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This booklet is brought to you by the AAAS/Science Business Office
Institute of Human Virology
Commemorating a
Q u a r te r Ce n t u r y o f
H I V/A I D S R e s e a rc h
Contents
Introductions
2
A Retrovirus Retrospective
3
The Once and Future Robert C. Gallo
Sean Sanders
E. Albert Reece
Science Articles
4 Detection, Isolation, and Continuous Production
of Cytopathic Retroviruses (HTLV-III) from Patients
with AIDS and Pre-AIDS
Mikulas Popovic, M.G. Sarngadharan, Elizabeth Read, et al.
10 Frequent Detection and Isolation of Cytopathic Retroviruses
(HTLV-III) from Patients with AIDS and at Risk for AIDS
Robert C. Gallo, Syed Z. Salahuddin, Mikulas Popovic, et al.
14 Serological Analysis of a Subgroup of Human T-Lymphotropic
Retroviruses (HTLV-III) Associated with AIDS
Jörg Schüpbach, Mikulas Popovic, Raymond V. Gilden, et al.
18 Antibodies Reactive with Human T-Lymphotropic
Retroviruses (HTLV-III) in the Serum of Patients with AIDS
M. G. Sarngadharan, Mikulas Popovic, Lilian Bruch, et al.
Perspectives
From Cause
to Care,
HIV/AIDS
Research at
Twenty-Five
24
The National Cancer Institute’s Role in AIDS and Cancer
27
Understanding Causation Leads to Treatment
29
On the Discovery of Human Retroviruses: Interleukin-2;
HTLV-I and HTLV-II; Isolation of HIV-1
Robert Wiltrout, John Niederhuber
Samuel Broder
Anders Vahlne
32
An HIV Vaccine—When and How?
José Esparza, Francine E. McCutchan, George K. Lewis, Robert C. Gallo
35
New Directions in HIV Therapy and Prevention—
The Next 25 Years
Robert R. Redfield, William A. Blattner
38
Cancers and HIV Infection: An Evolving Story
41
The Interface of Cancer and HIV/AIDS Vaccines:
Both Preventive and Therapeutic
Robert Yarchoan, C. David Pauza
Jeffrey Schlom, Douglas Lowy, John Schiller
The Institute of
Human Virology
725 W. Lombard St.
COVER Artist’s view of a human T cell as a globe, with the chemokine CCL3L1 shielding the cell from
infection by HIV-1 (circles with red spikes) by virtue of its interaction with the HIV coreceptor CCR5
(yellow). CCL3L1 is represented by green circles emanating from green bands on chromosome 17,
with the intensity indicating differences in gene dose (fluorescence in situ hybridization courtesy of
Robin Leach). [Image: S. K. Ahuja and D. Baker]
This booklet supported in part by a grant from the Bill & Melinda Gates Foundation.
Baltimore, MD 21201
Editor: Sean Sanders, Ph.D.; Copy Editor: Robert Buck; Designer: Amy Hardcastle
Guest Editor: C. David Pauza, Ph.D.; Editorial Assistance: Lauren Moscato
www.ihv.org
© 2009 by The American Association for the Advancement of Science. All rights reserved.
1 May 2009
A Retrovirus Retrospective
The Once and Future Robert C. Gallo
Twenty-five years ago a series of four papers containing the first description of the
human inmmunodeficiency virus (HIV)—then called HTLV-III—were published in
the journal Science. Representing the work of Dr. Robert Gallo, his colleagues and
collaborators, these research articles had an impact on the scientific and health
community the ripples of which are still being felt today.
To commemorate the anniversary of this important event, Science has
collaborated with the University of Maryland School of Medicine and Institute of
Human Virology, and the National Cancer Institute at the National Institutes of
Health, to bring you this educational booklet. It collects in one place not only those
first four seminal papers, but also a series of original essays from thought leaders
in the field of HIV/AIDS research, some of whom worked with Dr. Gallo in his
laboratory at NCI when the first retroviruses were discovered.
