and other
acute fulminant
- disease states and more profoundly affects the course
of its disease, it
- can be used to determine the prognosis of these diseases,
which information
- can be utilized for designing therapy regimes. Without
being bound by any
- proposed mechanism, it is believed that antibodies against
ORF-1 (see
- below) may react against CD4.sup.+ lymphocytes resulting
in an
- auto-antibody response against CD4 on T cells thus enhancing
the cytopathic
- effects of HIV-1 on T cells.
-
-
- I. DNA Characteristics of M. fermentans incognitus
-
- M. fermentans incognitus was originally isolated from
Kaposi sarcoma tissue
- of an AIDS patient. The DNA genome of the M. fermentans
incognitus is
- greater than 150 kilobase (kb) pairs and carries repetitive
sequences. An
- 8.6 kb pair cloned probe (psb-8.6) and a 2.2 kb pair
cloned probe (psb-2.2)
- of M. fermentans incognitus detected specific sequences
of DNA in Sb51
- cells and M. fermentans incognitus infected cells, but
not in DNA of
- uninfected NIH/3T3 cells.
-
- The cloned probes (psb-8.6 and psb-2.2) can be obtained
from an EcoRI
- partial digest of M. fermentans incognitus enriched DNA
which is cloned
- into bacteriophage lambda charon 28. The lambda-recombinant
clones are
- screened by differential plaque hybridization with .sup.32
P-labeled DNA
- derived from gradient banded M. fermentans incognitus.
The insert of the
- phage clone is then recloned into the EcoRI site of Bluescript
KS (M
- 13.sup.-) vector (Stratogene) to produce the cloned probes,
psb-8.6 and
- psb-2.2.
-
- By nucleic acid analysis, the M. fermentans incognitus
has been compared
- with large DNA viruses of the herpes group such as herpes
simplex virus
- type I and II (HSV-I and II), human cytomegalovirus (CMV),
Epstein-Barr
- virus (EBV), Varicella-Zoster virus (VZV) and human B-lymphocytic
virus
- (HBLV) or human herpesvirus-6 (HHV-6), vaccinia virus,
Herpesvirus saimiri
- (HVS) of monkeys and mouse cytomegalovirus (MCMV). Part
of the M.
- fermentans incognitus genomic DNA has been molecularly
cloned. The entire
- sequence of a cloned M. fermentans incognitus psb-2.2
DNA has been obtained
- and is shown as SEQ ID NO:2.
-
- To obtain the genetic materials of M. fermentans incognitus,
the Kaposi's
- sarcoma tissue is minced into small pieces and treated
with collagenase.
- The tissue suspension is then treated with a proteinase,
such as proteinase
- K. Genetic materials are obtained after phenol extraction,
- phenol/chloroform/ isoamylalcohol extraction, and chloroform/isoamylalcohol
- extraction. High molecular weight DNA is visibly observed
after ethanol
- precipitation of the genetic materials. The genetic materials
are dissolved
- and contain high molecular weight DNA and RNA of various
sizes.
-
- The isolated genetic materials from Kaposi's sarcoma
are utilized to
- transfect NIH/3T3 cells or other proper recipient cells
in accordance with
- the procedure of Graham, F. L., et al., Virology 52,
456 (1973). In this
- procedure, the nucleic acid is precipitated with calcium
phosphate and
- incubated with NIH/3T3 cells. The precipitated nucleic
acid is removed and
- the cells trypsinized. The trypsinized cells are reseeded
and treated with
- glycerol before splitting, as described by Copeland,
N. G., et al., Cell
- 16, 347 (1979). The subcultures are fed with Dulbecco's
medium with fetal
- bovine serum (FBS) and re-fed at three- to four-day intervals.
-
- Foci of morphologically transformed cells become evident
in about two
- weeks. The phenotypical transformation is characterized
by rapid overgrowth
- of the transfected cells which pile up in multilayers
and form grossly
- visible foci. Transformation efficiency is about 0.01-0.02
identifiable
- foci per microgram of donor nucleic acid. Transformed
colonies are
- harvested after three weeks, and are cultured in monolayers.The
DNA of
- transformants contain human repetitive DNA sequences.
-
- Genetic materials are isolated from the primary transfectants
as previously
- described, and used to transfect fresh NIH/3T3 cells.
Transformation is
- again seen using the genetic materials with a slightly
higher
- transformation efficiency. This demonstrates that the
genetic materials
- isolated from tissues of AIDS patients contain active
transforming
- elements. This is the first description ever of mycoplasmal
DNAtransfecting
- cells.
-
- The nucleotide sequence of the M. fermentans incognitus
EcoRI 2.2 kb DNA
- (plasmid psb 2.2) is shown in SEQ ID NO:2. This plasmid
has a segment of
- unique sequences which occurs repeatedly in the M. fermentans
incognitus
- genome.
-
- By sequence analysis, a genetic element of 1405 base
pairs (SEQ ID NO:3)
- with unique structural characteristics was identified.
These unique
- structural characteristics strongly resemble bacterial
insertion sequence
- (IS) elements. The IS-like element occurs repeatedly
in the M. fermentans
- incognitus genome.
-
- In analyzing the M. fermentans incognitus EcoRI 2.2 kb
DNA, one pair of
- inverted repeats (IR) consisting of 29 bp with seven
mismatches was found.
- These IR are SEQ ID NO:4 (left IR) and SEQ ID NO:5 (right
IR). Immediately
- outside and flanking these 29-bp IR is a 3-bp direct
repeat (DR), TTT. The
- element framed by these two 29-bp IR contains 1405 bp
(SEQ ID NO:3). Many
- pairs of IR that have eight or more contiguous nucleotides
are also found
- within this 1405-bp element. There are two potential
stem-and-loop (s&1)
- structures, L and R, in the element (see FIG. 5) (SEQ
ID NO:3).
- L(.DELTA.G=-16.8 kcal/mol) is located exactly at the
left terminus of the
- element, while R (.DELTA.G=-14.4 kcal/mol) is located
very near the right
- terminus. Both of the potential s&1 structures are
followed by a stretch of
- T residues pointing toward the interior of the element.
These s&1
- structures with T resides strongly resemble transcription
terminators
- (Rosenberg and Court, Annu. Rev. Genet., 13 319 (1979),
which would prevent
- transcription from the outside into the element (Syvanen,
Annu. Rev.
- Genet., 18 271 (1984)). The structures may also be responsible
for the
- strong polarity of this element (Grindley and Reed, Annu.
Rev. Biochem., 54
- 863 (1985)). Similar transcription terminators have been
found at the
- termini of several bacterial IS elements. These unique
structures are
- probably maintained for specific benefit of the IS elements
and play an
- important role in the regulation of transposition.
-
- Mycoplasma DNAs are extremely rich in A and T. It has
already been shown in
- the codon usage of ribosomal protein genes of M. capricolum
that synonymous
- nucleotide substitution and conservative amino acid substitution
can occur
- (Muto et al., Nucleic Acids Res., 12 8209 (1984)). It
has also been
- reported that TGA, instead of being a stop codon, is
a Trp codon in many
- species of mycoplasma (Yamao et al., Proc. Natl. Acad.
Sci. USA, 82 2306
- (1985)); Inamine et al., J. Bacteriol., 172 504 (1990)).
According to this
- unique character of codon usages in mycoplasma, three
potential ORFs,
- ORF-1, ORF-2, and ORF-3 (SEQ ID NO:6, 7 and 8, respectively)
have been
- identified in the 2.2-kb DNA. ORF-1 and ORF-2 are located
inside the
- element and ORF-3 is located on the complementary strand
100-bp away from
- the element.
-
- ORF-1 (SEQ ID NO:6) begins immediately after the s&1
structure L at
- nucleotide 176 and ends at nucleotide 604, and could
encode a protein of
- 143 amino acids (SEQ ID NO:9). There is a possible Shine
Delgarno (SD)
- sequence, AAGGGG (nucleotides 161-166), which precedes
the start codon of
- ORF-1 by 9-bp, and is located inside the s&1 structure
L (FIG. 5, SEQ ID
- NO:2 and 3, respectively). There is no consensus sequence
for the -10 and
- -35 promoter regions, however, the left IR may provide
a promoter function
- which has been previously shown in the E. coli IS1 element
(Machida et al.,
- J. Mol. Biol., 177 229 (1984)).
-
- ORF-2 (SEQ ID NO:7) begins at nucleotide 1149 and ends
at nucleotide 1457,
- immediately in front of the s&1 structure R, and
could encode a protein of
- 103 amino acids (SEQ ID NO:10). There is a promoter-like
region which has a
- -35 region (TTGATT) at nucleotides 1090-1095 and a -10
region (TAGGTT) at
- nucleotides 1114-1119 located upstream from ORF-2 (FIG.
5, SEQ ID NO:2 and
- 3, respectively). ORF-3 (SEQ ID NO:8), between nucleotide
1912 and 1637 (on
- the complementary strand), could encode a 92-amino acids
protein (SEQ ID
- NO:11) (FIG. 5, SEQ ID NO:2 and 3, respectively).
