US Army Mycoplasma Fermentans Incognitus Patent
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:
References Cited [Referenced By]
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
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.
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
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
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
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
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
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
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
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
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.
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.
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.
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.
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.
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.
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
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
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
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
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.
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
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
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
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
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.
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
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
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.
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
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
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.
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
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 - ______________________________________
Direct Isolation of AIDS-associated Myoplasma From Infected Tissues of
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
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.
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
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.
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.
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
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
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.
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,
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
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
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
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.
Identification of M. fermentans incognitus Infected Cells in Tissues of
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.
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