Following the Science papers, accounts from Wiltrout and Niederhuber, as well
as ex-director of NCI, Samuel Broder, lay out NCI’s role in AIDS research, from
discovery of HIV all the way to the current state of vaccine research; Anders
Vahlne takes a slightly different tack on these early discoveries, seen from a more
technical perspective. The remaining four essays delve deeper into the issue of
HIV vaccines and the impact of the virus on cancer. Esparza, McCutchan, Lewis,
and Gallo emphasize the importance of pursuing a vaccine for HIV, despite the
recent setbacks, while Redfield and Blattner look at a broader landscape of
therapies, taking the position that potential treatments are most likely to be found
at the intersection of the virus with its host. Yarchoan and Pauza raise the specter
of a growing incidence of cancer in the HIV-positive population as a side effect of
increasingly effective antiretroviral treatment regimens. In the final essay, Schlom,
Lowy, and Schiller pick up this theme, arguing that cancer vaccine research can
inform us in the development of vaccines against HIV; further, HIV-related cancers
may be valid targets for cancer vaccines.
It is clear that, although progress has been made in the quarter century since
the discovery of HIV, there is still much to be achieved. With the concerted effort
to develop a vaccine, it seems likely that success will eventually, must eventually,
be achieved. The questions, especially for those in developing nations who
have limited access to antiretrovirals, are how soon can this happen and will the
treatment be both affordable and effective?
On May 4, 1984, Dr. Robert Gallo, currently the director of the Institute of Human
Virology at the University of Maryland School of Medicine, and his collaborators
published a series of four remarkable papers in the journal Science, which
demonstrated convincingly that a retrovirus they and others had isolated, called HTLVIII, was the cause of a new and deadly epidemic that was just beginning to sweep
across the nation and the world: acquired immunodeficiency syndrome,
or AIDS.
A quarter century later, AIDS has killed millions of people worldwide. Currently an
estimated 33 million people—roughly equal to the entire population of the state of
California—are infected with HIV. Without Dr. Gallo’s contributions, that toll, as horrible
as it is, undoubtedly would be much greater. The discovery of the cause of AIDS led
not only to the development of an assay for detecting the virus in blood samples by Dr.
Gallo’s group, but also to methods for keeping the viral infection under control. Today,
with adequate intervention and therapy, HIV infection is a chronic, but survivable disease.
The four original papers of Gallo and colleagues have been reprinted in this special
Science booklet commemorating the 25th anniversary of their landmark discovery.
The essays that follow, written by long-time collaborators and colleagues of Dr. Gallo,
discuss the historical significance of his initial discoveries as well as their impact on the
future course of the treatment and prevention of HIV/AIDS.
Dr. Gallo’s story is far from over, however. Not long after discovering the virus,
he began devoting much of his career to caring for those infected with HIV and to
preventing its spread to vulnerable populations. In 1996, he and two of his long-time
scientific collaborators, Robert Redfield, M.D., and William Blattner, M.D., co-founded
the Institute of Human Virology in partnership with the State of Maryland and the City
of Baltimore. The institute’s mission was to create and develop a world-class center of
excellence focusing on the treatment, prevention, and, ultimately, a cure for those with
HIV/AIDS and other viral-related human diseases.
In 2006, the institute became part of the University of Maryland School of Medicine,
where it has continued to prosper and grow. Today, the institute has 220 full-time
employees, including 45 faculty engaged in basic or clinical research on HIV/AIDS,
other human viral diseases, and cancer. As one of the major partners in the President’s
Emergency Plan for AIDS Relief (PEPFAR), the institute currently delivers life-saving
medications and therapies to more than 5,000 HIV-infected men, women, and children
in Baltimore and almost a quarter million people living with HIV/AIDS at institutesupported clinics in Africa and the Caribbean.
In July 2007, the institute was awarded a grant from the Bill & Melinda Gates
Foundation for research into a preventive vaccine for HIV/AIDS and was recently
selected as one of only 10 centers worldwide to be part of the NIH-sponsored HIV
Vaccine Trials Network. Thus, it is now poised to play a significant role in accelerating
the pace of HIV vaccine development.
As we gather to celebrate and reflect on the 25th anniversary of Dr. Gallo’s historic
breakthrough, we congratulate him not only for his initial scientific discoveries, but also
his ongoing humanitarian efforts and his work to find an eventual vaccine—and cure—
for one of the worst plagues in human history.
Based on his past successes, we can only predict that his future and that of the institute
will be every bit as accomplished.
Sean Sanders, Ph.D.
