Methods for the propagation, isolation, identification, and...

Details Methods for the propagation, isolation, identification, and... Methods for the propagation, isolation, identification, and...

1995-06-06

2003-09-30

Scheiner, Laurie (Department: 1648)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving virus or bacteriophage

C424S188100, C424S208100

Reexamination Certificate

active

06627395

ABSTRACT:

The invention relates to antigens, means and methods for the diagnosis of lymphadenopathy and acquired immune deficiency syndrome.
The acquired immune deficiency syndrome (AIDS) has recently been recognized in several countries. The disease has been reported mainly in homosexual males with multiple partners, and epidemiological studies suggest horizontal transmission by sexual routes as well as by intravenous drug administration, and blood transfusion. The pronounced depression of cellular immunity that occurs in patients with AIDS and the quantitative modifications of subpopulations of their T lymphocytes suggest that T cells or a subset of T cells might be a preferential target for the putative infectious agent. Alternatively, these modifications may result from subsequent infections. The depressed cellular immunity may result in serious opportunistic infections in AIDS patients, many of whom develop Kaposi's sarcoma. However, a picture of persistent multiple lymphadenopathies has also been described in homosexual males and infants who may or may not develop AIDS. The histological aspect of such lymph nodes is that of reactive hyperplasia. Such cases may correspond to an early or a milder form of the disease.
It has been found that one of the major etiological agents of AIDS and of lymphadenopathy syndrom (LAS), which is often considered as a prodromic sign of AIDS, should consist of a T-lymphotropic retrovirus which has been isolated from a lymph node of a homosexual patient with multiple lymphadenopathies. The virus appears to be distinct from the human T-cell leukemia virus (HTLV) family (R. C. Gallo and M. S. Reitz, “J. Natl. Cancer Inst.”, 69 (No. 6), 1209 (1982)). The last mentioned virus has been known as belonging to the so-called HTLV-1 subgroup.
The patient was a 33-year-old homosexual male who sought medical consultation in December 1982 for cervical lymphadenopathy and asthenia (patient 1). Examination showed axillary and inguinal lymphadenopathies. Neither fever nor recent loss of weight were noted. The patient had a history of several episodes of gonorrhea and had been treated for syphilis in September 1982. During interviews he indicated that he had had more than 50 sexual partners per year and had travelled to many countries, including North Africa, Greece, and India. His last trip to New York was in 1979.
Laboratory tests indicated positive serology (immunoglobulin G) for cytomegalovirus (CMV) and Epstein-Barr virus. Herpes simplex virus was detected in cells from his throat that were cultured on human and monkey cells. A biopsy of a cervical lymph node was performed. One sample served for histological examination, which revealed follicular hyperplasia without change of the general structure of the lymph node. Immunohistological studies revealed, in paracortical areas, numerous T lymphocytes (OKT3
+
). Typing of the whole cellular suspension indicated that 62 percent of the cells were T lymphocytes (OKT3
+
), 44 percent were T-helper cells (OKT4
+
), and 16 percent were suppressor cells (OKT8
+
).
Cells of the same biopsed lymph node were put in culture medium with phytohemagglutinin (PHA), T-cell growth factor. (TCGF), and antiserum to human &agr; interferon (“The cells were grown in RPMI-1640 medium supplemented with antibiotics, 10
−5
M &bgr;-mercaptoethanol, 10 percent fetal calf serum, 0.1 percent sheep antibody to human &agr; interferon (neutralizing titer, 7 IU at 10
−5
dilution and 10 percent TCGF, free of PHA”). The reason for using the antiserum to &agr;-interferon was to neutralize endogenous interferon which is secreted by cells chronically infected by viruses, including retroviruses. In the mouse system, it had previously been shown that anti-serum to interferon could increase retrovirus production by a factor of 10 to 50 (F. Barre-Sinoussi et al., “Ann. Microbiol. (Institut Pasteur)” 130B, 349 (1979). After 3 days, the culture was continued in the same medium without PHA. Samples were regularly taken for reverse transcriptase assay and for examination in the electron microscope.