-
- A computer search of the National Biomedical Research
Foundation (NBRF)
- Protein Data Bank has revealed a 40% homology (49% with
conservative
- replacements) between a region of the deduced amino acid
sequence of ORF-1
- (SEQ ID NO:9; amino acid 101-140) and Streptococcus pyogenes
Pep M5 protein
- (amino acids 23-65). The biological function of antiphagocytosis
in this
- pathogenic bacteria is known to be associated with Pep
M5 protein (Fox,
- Bacteriol. Rev., 38 57 (1974)). The search also revealed
that 75% of the
- amino acids are identical between a region of the deduced
amino acid
- sequence of ORF-1 (SEQ ID NO:9, amino acid 117-128) and
the sequence in the
- extracelluar V4 domain of human T-cell surface glycoprotein
CD4 molecule
- (amino acid 319-329). Another extracellular domain (V1)
of the same CD4
- molecule is critical for recognition by HIV envelope
glycoprotein (Arthos
- et al., Cell, 57 469 (1989)). The significance of the
homologies of ORF-1
- with Pep M5 protein and the CD4 molecule on human T cells
is not clear at
- this time, but this 75% homology between the amino acid
sequence of ORF-1
- and CD4 is enough difference to result in the production
of antibody to the
- ORF-1 antigen. However, this antibody may then attack
both the ORF-1
- antigen and the CD4 receptors due to their similarity.
-
- In a similar analysis, a 43% homology (55% with conservative
replacements)
- between a region of the deduced amino acid sequence of
ORF-2 (SEQ ID NO:10,
- amino acid 18-74) and the deduced amino acid sequence
of the putative
- transposase of E. coli IS3 (SEQ ID NO:12, amino acid
189-245) was found. In
- addition, the ratio of basic to acidic amino acid in
protein predicted by
- ORF-2 is around 2. Thus, this basic protein encoded by
ORF-2 highly
- resembles the E. coli putative transposase which is believed
to be
- essential for transpositional recombination (Grindley
and Reed, Annu. Rev.
- Biochem., 54 863 (1985)). No significant homology was
found between ORF-3
- and sequences in the NBRF Protein Data Bank. Also there
is no significant
- homology between the nucleotide sequence of 2.2-kb DNA
(SEQ ID NO:2) and
- the nucleotide sequences in the GenBank database.
-
- It has been shown that this cloned DNA (psb-2.2; ID SEQ
NO:2) contains a
- unique sequence which occurs more than ten times in the
genome of M.
- fermentans incognitus (Lo et al., Am. J. Trop. Med. Hyg.,
40 213 (1989))
- (also FIG. 6). To precisely define the boundary of this
repetitive element,
- a series of ten oligos, B through K, were synthesized
and used as probes.
- Each probe contained 20-24 nucleotides of a specific
sequence from a
- selected segment in 2.2-kb DNA (FIG. 5). The nt positions
of the synthetic
- oligo, B through K, used as serial probes to identify
the boundary of the
- IS-like repetitive element in the M. fermentans incognitus
genome (see FIG.
- 4) as follows: B (1659-1678), C (1531-1550), D (1514-1533),
E (1454-1477),
- F (1228-1247), G (681-700), H (328-347), I (129-148),
J (115-135), and K
- (44-65) of SEQ ID NO:2. Among the ten oligos, D to I
are a series of
- representative sequences within the 1405-bp IS-like element,
and I and D
- represent sequences within the left and right terminal
IR, respectively. B,
- C, J, and K represent sequences outside the element.
Both J and C represent
- the sequence of the junction areas of the element and
actually carry a part
- of the sequence of the left and right IR, respectively.
Each of these
- synthetic oligo probes was end-labeled with .sup.32 P
and used individually
- to probe M. fermentans incognitus genomic DNA predigested
with either EcoRI
- or HindIII.
-
- The hybridization patterns of multiple bands produced
by probes D to I,
- which carry representative sequences of the various segments
in the IS-like
- element, were essentially the same. In EcoRI digestion,
there are eleven
- identical bands with sizes ranging from 2.20 to 8.90
kb (FIG. 6, D-I, lanes
- b). When using HindIII digestion, there are twelve identical
bands with
- sizes ranging from 1.95 to 9.10 kb (FIG. 6, D-I, lanes
a, b). This pattern
- of multiple hybridization bands matches exactly with
that produced when
- psb-2.2 DNA is nick-translated and used as a probe (FIG.
6A).
-
- In contrast, the probes B, C, J and K produced a completely
different
- pattern with only a single hybridization band of 2.2-kb
in EcoRI digestion
- or a 1.95-kb fragment in HindIII digestion (FIG. 6B,
C, J and K). Probes I
- (20-mer) and J (21-mer) overlap 7 nucleotides within
the left IR; the
- former produced the typical pattern of multiple bands
(FIG. 6I), however,
- the latter only produced a single band (FIG. 6J).
- It was also noted that probes D(20-mer) and C(20-mer)
overlap by 3
- nucleotides within the right IR; the former produced
the typical pattern of
- multiple bands (FIG. 6D), however, the latter only produced
a single band
- (FIG. 6C). Thus, the 1405-bp IS-like element (SEQ ID
NO:3) which is located
- between nucleotides 129 and 1533, is the repetitive element
which occurs
- more than ten times in the genome of M. fermentans incognitus.
This finding
- suggests that the IS-like element is a mobile element.
Such mobility
- suggests the use of this IS-like element as a means for
inserting other
- sequences into other cells (i.e. the IS-like-element
can be used as a
- cloning vector). The presence of mulitiple gene copies
may result from
- transposition.
-
- The evidence which supports the conclusion that the 1405-bp
element is an
- IS-like element is: (1) the size of the element (1405-bp)
being in the
- range of previously identified bacterial IS elements
(800-2500 bp); (2) the
- presence of 29-bp IR, with seven mismatches located at
both of the termini
- of the element; (3) the presence of a 3-bp DR immediately
flanking outside
- both of the terminal IR; (4) two ORFs (ORF-1 and ORF-2)
which could
- potentially encode two basic proteins; part of the deduced
amino acid
- sequence of ORF-2 being homologous to part of the putative
transposase of
- IS3, and (5) the presence of multiple copies in the genome
of M. fermentans
- incognitus. Several other unique structural features
found in the 1405-bp
- element which are also present in bacterial IS elements
are: (i) the s&1
- structure located close to at least one terminus; (ii)
the presence of a
- large number of sequences with properties of IR, and
(iii) part (9 bp) of
- the sequence in one of the terminal IR found again as
a repeat sequence
- (either direct or indirect) near the other terminal IR
(see SEQ ID NO:2 &
- 3).
-
-
- J. Detection of M. fermentans incognitus DNA by PCR
-
- A polymerase chain reaction (PCR) assay to detect M.
fermentans was
- designed on the basis of specific nucleotide (nt) sequences
found at one
- terminus of the cloned incognitus strain of M. fermentans
DNA psb-2.2 (SEQ
- ID NO:2). Primers (RS47 (SEQ ID NO:13) and RS49 (SEQ
ID NO:14)) were chosen
- to produce an amplified DNA fragment of 160 bp. (See
Examples 16 and 19.)
- The PCR assay detected very specifically the mycoplasmas
of M. fermentans
- species but not other human or hon-human mycoplasmas,
bacteria or
- eucaryotic cell DNA that we tested. However, this highly
specific assay
- using these primers failed to detect some mycoplasmas
of the M. fermentans
- species. Ten fg of DNA consistently yielded a positive
160 bp amplified
- band in DNA isolated from the incognitus strain of M.
fermentans, from a
- strain (k7) previously islated from the bone marrow of
a patient with
- leukemia/lymphoma and from other M. fermentans strains
(MT-2) isolated from
- contaminated human lymphocyte cultures. A thousand fold
higher amount of
- DNA (10 pg) isolated from the prototype strain of M.
fermentans (PG-18, and
- ATCC #19989) as well as DNA from two recent clinical
isolates from patients
- with AIDS tested negative for the diagnostic DNA fragments.
Thus, the
- specific gene arrangement used in this PCR assay was
apparently not
- universally present in the DNA of all M. fermentans species.
-
- A more sensitive PCR assay which is able to detect all
the different
- strains or clinical isolates of M. fermentans, yet remains
highly selective
- or specific, was then developed based on the presence
of multiple copies of
- an insertion sequence-like (IS-like) genetic element
in M. fermentans. The
- actual copy number of the IS-like element found in the
genomes of different
- strains or isolates of M. fermentans may vary and range
from 5 to more than
- 10 copies. A new set of primers (RW004 (SEQ ID NO:15)
and RW005 (SEQ ID
- NO:16)) used to produce an amplified fragment of 206
bp in our new PCR
- assay.
-
- Using the new set of primers and RW006 (SEQ ID NO:17)
as a probe, the
- reaction consistently detected 1 fg of DNA in all M.
fermentans species
- tested (FIG. 7) including the prototype strain PG-18
and new clinical
- isolates from patients with AIDS, whose DNA (up to 10-pg)
tested negative
- in the PCR reaction using the old set of primers. Sensitivity
of this newly
- developmed PCR assay was further verified by successfully
detecting 1 fg of
- the M. fermentans DNA in the presence of 1 ug of non-specific
human
- background DNA. Specificity of the reaction has also
been examined by
- attempting to amply the DNAs isolated from other human
or non-human
- mycoplasmas, common tissue culture contaminating mycoplasmas,
Gram-positive
- or Gram-negative bacteria, mouse, monkey and human cell
culture and/or
- tissue. The reaction does not produce the specific 206
bp DNA fragment.
-
- The present study shows that we have developed a highly
selective assay to
- detect M. fermentans by PCR with remarkable sensitivity.