Commercial Editor, Science
E. Albert Reece, M.D., Ph.D., M.B.A.
Vice President for Medical Affairs, University of Maryland
Bowers Distinguished Professor and Dean, University of Maryland School of Medicine
Detection, Isolation, and Continuous
Production of Cytopathic Retroviruses
(HTLV-III) from Patients with AIDS
and Pre-AIDS
Mikulas Popovic, M. G. Sarngadharan, Elizabeth Read, Robert C. Gallo
A cell system was developed for the reproducible detection of human T-lymphotropic
retroviruses (HTLV family) from patients with the acquired immunode­ficiency syndrome (AIDS)
or with signs or symptoms that frequently precede AIDS (pre-AIDS). The cells are specific clones
from a permissive human neoplastic T-cell line. Some of the clones permanently grow and
continuously produce large amounts of virus after infection with cytopathic (HTLV-III) variants
of these viruses. One cytopathic effect of HTLV-III in this system is the arrangement of multiple
nuclei in a characteristic ring formation in giant cells of the infected T-cell population. These
structures can be used as an indicator to detect HTLV-III in clinical specimens. This system opens
the way to the routine detection of HTLV-III and related cytopathic variants of HTLV in patients
with AIDS or pre-AIDS and in healthy carriers, and it provides large amounts of virus for detailed
molecular and immunological analyses.
E
pidemiologic data suggest that the acquired immunodeficiency syndrome
(AIDS) is caused by an infectious agent
that is horizontally transmitted by inti­mate
contact or blood products (1-3). Though the
disease is manifested by op­portunistic infections, predominantly Pneumocystis carinii
pneumonia (4), and by Kaposi’s sarcoma (5),
the underlying disorder affects the patient’s
cell-mediat­ed immunity (6), resulting in absolute lymphopenia and reduced subpopulations
of helper T lymphocytes (OKT4+). Moreover,
before a complete clinical manifestation of the
disease occurs, its prodrome, pre-AIDS, is frequently char­acterized by unexplained chronic
lymphadenopathy or leukopenia involving
helper T lymphocytes (5, 6). This leads to the
severe immune deficiency of the patient and
suggests that a specific sub­set of T cells could
be a primary target for an infectious agent.
Although pa­tients with AIDS or pre-AIDS are
often chronically infected with cytomegalovi­
rus (7) or hepatitis B virus (8), for vari­ous
reasons these appear to be opportu­nistic or coincidental infections. We have proposed that
AIDS may be caused by a virus from the family of human T-cell lymphotropic retroviruses
(HTLV) (9) that includes two major, wellcharac­terized subgroups of human retrovir­
uses, called human T-cell leukemia-lymphoma viruses, HTLV-I (9-12) and HTLV-II (9,
11, 13). The most common isolate, HTLV-I, is
obtained mainly from patients with mature Tcell malig­nancies (9, 12). Seroepidemiological
studies, the biological effects of the virus in vitro, and nucleic acid hybridization data indicate
that HTLV-I is etiologically associated with the
T-cell malignancy of adults that is endemic in
certain areas of the south of Japan (14), the
Caribbean (15), and Africa (16). HTLV-II was
first isolated from a patient with a T-cell variant of hairy cell leukemia (13). To date, this is
the only reported isolate of HTLV-II from a patient with a neoplas­tic disease. Virus isolation
and seroepi­demiological data show that both
HTLV-I and HTLV-II can sometimes be found
in patients with AIDS (17).
That a retrovirus of the HTLV family might
be an etiological agent of AIDS was suggested
by the findings (i) that another retrovirus, feline leukemia virus, causes immune deficiency in cats (18); and that (ii) retroviruses of the
HTLV family are T-cell tropic (12, 19); (iii)
preferentially infect helper T cells (OKT4+)
(12, 19); (iv) have cytopathic effects on various
human and mammali­an cells, as demonstrated
by their induc­tion of cell syncytia formation
(20); (v) can alter some T-cell functions (21);
(vi) can in some cases selectively kill T cells
(22); and (viii) may be transmitted by intimate
contact and blood products (9). Also consistent with an HTLV etiology were the results of
Fig. 1. Light and electron microscopic examination of clone H4/HTLV-III. (a) H4/HTLV-III cells were
characterized by the presence of large multinucleated cells that showed, with Wright-Giemsa staining,
a characteristic arrangement of their nuclei (x350). (b) Electron micrograph of the cells showing the
presence of extracellular viral particles (x60,000).