After 15 days of culture, a reverse transcriptase activity was detected in the culture supernatant by using the ionic conditions described for HTLV-I (B. J. Poiesz et al. “Proc. Natl. Acad. Sci. U.S.A.” 77, 7415 (1980)). Virus production continued for 15 days and decreased thereafter, in parallel with the decline of lymphocyte proliferation. Peripheral blood lymphocytes cultured on the same way were consistently negative for reverse transcriptase activity, even after 6 weeks. Cytomegalovirus could be detected, upon prolonged co-cultivation with MRC5 cells, in the original biopsy tissue, but not in the cultured T lymphocytes at any time of the culture.
The invention relates to the newly isolated virus as a source of the above said antigen which will be defined later.
The newly isolated virus, which will hereafter be termed as LAV
1
, will however be described first.
The virus is transmissible to cultures of T lymphocytes obtained from healthy donors. Particularly virus transmission was attempted with the use of a culture of T lymphocytes established from an adult healthy donor of the Blood Transfusion Center at the Pasteur Institute. On day 3, half of the culture was co-cultivate with lymphocytes from the biopsy after centrifugation of the mixed cell suspensions. Reverse transcriptase activity could be detected in the supernatant on day 15 of the coculture but was not detectable on days 5 and 10. The reverse transcriptase had the same characteristics as that released by the patient's cells and the amount released remained stable for 15 to 20 days. Cells of the uninfected culture of the donor lymphocytes did not release reverse transcriptase activity during this period or up to 6 weeks when the culture was discontinued.
The cell-free supernatant of the infected co-culture was used to infect 3-day-old cultures of T lymphocytes from two umbilical cords, LC1 and LC5, in the presence of Polybrene (2 &mgr;g/ml). After a lag period of 7 days, a relatively high titer of reverse transcriptase activity was detected in the supernatant of both cord lymphocyte cultures. Identical cultures, which had not been infected, remained negative. These two successive infections clearly show that the virus could be propagated on normal lymphocytes from either new-borns or adults.
In the above co-cultures one used either the cells of patient 1 as such (they declined and no longer grew) or cells which had been pre-X-rayed or mitomycin C-treated.
The LAV
1
virus, or LAV
1
virus suspensions, which can be obtained from infected cultures of lymphocytes have characteristics which distinguish them completly from other HTLV. These characteristics will be referred to hereafter and, when appropriate in relation to the drawing. It shows curves representative of variation of reverse transcriptase activity and [
3
H] uridine activity respectively versus successive fractions of the LAV
1
virus in the sucrose gradient, after ultra-centrifugation therein of the virus contents of a cell-free supernatant obtained from a culture of infected lymphocytes.
The analysis of LAV
1
virus by resorting to reverse transcriptase activity can be carried out according to the procedure which was used in relation to virus from patient 1, on FIG.
1
. The results of the analysis are illustrated on FIG.
1
. Cord blood T lymphocytes infected with virus from patient 1 were labelled for 18 hours with [
3
H] urinde (28 Ci/mmole, Amersham; 20 &mgr;Ci/ml). Cell-free supernatant was ultra-centrifuged for 1 hour at 50,000 rev/min. The pellet was resuspended in 200 &mgr;l of NTE buffer (10 mM tris, pH 7.4, 100 mM NaCl, and 1 mM EDTA) and was centrifuged over a 3-ml linear sucrose gradient (10 to 60 percent) at 55,000 rev/min for 90 minutes in an IEC type SB 498 rotor . Fractions (200 &mgr;l) were collected, and 30 &mgr;l samples of each fraction were assayed for, DNA fi,A dependant polymerase activity with 5 mM Mg
2+
and poly(A)-oligo-(dT) as template primer; a 20-&

Affiliated with

Also associated with

Rating

Methods for the propagation, isolation, identification, and... does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Methods for the propagation, isolation, identification, and..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Methods for the propagation, isolation, identification, and... will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3092242