The assay detects
- all the different strains and the new clinical isolates
of M. fermentans
- that the previous PCR assay using primers RS47 and RS49
failed to detect
- and appears to be 10 times more sensitive. The limitation
of reaction
- sensitivity per assay for our current PCR is 0.1 to 1
fg M. fermentans DNA
- within a background of 1 ug of human DNA instead of 1
to 10 fg of microbe
- DNA in our previous PCR assay. Thus, a molecular technique
selectively
- detecting a single microorganism of M. fermentans is
available.
-
-
- K. Infection and Transfection with M. fermentans incognitus
-
- M. fermentans incognitus is isolated from the transformants,
such as Sb51.
- In general, Sb51 cell pellets are lysed by freezing and
thawing to release
- M. fermentans incognitus particles. The large M. fermentans
incognitus
- particles are pelleted through a sucrose barrier and
banded in a sucrose
- isopycnic gradient. The intact M. fermentans incognitus
particles have a
- density of about 1.17 to about 1.20.
-
- M. fermentans incognitus can be introduced into mice.
In general, the M.
- fermentans incognitus isolated from 5.times.10.sup.6
Sb51 cells is injected
- either intravenously or intraperitoneally into six-week-old
mice. Nude mice
- or immunocompetent mice can be infected. Infection of
nude mice with M.
- fermentans incognitus results in significant mortality
of the infected
- animals. Many symptoms similarly seen in patients with
AIDS are induced by
- the infected mice. Thus, at necropsy, the infected mice
often showed
- prominent systemic lymphadenopathy, neuropathy or lymphoid
depletion with
- varying degrees of plasmacytosis. Signs of immune deficiency
with profound
- cutaneous infection in some of the animals were noted.
Disseminated
- pruritic skin rashes were also common. There were proliferative
lesions of
- spindle cells in the cutaneous tissue and deep viscera.
The immunocompetent
- mice (Balb/c) infected by M. fermentans incognitus were
found to be
- subsequently infected by Pneumocystis carinii, which
is evidence of the
- immunnodeficient state of these infected animals.
-
- Similar diseases are transmitted from animal to animal
by injecting
- filtrated lysates of spleen, lymph nodes or whole blood
from the
- diseasedanimals. M. fermentans incognitus is also identified
in the
- cytoplasm of the cytopathic cells. Some of the infected
mice were found to
- produce prominent antibody against M. fermentans incognitus.
-
- When silver leaf monkeys are inoculated with M. fermentans
incognitus, the
- monkeys show wasting syndromes and die within seven to
nine months after
- inoculation. At necropsy, the monkeys do not show evidence
of opportunistic
- infections, acute inflammatory lesions or malignancy.
M. fermentans
- incognitus-specific DNA can be directly detected in necropsy
tissues of the
- monkeys, by use of polymerase chain reaction method.
M. fermentans
- incognitus infection can be identified in spleen tissue,
liver tissue,
- kidney tissue and brain tissue of the monkeys. Some of
the infected monkeys
- produced antibody to M. fermentans incognitus.
-
- L. Detection of M. fermentans incognitus Antigens
-
- The M. fermentans incognitus pathogen is useful for the
detection of
- antibodies in the sera of patients or animals infected
with M. fermentans
- incognitus. Some of these patients who are infected with
M. fermentans
- incognitus will be patients who have been diagnosed as
having AIDS or
- ARC,Cchronic Fatigue Syndrome, Wegener's Disease, Sarcoidosis,
respiratory
- distress syndrome, Kibuchi's disease, antoimmune diseases
such as Collagen
- Vascular Disease and Lupus and chronic debilitating diseases
such as
- Alzheimer's Disease. In one procedure, presistently M.
fermentans
- incognitus infected cells are grown in low cell density
on sterile glass
- slides. Sera from suspected patients, and normal subjects
are examined in
- an immunoperoxidase staining procedure such as that described
by Hsu, S-M.,
- et al., Am.J.Clin.Path. 80, 21 (1983). Using this assay,
23 of 24 sera from
- AIDS patients showed strong positivity. Serum from the
other AIDS patient
- showed weak positivity. Twenty-six of 30 sera from non-AIDS
normal subjects
- showed no reactivity. The other four non-AIDS patients
showed mild to weak
- reactivity, but much weaker than that of AIDS patients.
In addition, some
- of the sera from experimentally infected animals, as
described above, also
- contained antibodies which reacted with the persistently
M. fermentans
- incognitus-infected cells in this assay procedure. Similarly,
M. fermentans
- infected cells can also be used in this procedure to
detect antibodies in
- sera of infected patients as a result of homologous antigens.
-
- In addition to this procedure, any other procedure for
detecting an
- antigen-antibody reaction can be utilized to detect antibodies
to M.
- fermentans incognitus or M. fermentans in the sera of
AIDS patients or
- patients with ARC. Such procedures include, but are not
limited to, ELISA,
- Western-blot, direct or indirect immunofluorescent assay
and
- immunoradiometric assay. Such assay procedures for the
detection of
- antibodies in sera of AIDS patients or patients with
ARC have been
- described in U.S. Pat. No. 4,520,113, incorporated herein
by reference,
- which uses HTLV-III/LAV as the antigen. Similar procedures
employing M.
- fermentans incognitus or M. fermentans can be used. A
diagnostic kit for
- the detection of M. fermentans incognitus-specific or
M.
- fermentans-specific antibodies can be prepared in a conventional
manner
- using M. fermentans incognitus or M. fermentans. It is
expected that assays
- utilizing these techniques, especially Western-blot,
will provide better
- results, particularly fewer false-positives.
-
- A final procedure for detecting the presence of M. fermentans
incognitus or
- other M. fermentans strains in suspected patients is
by testing for DNA in
- conventional methods, preferably using probes based on
the sequence of the
- IS-like element (SEQ ID NO:3). A preferred method is
the PCR assay
- described above.
-
-
- M. Production of Antibodies to M. fermentans incognitus.
-
- Antibodies against M. fermentans incognitus (or M. fermentans)
can be
- produced in experimental animals such as mice, rabbits
and goats, using
- standard techniques. Alternatively, monoclonal antibodies
against M.
- fermentans incognitus (or other strains of M. fermentans)
antigens can be
- prepared in a conventional manner. Homologous antibodies
are useful for
- detecting antigens to M. fermentans incognitus in infected
tissues such as
- lymph nodes, spleen, Kaposi's sarcoma, lymphoma tissue,
brain and
- peripheral blood cells, as well as sera, of patients
with AIDS. Any
- procedure useful for detecting an antigen-antibody reaction,
such as those
- described above, can be utilized to detect the M. fermentans
incognitus
- antigens in tissues of patients infected by the mycoplasma.
-
- Rabbit antiserum has been prepared using M. fermentans
incognitus. The
- antiserum positively immune stains brain and lymph node
tissue from AIDS
- patients. To produce the antiserum, sucrose gradient
banded M. fermentans
- incognitus or any form of concentrated mycoplasma is
used with complete
- adjuvant and administered to rabbits by intraperitoneal
and subcutaneous
- injections at multiple sites. Serum collected from the
rabbits is then
- preabsorbed with NIH/3T3 cells, mouse liver powder and
normal human
- peripheral mononuclear cells isolated from Ficoll-Hypaque
gradients.
- Monoclonal antibodies may also be prepared by conventional
procedures.
-
- The antibodies are useful for detecting cells which have
been infected by
- M. fermentans incognitus. This capability is useful for
the isolation of M.
- fermentans incognitus from other tissues. For example,
additional M.
- fermentans incognitus can be isolated by co-cultivating
infected tissue
- from patients with AIDS and a suitable recipient cell
line or cells, such
- as lymphocytes. The infected cells are assayed or recognized
by the
- antibody, and M. fermentans incognitus can be obtained
from the infected
- cells as described above. An affinity column can also
be prepared using the
- antibodies and used to purify the M. fermentans incognitus
from the
- infected cell lysate.
-
-
- N. Vaccines
-
- The M. fermentans incognitus pathogen, antigens of M.
fermentans incognitus
- or homologous antigens of other M. fermentans strains
can be utilized as a
- vaccine in a conventional manner to induce the formation
of protective
- antibodies or cell-mediated immunity. The antigens can
be isolated from M.
- fermentans incognitus (or other strains) or can be produced
by conventional
- recombinant DNA techniques. The vaccines are prepared
by usual procedures,
- such as by in vitro cell cultures, by recombinant DNA
techniques, and by
- application of the usual and prescribed controls to eliminate
bacterial
- and/or viral contaminations, according to well known
principles and
- international standard requirements.
- Preferably an inactivated, i.e., attenuated or killed,
vaccine is utilized.
- The M. fermentans incognitus pathogen is isolated from
the infected cells
- grown in monolayers. M. fermentans incognitus is killed
by known procedures
- or modifications thereof, e.g., by the addition of betapropiolactone,
- Formalin or acetylethyleneimine, by ultraviolat radiation,
or by treatment
- with psoralen or psoralen derivatives and long-wavelength
ultraviolet
- light. Alternatively, M. fermentans incognitus is attenuated
by
- conventional techniques and isolated.
-
- The vaccine of the invention may contain one or more
suitable stabilizers,
- preservatives, buffering salts and/or adjuvants. The
vaccine may be
- formulated for oral or parenteral administration. Compositions
in the form
- of an injectable solution contain a proper titer of M.
fermentans
- incognitus as the active ingredient, and may also contain
one or more of a
- pH adjuster, buffer, stabilizer, excipient and/or an
additive for rendering
- the solutions isotonic. The injectable solutions may
be prepared to be
- adapted for subcutaneous, intramuscular or intravenous
injection by
- conventional techniques. If desired, the solutions may
be lyophilized in a
- usual manner to prepare lyophilized injections.