Essex and Lee and their colleagues showing
the presence of antibodies to cell membrane
antigens of HTLV-infected cells in serum
samples from more than 40 percent of patients
with AIDS (23). This antigen has since been
defined as part of the envelope of HTLV (24).
The more frequent detection in AIDS patients
of antibodies to a mem­brane protein rather
than to HTLV-I internal structural core proteins (25), to­gether with the low incidence of
isola­tions of HTLV-I or HTLV-II from AIDS
patients, also suggested that a new vari­ant of
HTLV might be present.
The original detection and isolation of
HTLV-I were made possible by the dis­covery
of T-cell growth factor (TCGF) (26), also
called interleukin 2 (IL-2), which stimulates
the growth of different subsets of normal and
neoplastic mature T cells (27), and by the development of sensitive assays for reverse transcriptase (RT), an enzyme characteristic of
retro­viruses (28). The procedures used previ­
ously for the transmission and continu­ous production of HTLV-I and -II were first worked
out in mammalian cells transformed by avian
sarcoma virus (29). These methods involved
cocultivation of the transformed cells with
cells permissive for the particular virus strain.
Normal human T cells in cocultivation experiments preferentially yielded HTLV of both
subgroups. Some of these viruses showed an
immortalizing (transforming) capability for
certain target T cells (9, 12). We thought that
HTLV variants that have cytopathic effects
on their target cells but do not immortalize
them might be more important in the cause of
AIDS. In fact, such variants were frequently
but only transiently detected when nor­mal T
cells were used as targets in cocultivation or
cell-free transmission experiments. This transience was our main obstacle to the isolation
of these cytopathic variants of HTLV from
patients with AIDS or pre-AIDS. We subsequently found a cell line that is highly susceptible to and permissive for cytopathic variants
of HTLV. This cell line can grow permanently
after infection with the virus. We report here
the establishment and characterization of this
new immortalized T-cell population and its
use in the isolation and continuous high-level
production of HTLV variants from patients
with AIDS and pre-AIDS.
Several neoplastic human cell lines established in vitro were assayed for susceptibility to infection with HTLV-I and -II and with
many of the more cytopathic retroviruses
isolated from AIDS patients (30). One neoplastic aneuploid T-cell line, derived from an
adult with lymphoid leukemia, was found to
be susceptible to infection with the new cytopathic virus isolates. This cell line, termed
HT, has produced HTLV-vari­ants in sufficient
quantities to permit the development of specific immunologic re­agents and nucleic acid
probes that can be used to characterize new
isolates and compare them with HTLV-I and
HTLV-II (30). These cytopathic variants differ
from HTLV-I and -II not only in their biologi
Table 1. Response of cloned T-cell populations to infection with HTLV-III. Single-cell clones were
isolated as described (34, 35) from a long-term cultured aneuploid HT cell line exhibiting mature T-cell
phenotype [OKT3+ (62 percent), OKT4+ (39 percent), and OKT8- ] as deter­mined by cytofluorometry
with a fluorescence-activated cell sorter. The cultures are routinely maintained in RPMI 1640 medium
containing 20 percent fetal calf serum (FCS) and antibiotics. The terminal cell density of the parental cell
culture, seeded at a concentration of 2 x 105 cells per milliliter of culture media, was in the range of 106
to 1.5 x 106 cells per milliliter after 5 days of culture.
Fig. 2. (a) Continuous HTLV-III production from H4/HTLV-III in long-term culture was characterized by
fluctuation in the amount of released virus as assessed by RT activity in the culture fluid (for details,
see Table 1 and Fig. 2b). Viability of the infected cells was in the range of 60 to 90 percent, (b) Sucrose
density gradient banding of HTLV-III showed the highest particulate RT activity at a density of 1.16 g/ml.