-
- The dosage of M. fermentans incognitus administered will,
of course, depend
- on the mode of administration and the interval of administration.
An
- optimal dose of the active ingredient and an optimal
interval of
- administration can easily be determined by routine preliminary
tests known
- in the art.
-
- The antigens of mycoplasmas such as other strains of
M. fermentans which
- share antigenic determinants with M. fermentans incognitus
can also be used
- as vaccines to induce the formation of protective antibodies
or
- cell-mediated immunity to M. fermentans incognitus. It
has been found that
- antigens of other mycoplasmas share many antigenic determinants
with M.
- fermentans incognitus, but lack the pathogenicity of
M. fermentans
- incognitus. One such mycoplasma which can then be used
in a vaccine against
- M. fermentans incognitus is M. fermentans. Other mycoplasmas
useful in
- vaccines against M. fermentans incognitus can be determined
using
- conventional techniques for comparing nucleotide sequences
for sequence
- homology.
-
-
- O. Other Disease States in Which M. fermentans incognitus
Has Been Implicated
-
- In addition to AIDS, M. fermentans incognitus has been
implicated in a
- number of other Disease states including Chronic Fatigue
Syndrome,Wegener's
- Disease, Sarcoidosis, respiratory distress syndrome,
Kikuchi's disease,
- autoimmune diseases such as Collagen Vascular Disease
and Lupus, and
- chronic debilitating diseases such as Alzheimer's Disease.
M. fermentans
- incognitus may be either a causative agent of these diseases
or a key
- co-factor in these diseases.
-
-
- P. Treatment of M. fermentans incognitus Infection
-
- M. fermentans incognitus is known to be sensitive to
a number of
- antibiotics, including doxycycline, quinalones such as
ciprofloxacin,
- chloramphenicol and tetracycline. Therefore, effective
treatment of any of
- the above implicated diseases should include administration
of antibiotics
- to which M. fermentans incognitus is sensitive.
-
- When using the effective antibiotics as the active ingredients
of
- pharmaceutical compositions, the pharmaceutical compositions
may be
- administered by a variety of routes including oral, intravenous,
aerosol
- and parenteral. The amount of active ingredient (antibiotic)
necessary to
- treat an M. fermentans incognitus infection will depend
on the body weight
- of the patient, but will usually be from about 0.001
to about 100 mg/kg of
- body weight, two to four times daily.
-
-
- Q. Enhancement of HIV-1 Cytocidal Effects in CD4.sup.+
Lymphocytes by M.
- fermentans incognitus.
-
- Coinfection with Mycoplasma fermentans (incognitus strain)
enhances the
- ability of human immunodeficiency virus type-1 (HIV-1)
to induce cytopathic
- effects on human T lymphocytes in vitro.
- Syncytium formation of HIV-infected T cells was essentially
eliminated in
- the presence of M. fermentans (incognitus strain), despite
prominent cell
- death. However, replication and production of HIV-1 particles
continued
- during the coinfection. Furthermore, the supernatant
from cultures
- coinfected with HIV-1 and mycoplasma may be involved
in the pathogenesis of
- acquired immunodeficiency syndrome (AIDS).
-
- Abstract from Science 251, 1074 (1991). Since the presence
of M. fermentans
- incognitus is most often associated with AIDS and other
acute fulminant
- disease states and more profoundly affects the course
of its disease, it
- can be used to determine the prognosis of these diseases,
which information
- can be utilized for designing therapy regimes.
-
- The presence of M. fermentans incognitus in patient tissue
or cell sample
- is determined by conventional techniques such as immunoassays,
PCR analysis
- and DNA hybridizations as more fully described herein.
- The present invention is further illustrated by reference
to the following
- examples. These examples are provided for illustrative
purposes, and are in
- no way intended to limit the scope of the invention.
-
-
- EXAMPLE 1
-
- Isolation of Genetic Materials from AIDS Patients and
Cell Culture
-
- Kaposi's sarcoma tissue was obtained from a patient with
AIDS who died of
- fulminant Pneumocystis carinii pneumonitis. At autopsy,
extensive Kaposi's
- sarcoma involving skin, both lungs, parietal pleura,
gastrointestinal
- tract, pancreas, liver, kidney and lymph nodes was found.
The tissue used
- to extract genetic material was derived from Kaposi's
sarcoma in the
- patient's retroperitoneal lymph nodes, five to six hours
after death.
- Permanent paraffin sections confirmed near-total effacement
of lymph node
- architecture by Kaposi's sarcoma.
-
- Splenic tissue was obtained from a second patient with
AIDS who also died
- of P. carinii pneumonitis. No tumor (i.e., Kaposi's sarcoma
or lymphoma)
- was identified at autopsy. Paraffin sections of the splenic
tissue used to
- extract genetic material showed congestion and lymphocyte
depletion.
-
- The splenic or Kaposi's sarcoma tissue (1-2 g) was minced
into small pieces
- and treated with collagenase (5 mg/ml) in 1 ml phosphate-buffered
saline
- (PBS) at 37.degree. C. for 15 minutes. The tissue suspension
was then
- treated with proteinase K (250 g/ml) in 10X volume of
150 mM NaCl, 10 mM
- Tris (pH 7.5), 0.4% SDS, at 65.degree. C. for 30 minutes
and at 37.degree.
- C. for ten hours. Phenol extraction (twice) followed
by
- phenol/chloroform/isoamylalcohol (25:24:1) and chloroform/isoamylalcohol
- (24:1) extraction were used to purify genetic material.
Grossly visible
- high molecular weight DNA was easily observed after ethanol
precipitation.
- The genetic materials were redissolved in aqueous phase
(1 mM Tris, 1 mM
- EDTA) after overnight air-drying. The recovered genetic
materials contained
- high molecular weight DNA and 30-40% RNA of various size.
The procedures of
- isolating genetic materials from the cultures of the
primary transformants
- and normal human fibroblasts (ATCC, CRL-1521) were similar.
The pellets of
- 10-20.times.10.sup.6 cells were mixed directly with 10X
volume of
- proteinase K (250 g/ml) in the same buffer without collagenase
treatment.
-
-
- EXAMPLE 2
-
- Transfection of NIH/3T3 Cells
-
- The transfection procedures were slightly modified from
that of Graham et
- al., supra. Approximately 30 micrograms of nucleic acid
isolated from
- Kaposi's sarcoma tissue, splenic tissue, normal human
fibroblast, or salmon
- sperm were precipitated with calcium phosphate in each
60 mm Petri dish
- culture (containing about 5.times.10.sup.5 NIH/3T3 cells).
The DNA
- precipitate was removed after cells were incubated at
37.degree. C. for 12
- hours. After an additional 24 hours, each plate of cells
was trypsinized
- and reseeded into four to five 60 mm Petri dishes. The
cells received five
- minutes of 15% glycerol treatment in 10% fetal bovine
serum (FBS, Gibco)
- Dulbecco's modified Eagle's medium (DMEM) before the
splitting as described
- by Copeland et al., supra. The subcultures were fed with
Dulbecco's medium
- with 5% FBS and re-fed with this medium at intervals
of three to four days.
- Foci of morphologically transformed cells became evident
in two weeks.
- Colonies were harvested after three weeks.
-
- NIH/3T3 cells transfected with genetic material derived
from both spleen
- and Kaposi's sarcoma tissue of AIDS patients produced
morphologically
- transformed colonies which were visible within two weeks.
The phenotypical
- transformation was characterized by the rapid overgrowth
of the transfected
- cells which piled up in multilayers and formed grossly
visible foci.
- Transformation efficiency was approximately 0.01 to 0.02
identifiable foci
- per microgram of donor nucleic acid. In contrast, no
transformed foci were
- identified in parallel cultures using DNA from salmon
sperm or nucleic acid
- from human fibroblasts. The transformants were recovered
from these
- phenotypically malignant foci after two weeks and cultured
in monolayers.
- Transformants retained their tendency of piling up in
multilayers and
- reached more than three-fold higher cellular density
than normal NIH/3T3
- fibroblasts.
-
-
- EXAMPLE 3
-
- Confirmation of NIH/3T3 Cell Transformation
-
- To confirm that transformation of the NIH/3T3 cells was
mediated by active
- transforming genetic elements, the primary transformants'
capacity to
- transmit their malignant phenotypes of rapid cell growth
and pile-up (lack
- of cell-cell contact inhibition) in high cellular density
in subsequent
- cycles of transfection was examined. Thus, a second cycle
of transfection,
- as described above, was performed using genetic material
which was isolated
- as previously described from some of the primary transfectants.
A higher
- efficiency of transformation was observed in the second
cycle of the
- transfection assay (up to 0.05 foci per microgram of
donor nucleic acid).
- These results indicate that genetic materials isolated
from spleen and
- Kaposi's sarcoma tissues of the AIDS patients contained
active transforming
- elements that induce malignant transformation of rapid
cell growth upon
- transfection and retransfection of phenotypically normal
cells. DNA from
- first and second stages of transformation clones selected
for further
- studies were then characterized with respect to the presence
of human DNA
- repetitive sequences by probing with .sup.32 P nick-translated
Blur
- 8-plasmid. No human repetitive DNA sequences were detected
in these
- transformants.
-
-
- EXAMPLE 4
-
- Analysis of Transformants
-
- Normal NIH/3T3 and transformant clones were all routinely
maintained in
- monolayer cultures with 10% FBS-supplemented Dulbecco's
media. Autoclavable
- slides (Cell-line Asso. Inc.) were previously sterilized
and overlaid with
- trypsinized cell suspension (1.times.10.sup.5 cells/ml)
in square petri
- dishes. The cultures were incubated at 37.degree. C.
in a 5% CO.sub.2
- incubator for 48 to 72 hours. The culture slides were
washed three times
- with cold PBS, air-dried and stored at 4.degree. C. Immunocytochemistry
was
- performed within two to three days on these stored slides.
-
- The monolayers were scraped directly from the cultures.
The cells were
- harvested by centrifugation of 1,000 rpm for 10 minutes.
The cell pellets
- were fixed overnight at 4.degree. C. in 2.5% glutaraldehyde
in phosphate
- buffer and post-fixed with 1% OsO.sub.4. The fixed tissues
were then
- processed by standard methods and embedded in Maraglass
655. The grids with
- ultra-thin sections were double-stained with uranylacetate
and lead
- citrate. The specimens were then examined under an electron
microscope with
- 60 kv or 100 kv voltage. Negative staining of the virus-like
particles in
- the culture supernatants was performed. Briefly, the
particles in the
- culture supernatant were pelleted through a 5 ml 20%
sucrose barrier in
- SW41 centrifugation tubes, at 40,000 rpm for one hour.
The pellets were
- then resuspended in 1/50 to 1/100 volume of Tris-normal
saline (pH 7.4,
- 0.05M Tris). The suspensions were directly put on formvar
coated grids and
- negatively stained with 2% phosphotungstic acid (PTA)
(pH 7.2).
-
- Two of the transformants (Sb51 and Kb43, from different
patients) were
- studied in detail. These two transformants were obtained
from the second
- cycle transfections with genetic materials from Kaposi's
sarcoma spleen and
- tissues, respectively. Sb51 cells persistently infected
with M. fermentans
- incognitus were deposited with the ATCC under No. CRL
9127 under the
- Budapest Treaty on Jun. 17, 1986. The cells grew in high
cellular density
- with no significant cytopathic changes. However, occasional
lytic plaques,
- with cells showing cytopathic changes, were noted after
the transformants
- reached saturated density.
-
- Many physiologic factors, including incubation temperatures
and culture
- media, were found to affect the degree of lytic plaque
formation. For
- example, a reduction in the temperature to 32.degree.
C. results in higher
- lytic plaque formation. Sb51 cells tended to pile-up
in a monolayer
- culture. Foci of rapid cell overgrowth and pile-up into
multicellular
- layers can best be appreciated under low-power light
microscopy with a dark
- background. Cytopathic changes commonly occurred in the
centers of the high
- cell density foci. Detachment of the cytolytic cells
in the center of
- hyperplastic foci was evident. There were prominent cytopathic
effects
- among the densely-packed cells on the peripheral edges
of the lytic plaque.
-
- These cells rounded up and appeared smaller in size with
a shrunken
- configuration.
-
- The monolayers of Sb51 and Kb43 which showed significant
cytopathic changes
- in at least 30% of cells were examined by electron microscopy.
-
- In those cells undergoing cytopathic changes numerous
M. fermentans
- incognitus cells were seen, mainly in the cytoplasm of
disrupted cells.
- Early cytopathic changes showing nuclear chromatin condensation
and
- margination was seen at 15,000X magnification. Accumulation
of M.
- fermentans incognitus nucleocapsids within the nucleus
is prominent.
- Numerous M. fermentans incognitus particles of different
maturation stages
- were seen in the cytoplasm at 45,800X magnification.
Most of the mature M.
- fermentans incognitus cells in the cytoplasm are lined
up along the plasma
- membrane while others are free. The M. fermentans incognitus
cells were
- roughly spherical enveloped particles of heterogenous
sizes. The majority
- of mature M. fermentans incognitus cells were 140-280
nm, with an overall
- range of 100-900 nm. The intact M. fermentans incognitus
particle had a
- well-defined outer limited membrane about 8 nm thick
and tightly packed
- internal nucleocapsids. Occasionally, the nucleocapsids
were seen to
- condense into compact cores inside the M. fermentans
incognitus cell.
- Although the M. fermentans incognitus outer envelope
was well-defined and
- thick, it was not rigid. Elongated, ovoid, and pleomorphic
forms with
- protrusions were not uncommonly identified among the
M. fermentans
- incognitus cells (at 45,800X magnification).
-
- To further confirm the ultrastructure and morphology
of M. fermentans
- incognitus, the unsectioned M. fermentans incognitus
were examined by
- pelleting M. fermentans incognitus particles from Sb51
and Kb43 culture
- supernatants through a 20% sucrose gradient barrier.
The particles were
- resuspended in Tris-normal saline at 1/100 of original
volume. The
- precipitated particles were directly examined under electron
microscopy
- following negative stainings with PTA. Some preparations
of the intact M.
- fermentans incognitus particles were briefly fixed with
0.5% Formalin to
- preserve the M. fermentans incognitus morphology as well
as to avoid
- possible infectious problems in the laboratory. The negative
staining
- preparations of M. fermentans incognitus usually revealed
more surface
- detail together with their internal structure. There
was some heterogeneity
- in both particle size and shape. Some M. fermentans incognitus
cells often
- appeared to be elongated or had irregular bulging protrusions
(when viewed
- at 101,800X magnification. The internal component consisted
of strands
- arranged more or less parallel to each other and to the
long axis of the
- particle. The internal nucleocapsid strands appeared
to be better preserved
- in the particles fixed with low concentrations of Formalin.
The
- well-defined envelope revealed inconspicuous spikes on
the external
- surface. At high magnification (370,000X), M. fermentans
incognitus
- demonstrated complex membranous envelopes. The released
nucleocapsids
- appear to be uncoiled.
-
-
- EXAMPLE 5
-
- PCR Assay for M. fermentans incognitus
-
- An assay of urine sediments prepared in Example 6 is
illustrative of a PCR
- assay. The amplification of selective DNA sequences was
performed with
- thermostable Taq DNA polymerase (Native Taq; Perkin Elmer
Cetus, Norwalk,
- Conn.) (10) in the automated Perkin-Elmer Cetus DNA thermal
cycler
- (Norwalk, Conn.). One ml of urine sediment prepared and
filtered as
- described in Example 6 was first centrifuged at 1,500
x g for 15 min.
- Nine-hundred ul of the supernatant was removed. Proteinase
K was added to
- the remaining 100 ul sample (final concentration of 200
ug/ml) and the
- sample was digested at 56.degree. C. for 2 hrs. Before
PCR analysis the
- digested samples were heated at 95.degree. C. for 10
min. Each 10 ul urine
- sediment sample to amplified was adjusted to a total
volume of 160 ul with
- PCR buffer containing a final concentration of 50 mM
KCl, 20 mM Tris-HCl
- (pH 8.3), 1 mM MgCl.sub.2, 0.001% gelatin, each primer
(RW004 (SEQ ID
- NO:15) and RW005 (SEQ ID NO:16) (R. Y-H Wang et al.,
Abstr. Gen. Meet. Am.
- Soc. Microbio. 1991, G-5, p. 134) at 0.5 uM, each dNTP
at 250 uM and 2.5 U
- of Taq DNA polymerase. It has been found that these primers
are preferred
- over the RS47 and RS49 primers used in PCR assays below
(Example 16 and
- 19). The samples were overlaid with 3 drops of mineral
oil (50 ul). Samples
- were denatured at 94.degree. C. for 35 sec, annealing
of primers at
- 56.degree. C. for 45 sec and extension at 72.degree.
C. for 1 min. The
- annealing time was increased by one sec/cycle during
the amplification.
-
- After the final cycle, the annealing time was increased
to 5 min, followed
- by extension for 5 min. Twenty ul aliquots from each
amplified sample were
- removed and analyzed on a 6% polyacrylamide gel in 1.times.
- Tris-borate-EDTA buffer (Maniatis et al., Molecular Cloning:
a laboratory
- manual. Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y. (1982)).
- The gels were stained with ethidium bromide and the DNA
visualized by UV
- fluorescence. The fractionated DNA was electroblotted
onto a Zeta-Probe
- membrane (Bio-Rad, Richmond, Calif.) at 100 volts for
2 hrs., in 0.5.times.
- Tris-acetate-EDTA buffer (Maniatis et al., supra), followed
by denaturation
- and fixation in 400 mM NaOH, 2 mM EDTA for 10 min. at
room temperature. The
- Zeta-Probe membrane was rinsed 3 times with 2.times.
SSC in 20 mM Tris-HCl
- (pH 7.5) and air dried for 10 min. Prehybridization was
carried out in 30%
- formamide, 4.times. SSC, 5.times. Denhardt's, 20 mM Tris-HCl,
(pH 7.5), 2
- mM EDTA, 1% SDS and 350 ug/ml of denatured salmon sperm
DNA at 30.degree.
- C. Hybridization was in the same mixture but contaiing
the oligonucleotide
- probe RW006 (SEQ ID NO:17) (Wang et al., Abstr. Gen.
Meet. Am. Soc.
- Microbiol. 1991, G-5. p. 134) which was 5'end labeled
with .sup.32 P-ATP,
- and was conducted overnight at 30.degree. C. After hybridization
the
- membrane was washed at 45.degree. C. in 2.times. SSC,
0.5% SDS four times
- (30 min. each).