A cell-free virus concentrate from a culture of H4/HTLV-III was layered on a 20 to 60 percent (by weight)
sucrose gradient in 10 mM tris-HCl (pH 7.4) containing 0.1M NaCl and 1 mM EDTA and centrifuged
overnight at 35,000 rev/min in a Spinco SW47 rotor. Fractions of 0.7 ml were collected from the bottom
of the gradient and portions were assayed for RT (•) with (dT)15 • (A)n being used as the primer template
and Mg2+ as the divalent cation according to the methods described earlier (10, 28). Density of sucrose
(X) was determined by refractive index measurements.
*Cell smears were prepared from cultures 6 and 14 days after infection and stained with Wright-Giemsa.
Cells with more than five nuclei were considered to be multinucleated. Cloned cells from uninfected
cultures also contained some multinucleated giant cells; however, the arrangement of the multiple nuclei
in a characteristic ring formation (see Fig. 1a) was lacking and the number of these cells was much less
(0.7 to 10 percent). †Cells were washed with phosphate-buffered saline (PBS) and resuspended in the
same buffer at concentration 106 cells per milliliter. Approximately 50 µl of cell suspension was spotted
on a slide, air dried, and fixed in acetone for 10 minutes at room temperature. Slides were stored at
-20°C until use. Twenty microliters of either rabbit antiserum to HTLV-III (diluted 1:2000 in PBS) or serum
from the patient (E.T.) diluted 1:8 in PBS was applied to cells and incubated for 50 minutes at 37°C. The
fluorescein-conjugated antiserum to rabbit or human immunoglobulin G was diluted and applied to the
fixed cells for 30 minutes at room temperature. Slides were then washed extensively before microscopic
examinations. The uninfected parental cell line as well as the clones were consistently negative in these
assays. ND, not done. ‡Virus particles were precipitated from cell-free supernatant as follows: 0.3 ml of
4M NaCl and 3.6 ml of 30 percent (weight to volume) polyethylene glycol (Carbowax 6000) were added
to 8 ml of harvested culture fluids and the suspension was placed on ice overnight. The suspension was
centrifuged in a Sorvall RC-3 centrifuge at 2000 rev/min at 4°C for 30 minutes. The precipitate was
resuspended in 300 µl of 50 percent (by volume) glycerol (25 mM tris-HCl, pH 7.5, 5 mM dithiothreitol,
150 mM KCl, and 0.025 percent Triton X-100). Virus particles were disrupted by addition of 100 µl
of 0.9 percent Triton X-100 to 1.5M KCl. Reverse transcriptase assays were performed as previously
described (10, 28) (see comments to Fig. 2b) and expressed in counts per minute per milliliter of
culture medium.
cal effects but also in several im­munological
assays and in their morphol­ogy (31). They
nevertheless have many properties similar to
HTLV-I and -II. For example, they are T4 lymphotropic, they have a similar RT (30), they
cross-react with several structural proteins in
heterologous radioimmune assays with serum
from AIDS patients and with antisera to the
virus raised in animals (31), and they induce
syncytia. These new HTLV isolates are collec
tively designat­ed HTLV-III, although it is not
yet proved that they are identical.
The cell line HT was tested for HTLV before
being infected in vitro and was negative by all
criteria including lack of proviral sequences
(32). Continuous pro­duction of HTLV-III
was obtained after repeated exposure of parental HT cells (3 x 106 cells pretreated with
polybrene) to concentrated culture fluids
harvested from short-term cultures of T cells
(grown with TCGF) obtained from pa­tients
with AIDS or pre-AIDS. The con­centrated
fluids were first shown to con­tain particle-associated RT. When cell proliferation declined,
usually 10 to 20 days after exposure to the
culture fluids, the fresh (uninfected) HT cells
were add­ed to the cultures. Culture fluids from
the infected parental cell line were posi­tive for
particulate RT activity, and about 20 percent of
the infected cell population was positive in an
indirect immune fluorescence assay in which
we used serum from a hemophilia patient with
pre-AIDS (patient E.T.). Serum from E.T. also
contained antibodies to proteins of disrupted
HTLV-III (33) but did not react with cells infected with HTLV-I or HTLV-II.