-
- Forty-three urine sediments obtained from 40 HIV positive
patients and 50
- urine sediments obtained from HIV negative healthy control
individuals were
- tested for the presence of M. fermentans specific DNA
sequences by using
- the PCR assay. Primer pairs of synthetic oligonucleotides,
designated RW004
- (SEQ ID NO:15) and RW005 (SEQ ID NO:16) containing specific
sequences
- within the insertion sequence (IS)-like genetic element
found in multiple
- copies in M. fermentans mycoplasmas were used to amplify
a 206 bp segment
- of the IS-like DNA. Ten of 43 urine sediments obtained
from HIV positive
- patients with varying stages of AIDS disease, tested
positive for the
- presence of M. fermentans DNA. In contrast, none of the
50 urine sediments
- obtained from HIV negative non-AIDS controls tested positive.
FIG. 8 shows
- the PCR results of representative samples from HIV negative
controls (FIG.
- 8, lanes b and c) and HIV positive patients' urine sediments
(FIG. 8, lanes
- d-m). Lane n contained one femtogram M. fermentans incognitus
DNA diluted
- into one microgram of human placental DNA and lane o
contained pUC18 DNA
- digested with MspI, serving as size markers. A distinct
band could be
- observed in the ethidium bromide stained gel at a position
corresponding to
- the 206 bp fragment amplified in M. fermentans control
DNA (FIG. 8A, lane
- n), and in positively amplified AIDS patients' urine
sediments (FIG. 8A,
- lanes d-f, h, k and l). The RW006 (SEQ ID NO:17) probe
hybridized strongly
- to all positively amplified samples (FIG. 8B, lanes d-f,
h, k, l, and n).
-
- Using a similar procedure, M. fermentans species including
the prototype
- strain PG-18 and new clinical isolates from patients
with AIDS, which had
- tested negative in previous PCR reactions were analyzed
in a PCR reaction
- using RW004 (SEQ ID NO:15) and RW006 (SEQ ID NO:16) as
primers. The assay
- consistently deteted 1 fg of DNA in all species (FIG.
7). Specificity of
- the reaction has also been examined by attempting to
amply the DNAs
- isolated from other human or non-human mycoplasmas, common
tissue culture
- contaminating mycoplasmas, Gram-positive or Gram-negative
bacteria, mouse,
- monkey and human cell culture and/or tissue. The reaction
does not produce
- the specific 206 bp DNA fragment
- (Table 1).
-
- TABLE 1
- ______________________________________
- SPECIFICITY OF PCR FOR M. FERMENTANS
- USING UNIQUE SEQUENCES WITHIN THE
- IS-LIKE GENETIC ELEMENT
- Concentration
- of DNA
- Sources tested Positivity
- ______________________________________
- Mycoplasmas
- M. fermentans
- ATCC 19989 1 fg +
- incognitus strain 1 fg +
- PG-18 1 fg +
- K-7 1 fg +
- MT-2 1 fg +
- and nine clinical isolates
- 1 fg +
- M. hominis (ACTCC 15488)
- 1 ng -
- M. orale (ATCC 23714)
- 1 ng -
- and one clinical isolate
- 1 ng -
- M. salivarium (ATCC 23064)
- 1 ng -
- and two calinical isolates
- 1 ng -
- M. buccale 1 ng -
- M. pneumoniae (ATCC 15531)
- 1 ng -
- M. genitalium (ATCC 33530)
- 1 ng -
- M. arginini (ATCC 23838)
- 1 ng -
- M. pirum 1 ng -
- M. alvi 1 ng -
- M. moatsii 1 ng -
- M. sualvi 1 ng -
- M. iowae 1 ng -
- M. arthritidis 1 ng -
- M. hyorhinis (ATCC 17981)
- 1 ng -
- Acholeplasma laidlawii (ATCC 23206)
- 1 ng -
- Ureaplasma urealyticum
- 1 ng -
- (ATCC 27618)
- Bacteria
- E. coli 1 ug -
- Streptococcus pneumoniae
- 1 ug -
- Clostridium perfringens
- 1 ug -
- Mouse
- NIH/3T3 1 ug -
- Spleen (Balb/c) 1 ug -
- Liver (Balb/c) 1 ug -
- Brain (Balb/c) 1 ug -
- Monkey
- Vero cells (ATCC CCL18)
- 1 ug -
- Spleen (green monkey)
- 1 ug -
- Liver (green monkey)
- 1 ug -
- Brain (green monkey)
- 1 ug -
- Human
- CCRF-cem (ATCC CCL119)
- 1 ug -
- Placenta (nl. delivery) 4X)
- 1 ug -
- PBMC (nl. donor) 50X
- 1 ug -
- ______________________________________
-
-
- EXAMPLE 6
-
- Direct Isolation of AIDS-associated Myoplasma From Infected
Tissues of AIDS
- Patients
-
- Urine was collected in sterile containers and concentrated
10-fold by
- centrifugation (3000.times.g for 15 min. at 4.degree.
C.) and resuspended
- in 1/10 of the original urine. The resulting urine sediments
were diluted
- 1:10 in modified SP-4 media (Lo et al. (1989(a), Am.
J. Trop. Med. Hyg. 41:
- 586-600) and then filtered through a 0.22 um filter.
-
- The filtered urine sediments (10 ml), previously diluted
in modified SP-4
- media, were cultured in 25 cm.sup.2 tissue culture flasks
and also cultured
- with a further 1:10 aerobically and in GasPak jars (BBL,
Microbiology
- Systems, Cockeysville, Md.) anaerobically. Flasks showing
a color change
- were subcultured to modified SP-4 agar to confirm the
mycoplasma growth.
- Speciation of various mycoplasma colonies obtained was
assayed by
- immunofluorescence of colonies on agar using species-specific
- FITC-conjugated antibodies (Del Guidice et al. (1967),
J. Bacteriol.
- 93:1205-1209).
-
- Restriction endonuclease cleavage and Southern blot hybridization
of
- genomic DNA from prototype strains and new clinical isolates
of M.
- fermentans was carried out basically as previously described
(Lo et al.
- (1989a), supra; Lo et al. (1989b). Am. J. Trop. Med.
Hyg. 41:213-226). DNA
- was isolated from cultures of each isolate or strain
of M. fermentans,
- purified by standard methods, and digested with either
EcoRI or HindIII
- restriction enzymes (Gibco-BRL, Gaithersburg, Md.). The
enzyme digests of
- NDA, after electrophoresis in 1% agarose, were transferred
to a Zeta-Probe
- membrane by the Southern blot method. Each filter was
prehybridized in 50%
- formamide, 4 x SSC, 5 x Denhardts', 20 mM Tris-HCl (pH
7.5), 2 mM EDTA, 1%
- SDS, and 250 ug/ml denatured salmon sperm DNA for at
least 4 hrs at
- 42.degree. C. and hybridized with .sup.32 P nick-translated
psb-2.2 DNA (Hu
- et al. (1990). Gene 93:67-72) at 42.degree. C. in the
prehybridization
- solution as described, (Lo et al. (1989b), supra). After
hybridization the
- blots were washed at 55.degree. C. in x 2 SSC, 0.5% SDS,
10 mM Tris-HCl (pH
- 7.5) for 120 min. with 4 changes and then washed at 50.degree.
C. in 0.5 x
- SSC, 0.1% SDS for 60 min. with 2 changes before autoradiography
(Lo et al.
- (1989b), supra).
-
- M. fermentans was isolated and grown in modiifed SP-4
media from 3 of the
- AIDS patients' urine sediments which tested positive
in the PCR assay of
- Example 5. DNA was prepared from cultures of the new
clinical isolates and
- compared with that of representative M. fermentans strains
in Southern blot
- analysis. The DNASs were digested with EcoRI (a lanes)
or HindIII (b
- lanes), fractionated in an agarose gel and hybridized
with .sup.32
- P-labeled psb-2.2 (FIG. 9). Lane m is HindIII digested
lambda phage DNA
- used as marker of 23.1, 6.6, 4.4, 2.3 and 2.0 kb, respectively.
The new
- clinical isolates (FIG. 9, D and E) have similar but
distinct restriction
- enzyme patterns from K7 strain (FIG. 9,A) PG18 prototype
strain (FIG. 9,B),
- original M. fermentans incognitus (FIG. 9,C) which indicates
that they are
- indeed independent isolates.
- M. fermentans mycoplasmas were successfully isolated
and grwon in
- mycoplasma culture from 3 urine sediments derived from
2 HIV positive
- individuals (Table 1). Five Ureaplams Urealyticum and
two M. hominis were
- also isolated from the 43 cultures of AIDS patients'
urine sediments. Fifty
- urine sediments similarly prepared from age-matched HIV
negative healthy
- controls did not grow M. fermentans mycoplasmas. In this
study, 23
- Ureaplasma Urealyticum and M. hominis were isolated from
the 50 control
- urine sediments (Table 2).
-
-
- TABLE 2
- ______________________________________
- Isolation of Different Species of
- Mycoplasma and Ureaplasma urealyticum
- from Urine of HIV positive AIDS patients
- and HIV negative non-AIDS controls
- Source of Urine
- HIV Positive HIV Negative
- Species AIDS Patients
- Controls
- ______________________________________
- M. fermentans 3/43.sup.a (7.0%).sup.b
- 0/50 (0%)
- M. fermentans 2/43 (4.7%) 1/50 (2.0%)
- U. urealyticum
- 5/43 (11.6%) 23/50 (46.0)%
- ______________________________________
- .sup.a Number of isolates over number of samples cultured
- .sup.b Percentage of isolation
-
-
- EXAMPLE 7
-
- Isolation and Gradient Banding of M. fermentans incognitus
-
- Sb51 cells grown as monolayers were briefly trypsinized
and pelleted by
- centrifugation at 1,000 rpm for 10 minutes. The cell
pellet was resuspended
- with an equal volume of Dulbecco's medium. The cells
were lysed by five
- cycles of freezing and thawing to release the cell-associated
M. fermentans
- incognitus particles. The particles were pelleted through
a 20% sucrose
- barrier in a SW41 centrifuge tube by centrifugation at
40,000 rpm for 45
- minutes. The particles were resuspended in PBS and banded
in a sucrose
- isopycnic gradient (20% to 60%). Electron micrographs
of the M. fermentans
- incognitus cells in the cytoplasm of degenerating Sb51
cells is shown in
- FIG. 10. The M. fermentans incognitus particles were
localized at a density
- of about 1.17 to about 1.20 (FIGS. 10(B) and 10(C)).
The M. fermentans
- incognitus particles were directly identified by electron
microscopy with
- PTA negative staining.
-
-
- EXAMPLE 8
-
- Production of Antibodies Against M. fermentans incognitus
-
- M. fermentans incognitus particles were isolated as described
in Example 7
- from 5.times.10.sup.6 Sb51 cells, and mixed with Freund's
adjuvant. Rabbits
- were injected with the immunogen twice at a two- to three-month
interval. A
- good antibody response to M. fermentans incognitus was
obtained after the
- second immunization.
-
-
- EXAMPLE 9
-
- Infection of Mice by M. fermentans incognitus
-
- M. fermentans incognitus was isolated as described in
Example 7, from
- 5.times.10.sup.6 Sb51 cells, and resuspended in a small
amount of PBS. The
- M. fermentans incognitus suspension was injected into
either a six-week-old
- NIH (Nu) male mouse or a six-week-old Balb/c male mouse.
The injection was
- performed either intravenously or intraperitoneally.
Sixty percent of the
- nude mice who received intravenous or intraperitoneal
injections of the M.
- fermentans incognitus preparation showed evidence of
skin rashes with areas
- of erythematous changes and conjunctivitis in 10 to 12
days. One animal
- also showed prominent periorbital edema. These signs
disappeared after two
- to three weeks. All the animals appeared to recover from
the acute
- infection. Two animals then developed pruritic skin rashes
after six weeks.
- These two animals and the other two animals died or became
too sick, and
- had to be sacrificed in three months. Therefore, 40%
of the animals had
- died in the first three months following injection. One
animal which did
- not develop recognizable skin lesions showed systemic
lymphadenopathy and
- paralysis. The animal appeared to be wasting and experienced
complete
- paralysis of its hind legs. One animal had several purplish
skin lesions
- which were slightly raised. At necropsy, all lymph nodes
in these animals
- showed lymphocyte depletion. Only very small lymph nodes
were identified on
- gross examination. In contrast, disseminated lymphadenopathy
was seen in
- the inguinal, axillary, cervical, mediastinal and mesentery
lymph nodes.
- The animal also developed hepatosplenomegaly. Histologic
sections of the
- lymph nodes revealed prominent plasmacytosis. Areas of
sinus histiocytosis
- were also noted. The plasma cell effaced normal lymph
node architecture and
- diffusely infiltrated the sinus. Lymph nodes in all the
other animals
- showed lymphocyte depletion. Only small lymph nodes could
be identified
- grossly.
-
- Histologic sections of purplish skin lesions revealed
spindle cell
- proliferation. The spindle cells appeared to infiltrate
cutaneous adipose
- tissue as well as underlying muscles. Extravasation of
red blood cells was
- seen in some areas. Mitotic figures were identified,
but not prominent.
- Histologic examination of the liver of the animal also
revealed spindle
- cell proliferation in the periportal areas. The homogeneous
tumor cells
- exhibited more epithelioid appearance. Numerous red blood
cells were
- trapped in the intercellular slits.
-
- Electron microscopic examination of the infiltrating
spindle cells in the
- skin lesions revealed cells with cytopathic changes.
An accumulation of M.
- fermentans incognitus nucleocapsids were seen in many
of the nuclei, and
- some in the cytoplasm. The morphology and the characters
of these M.
- fermentans incognitus nucleocapsids were similar to those
observed in Sb51
- cells previously described. Mature M. fermentans incognitus
cells were also
- identified in some of the disrupted cells. Both nucleocapsids
and M.
- fermentans incognitus cells were often seen in dilated
cisternae of smooth
- endoplasmic reticulum. Electron microscope studies of
the periportal
- spindle cell lesions in the liver similarly revealed
prominent infection of
- M. fermentans incognitus.
-
- Balb/c mice infected with the M. fermentans incognitus
also appeared to be
- sensitive to the M. fermentans incognitus pathogen. Three
of seven animals
- died in the first three months following infection. Two
more animals died
- in the fourth month following infection. None of the
control animals showed
- any disease in four months. Clinical evaluation of skin
rashes and
- lymphadenopathy while these animals were alive was much
more difficult. At
- necropsy, all of the animals were found to be lymphocyte-depleted.
The
- animals had very small lymph nodes and spleens. Lymph
nodes were often
- unrecognizable grossly. The lungs of these animals were
found to have
- severe pneumonitis. M-Ag and toluene blue staining revealed
P. carinii.
- Therefore, these animals were believed to be severely
immunodeficient. Two
- of the animals who survived for more than four months
were found to have
- antibody in their sera which recognized Sb51 cells but
not NIH/3T3 parental
- cells. Immunoperoxidase reaction of the sera showed positive
reactions in
- both the nuclei and the cytoplasm of Sb51 cells indicating
the presence of
- M. fermentans incognitus.
-
-
- EXAMPLE 10
-
- Infection of Non-Human Primates with the M. fermentans
incognitus
-
- Four silver leaf monkeys (presbytis cristatus) were inoculated
- (intraperitoneally) with partially purified M. fermentans
incognitus (see
- Example 7 above). All four monkeys displayed a wasting
syndrome as shown in
- FIG. 11, and died within seven to nine months. A control
monkey which had
- been inoculated with a preparation derived from normal
NIH/3T3 cells did
- not exhibit the wasting syndrome and did not die during
the seven- to
- nine-month period.
-
- The monkeys were followed daily for signs of illness,
and examined once
- every two weeks for body weight, body temperature and
general physical
- condition. Serial blood samples were also collected every
two weeks for
- blood cell counts and antibody and antigen assays.
-
- Two weeks after M. fermentans incognitus inoculation,
one monkey showed
- signs of a flu-like syndrome which persisted for six
weeks. This same
- monkey later developed facial/neck edema (between week
8 and week 12), poor
- skin tones, and dermatities associated with alopecia
(after week 18). This
- was the first monkey to succumb, expiring at the 29th
week after M.
- fermentans incognitus inoculation. The animal had apparently
been afebrile
- throughout the whole course, except at the time of the
16th week after M.
- fermentans incognitus inoculation.
-
- Body weights of all M. fermentans incognitus inoculated
monkeys fluctuated.
- However, a progressive weight loss was noted among these
animals in the
- last 14 weeks of the experiment (FIG. 11). No diarrhea
was detected for any
- of the animals. Two of the monkeys also had transient
lymphadenopathy at 4
- to 14 weeks and 4 to 20 weeks after M. fermentans incognitus
inoculation,
- respectively. Three monkeys appeared to have persistent
low grade fever in
- the earlier course of the experiment, but no significant
febrile response
- could be detected in the later stages (the last month).
The moribund
- animals revealed paradoxical hypothermia in the final
periods. One monkey
- revealed signs of tremor, rigidity and imbalance in the
terminal stage. The
- clinical signs strongly suggested a neurological illness.
Accurate
- assessment, however, was hampered by the obvious physical
weakness of the
- animal which may have been due to the prominent weight
loss.
-
- At necropsy, no malignant tumor or opportunistic infection
could be
- identified in any M. fermentans incognitus inoculated
animal.
- Histopathology of the lymph nodes obtained from these
monkeys revealed
- features of lymphocyte depletion. There was spindle cell
proliferation in
- the perinodal areas, but typical diagnosis of Kaposi's
sarcoma could not be
- made.
-
- One animal revealed persistent and significant leukocytosis
that lasted for
- about three months (between 16 to 28 weeks after inoculation).
In contrast,
- two other monkeys showed prominent leukopenia in the
terminal stage.
- Differential cell count revealed that their lymphocytes
were 448, and 410
- per microliter, respectively. Both red blood cell and
platelet counts
- fluctuated. Transient periods of low platelet counts
were observed during
- the course of the study for all animals. However, no
animal was
- thrombocytopenic in the terminal stage.
-
- To study if the M. fermentans incognitus inoculated animals
developed an
- immune response and produced specific antibodies, the
serum samples
- obtained from serial bleedings during the course of the
experiment were
- examined. Sucrose gradient-banded M. fermentans incognitus
was used as the
- antigen in the Western blot antibody analysis. Seroconversions
which were
- defined by definite changes of the immunoreactive patterns
and development
- of new reactive bands on the blot strips after M. fermentans
incognitus
- inoculation, occurred unusually late. Only one monkey
had a prominent
- antibody response, which however, occurred as late as
seven months after M.
- fermentans incognitus inoculation. Another monkey had
a transient antibody
- response for two months (six months to eight months after
M. fermentans
- incognitus inoculation) which apparently disappeared
in the terminal stage,
- one month before the animal expired. The other two monkeys
had a poor and
- very late immune response which again only occurred in
the terminal stage,
- 4 to 6 weeks before the animals expired. No antibody
response could be
- detected in the control monkey. Estimated molecular weights
for the newly
- developed major protein bands which revealed a positive
reaction with the
- first monkey's sera obtained seven months post M. fermentans
incognitus
- inoculation, were 97, 88, 84, 32.5 and 27.5 kilodaltons,
respectively.