The parental T-cell population was ex­
tensively cloned in order to select the most
permissive clones that would pre­serve high
rates of growth and virus production (for example, see clones 4 and 9 in Table 1). A total of 51 single-cell clones were obtained by
both capillary (34) and limited dilution (35)
techniques using irradiated mononuclear cells
from peripheral blood of a healthy donor as a
feeder. The clones were infected with HTLVIII by exposure to concentrated virus (2 x 106
cells of each clone and 0.1 ml of virus). Then
cell growth and mor­phology, expression of
cellular viral anti­gens, and RT activity in culture fluids were assessed 6 and 14 days after
infec­tion. Results for eight of these clones are
shown in Table 1. Although all of these clones
were susceptible to and permis­sive for the
virus, there were consider­able differences in
their ability to prolif­erate after infection. For
example, the cell number decreased by 10 to
90 per­cent from the initial cell count within 6
days after infection. The percentage of T cells
positive for viral antigens ranged from 10 to
80 percent, as determined by immune fluorescence assays with serum from patient E.T.
and with antiserum from rabbits infected repeatedly with disrupted HTLV-III. At 14 days
after infection, the total cell number and the
proportion of HTLV-III positive cells had increased in all eight clones. The virus positive
cultures consistently showed a high proportion
of round giant cells containing numerous nuclei (Fig. la). These cells resemble those induced by HTLV-I and -II (9) except that the
nuclei exhibit a characteristic ring forma­tion.
Electron microscopic examinations showed
that the cells released consider­able amounts of
virus (Fig. lb).
Both virus production and cell viabili­ty of
the infected clone H4 (H4/HTLV-III) were
monitored for several months. Although virus
Table 2. Isolation of HTLV-III from patients with AIDS and pre-AIDS.
*Cocultivation with H4 recipient T-cell clone was performed with fresh mononuclear cells from peripheral
blood of patients R.F. and S.N., respectively. For patients B.K. and L.S. cocultivation was performed with T
cells grown in the presence of exogenous TCGF (10 percent by volume) for 10 days. The ratio of recipient
to donor (patients’) cells was 1:5. The mixed cultures were maintained in RPMI 1640 medium (containing
20 percent FCS and antibiotics) in the absence of exogenous TCGF. H9 cells were also infected by exposing
the cells to concentrated culture fluids harvested from T-cell cultures of patient W.T. The cultures were
grown in the presence of exogenous TCGF for 2 weeks before the culture fluids were harvested and
concentrated. Cells of H9 clones were treated with polybrene (2 µg/ml) for 20 minutes and 2 x 106 cells
were exposed for 1 hour to 0.5 ml of 100-fold concentrated culture fluids positive for particulate RT
activity. †HTLV-III virus expression in cells infected by the coculture and cell-free methods was assayed
approximately 1 month after cultivation in vitro. Note a considerable fluctuation in HTLV-III expression.
For details of the RT and indirect immune fluorescence assays see Table 1.
production fluctuated (Fig. 2a), culture fluids
harvested and assayed at approximately 14day inter­vals consistently showed particulate
RT activity which has been followed for over
5 months. The viability of the cells, ranged
from 65 to 85 percent and the doubling time
of the cell population was approximately 30 to
40 hours (data not shown). Thus the data show
that this permanently growing T-cell population can continuously produce HTLV-III.
The yield of virus from H4/HTLV-III cells
was assessed by purification of con­centrated
culture fluids through a su­crose density gradient and assays of par­ticulate RT activity in
each fraction col­lected from the gradient. As
shown in Fig. 2b, the highest RT activity was
found at a density of 1.16 g/ml, which is similar to other retroviruses. The highest RT activity was found in the fractions with the largest
amount of virus, as de­termined by electron
microscopy. The actual number of viral particles deter­mined by this method was estimated
(36) to be about 1011 per liter of culture fluid.
We have used clones H4 and H9 for the
long-term propagation of HTLV-III from patients with AIDS and pre-AIDS. HTLV-III was
isolated from four pa­tients by the cocultivation method and from one patient by cell-free
infection of these T-cell clones (Table 2). The
trans­mission was monitored by RT activity,
electron microscopic examinations, and expression of viral protein. When the H4 cells
thus infected were fixed with ace­tone and
tested with rabbit antiserum to HTLV-III and
with serum from patients E.T., the percentage
of positive cells was between 5 and 80 percent. HTLV-III has also been isolated in our
laboratory from a total of 48 patients by the
more conventional methods for isolation of
HTLV (30). Some of these isolates have now
successfully been transmitted to the HT clones
for production and detailed analyses.