-
- M. fermentans incognitus antigens in the animals' sera
obtained during the
- course of the experiment were also measured. By sandwiched
radioimmunoassay
- using rabbit antiserum raised against M. fermentans incognitus
antigens,
- periodic M. fermentans incognitus antigenemia was detected
in the three
- monkeys which failed to produce a prominent antibody
response. The first
- monkey to succumb showed the most prominent, early and
persistent M.
- fermentans incognitus antigenemia.
-
- To further confirm that these animals inoculated with
M. fermentans
- incognitus suffered a fatal systemic infection by M.
fermentans incognitus,
- DNA obtained from various tissues taken at necropsy was
directly examined.
- In this study, the highly sensitive polymerase chain
reaction (PCR) method
- of selective DNA amplification was used. Primer pairs
(RS47 (SEQ ID
- NO:13)/RS49 (SEQ ID NO: 14)) of synthetic oligonucleotides
with M.
- fermentans incognitus-specific sequences and Taq DNA
polymerase were used
- for 35 reaction cycles of M. fermentans incognitus-specific
DNA
- amplification. The primer pairs RS47/RS49 were previously
shown to span the
- first 160 bp region at one terminal end of M. fermentans
incognitus DNA of
- psb-2.2 (SEQ ID NO:2). The presence of M. fermentans
incognitus-specific
- DNA in the amplified products was confirmed by blot hybridization
using
- synthetic oligonucleotide probe (RS48 (SEQ ID NO:1))
5' end-labeled with
- .sup.32 P. The typical positive hybridizations for M.
fermentans
- incognitus-specific DNA products revealed diagnostic
160 bp DNA fragments
- with sequence homology to RS48 (SEQ ID NO:1) representing
a central segment
- of the intervening sequences between RS47 (SEQ ID NO:13)
and RS49 (SEQ ID
- NO:14). In the PCR, M. fermentans incognitus DNA was
found in spleen,
- liver, brain and kidney of the M. fermentans incognitus
inoculated animals,
- but not in the tissues of the control animal.
-
- The necropsy tissues of two monkeys' livers as well as
a monkey which
- appeared to contain the most abundant amount of M. fermentans
incognitus
- DNA also stained positively with M. fermentans incognitus-specific
rabbit
- antiserum. Direct examination by electron microscopy
of these tissues
- revealed M. fermentans incognitus particles. Clusters
of M. fermentans
- incognitus particles could most frequently be found in
the cytoplasm of
- hepatocytes and degenerating Kuffer cells. The nearly
spherical particles
- were 140-280 nm in diameter, had well-defined outer limiting
membranes and
- a densely packed granular or thin tubular internal structure.
Occasionally,
- these M. fermentans incognitus particles were seen in
the nuclei of cells
- with prominent pathological changes. Some M. fermentans
incognitus
- particles were also noted in the extracellular tissue
matrix. The necropsy
- tissues of liver and apleen obtained from the control
monkey which did not
- contain M. fermentans incognitus DNA did not stain with
M. fermentans
- incognitus-specific antiserum and did not have similar
M. fermentans
- incognitus particles.
-
- In an attempt to reisolate M. fermentans incognitus from
M. fermentans
- incognitus-inoculated monkeys, the peripheral blood mononuclear
cells
- obtained from the moribund monkeys were co-cultivated
with normal human
- peripheral blood mononuclear cells (PBMC), NIH/3T3 cells
and monkey BSC
- cells. Supernatants of the cultures were assayed for
the presence of M.
- fermentans incognitus-specific antigens and DNA once
every week. The
- cultures were maintained for three months without evidence
of M. fermentans
- incognitus growth. All the cultures were also examined
for the presence of
- reverse transcriptase enzyme activity representing growth
of retroviruses.
- Homogenates of necropsy tissues such as liver and spleen
were also
- inoculated into NIH/3T3 cells and monkey BSC cells. No
M. fermentans
- incognitus was successfully recovered in any of these
attempts.
-
-
- EXAMPLE 11
-
- Detection of Antibodies Against M. fermentans incognitus
-
- Sera from AIDS patients and from normal subjects were
analyzed by the
- immunoperioxidase straining procedure as described by
Hsu et al., supra.
- Briefly, persistently infected Sb51 cells or normal NIH/3T3
cells were
- grown in low cell density on sterile glass slides. The
culture slides were
- fixed in acetone at room temperature for five minutes.
After washing in
- Tris-buffered saline (TBS), pH 7.6, 0.05M, the slides
were first incubated
- with 1% normal horse serum containing 100 g/ml avidin
(Sigma) for 30
- minutes, and then incubated with saturated solution of
biotin (Sigma) in
- TBS for an additional 15 minutes.
-
- This initial step has been shown to minimize any nonspecific
reaction
- derived from avidin-biotin-peroxidase complex (ABC).
The human antisera
- from AIDS patients or normal subjects were then used
at 1:200 dilution
- followed by biotin- labelled goat anti-human immunoglobulin
(Tago,
- Burlingame, Calif.) at 1:200 dilutions and ABC (Vector
Lab., Burlingame,
- Calif.). Each incubation step was conducted for 30 minutes
with extensive
- washing between steps. The color reaction was developed
in DAB-Ni-H.sub.2
- O.sub.2 solution and counterstained with methyl green.
Controls for the
- technique were performed by omitting the secondary antibody.
-
- Sera of patients with AIDS produced positive immunochemical
reactions with
- these infected cells, but not with normal NIH/3T3 cells
(FIGS. 12(C) and
- 12(B), respectively). The reaction appeared to be positive
in both nuclei
- and cytoplasm of Sb51 cells. However, many of the nuclei
stained
- significantly stronger than the cytoplasm. A population
of smaller round
- cells with apparently fewer cellular processes were found
to be most
- heavily stained. Using this assay, 23 of 24 sera from
AIDS patients,
- whether they presented with Kaposi's sarcoma, Kaposi's
sarcoma with
- opportunistic infections, or opportunistic infections
alone, were positive
- (Table 3). Serum from only one AIDS patient, with both
Kaposi's sarcoma and
- opportunistic infections, showed weak positivity. Twenty-six
of 30 non-AIDS
- normal human sera showed no reactivity to the infected
Sb51 cells. One such
- negative reaction is shown in FIG. 12(A). The other four
sera showed mild
- reactivity to these cells. However, staining intensity
was significantly
- less than that seen in the reactions of AIDS patients'
sera.
-
-
- TABLE 3
- ______________________________________
- Prevalence of Serum Antibodies to Sb51 Cells
- in AIDS Patients with Various Clinical Presentations
- Number
- Risk Group Positive for
- Male Antibodies
- Homo- Total to SB.sub.51
- Subjects sexual Other Number Cells**
- ______________________________________
- Patients with
- 23 1* 24 23
- AIDS
- Kaposi's 8 8 8
- sarcoma
- Opportunistic
- 5 1* 6 6
- infection
- Kaposi's 10 10 9
- sarcoma and
- opportunistic
- infections
- Normal 30 0**
- Controls
- ______________________________________
- *Female, sexual partner of bisexual males.
- **Four nonAids control sera showed mild reactivity; all
the other control
- sera did not elicit any reaction.
-
-
- EXAMPLE 12
-
- Identification of M. fermentans incognitus Infected Cells
in Tissues of
- AIDS Patients
-
- Lymph node, spleen, Kaposi's sarcoma and brain tissues
from AIDS patients
- were fixed in Formalin and processed in paraffin sections.
An
- immunoperoxidase assay, such as described in Example
11, was performed
- using antisera from mice or rabbits prepared as described
in Example 8 in
- place of the antisera from AIDS patients. M. fermentans
incognitus infected
- cells were identified in virtually all of the tissues
examined. Electron
- microscopy was performed to confirm the infection by
M. fermentans
- incognitus. Mature M. fermentans incognitus cells were
also seen in some of
- the cells of the infected tissues.
-
-
- EXAMPLE 13
-
- Transmission of Cell-Free M. fermentans incognitus
-
- Sb51 cells (about 2.times.10.sup.7 cells) were harvested
following
- trypsinization. The cell pellet was resuspended in 2
ml of RPMI-1640 media
- with 10% sorbitol (w/v). The suspension was then subjected
to five cycles
- of freezing and thawing followed by clarification of
cell debris as
- described above. Supernatant containing M. fermentans
incognitus was
- diluted in 20 ml of RPMI-1640 with 10% bovine calf serum
and filtered
- through a 0.22 micron filter. The filtered supernatant
was added to four
- 75-cm.sup.2 tissue culture flasks containing 70% to 80%
confluent normal
- NIH/3T3 cells, human embryo fibroblasts or monkey BSC
cells (about 5 ml of
- filtered supernatant were added to each flask). The infected
cultures were
- split one week later and replenished with fresh media.
The cultures were
- kept for an additional week. At the end of two weeks,
two flasks of cells
- were used for the next cycle of cell-free, M. fermentans
incognitus
- transmission. The other two flasks were used for DNA
extraction or antigen
- determination. Equal numbers of normal NIH/3T3 cells,
instead of Sb51
- cells, were cultured in parallel through each cycle of
cell-free M.
- fermentans incognitus transmission as controls.
-
-
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