A few T-lymphocyte retroviruses that differed from HTLV-I and -II but were associated
with lymphadenopathy syn­drome were detected earlier (37, 38). One such virus, called
LAV, was report­ed to be unrelated to HTLVI or -II (38). Moreover, serum samples from
37.5 percent of patients with AIDS were found
to react with it (38). In contrast, HTLV-III is
related to HTLV-I and -II (31, 39) and, by all
criteria, this new virus belongs to the HTLV
family of retroviruses. In ad­dition, more than
85 percent of serum samples from AIDS patients are reactive with proteins of HTLV-III
(33). These findings suggest that HTLV-III and
LAV may be different. However, it is possible
that this is due to insufficient character­ization
of LAV because the virus has not yet been
transmitted to a permanently growing cell line
for true isolation and therefore has been difficult to obtain in quantity.
The transient expression of cytopathic variants of HTLV in cells from AIDS patients and
the previous lack of a cell system that could
maintain growth and still be susceptible to and
permissive for the virus represented a major
obstacle in detection, isolation, and elucidation
of the precise causative agent of AIDS. The establishment of T-cell populations that continuously grow and produce virus after infection
opens the way to the routine detection of cytopathic variants of HTLV in AIDS patients and
provides the first opportunity for detailed immu­
nological (31, 33) and molecular analyses of
these viruses.
References and Notes
1. Centers for Disease Control Task Force on Kaposi’s
Sarcoma and Opportunistic Infections, N. Engl J.
Med. 306, 248 (1982).
2. J. P. Hanrahan, G. P. Wormser, C. P. Maguire, L. J.
DeLorenzo, G. Davis, ibid. 307, 498 (1982).
3. J. W. Curran et al., ibid. 310, 69 (1984).
4. “Pneumocystis pneumonia—Los Angeles,” Morbid.
Mortal. Weekly Rep. 30, 250 (1981).
5. “Kaposi’s sarcoma and pneumocystis pneumo­nia
among homosexual men—New York City and
California,” ibid., p. 305; A. E. Friedman-Klein et al.,
Ann. Int. Med. 96, 693 (1982).
6. M. Gottlieb et al., N. Engl. J. Med. 305, 1425 (1981);
J. Masur et al., ibid., p. 1431.
7. C. Urmacher, P. Myskowski, M. Ochoa, M. Kris, B.
Safai, Am. J. Med. 72, 569 (1982).
8. D. R. Francis and J. E. Maynard, Epidemiol. Rev. 1, 17
(1979); N. Clumeck et al., N. Engl. J. Med. 310, 492
(1984).
9. R. C. Gallo, in Cancer Surveys, L. M. Franks, L. M.
Wyke, R. A. Weiss, Eds. (Oxford Univ. Press, Oxford,
in press).
10. B. J. Poiesz et al., Proc. Natl. Acad. Sci. U.S.A. 77,
7415 (1980); M. Yoshida, I. Miyoshi, Y. Hinuma, ibid.
79, 2031 (1982).
11. M. S. Reitz, M. Popovic, B. F. Haynes, S. C. Clark, R. C.
Gallo, Virology 26, 688 (1983).
12. M. Popovic, et al., Science 219, 856 (1983).
13. V. S. Kalyanaraman et al., ibid 218, 571 (1982).
14. Y. Hinuma et al., Proc. Natl. Acad. Sci. U.S.A. 78,
6476 (1981); M. Robert-Guroff et al.., Science 215,
15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 975 (1982); V. S. Kalyanaraman et al.., Proc. Natl.
Acad. Sci. U.S.A. 79, 1653 (1982).
W. A. Blattner et al., Int. J. Cancer 30, 257 (1982).
W. C. Saxinger et al., in Human T-Cell Leuke­mia
Viruses, R. C. Gallo, M. Essex, L. Gross, Eds. (Cold
Spring Harbor Press, Cold Spring Harbor, N.Y., in
press).
R. C. Gallo et al., Science 220, 865 (1983); E. P.
Gelmann et al., ibid., p. 862; M. Popovic et al., in
preparation.
M. Essex, W. D. Hardy, Jr., S. M. Cotter, R. M.
Jakowski, A. Sliski, Infect. Immun. 11, 470 (1975);
W. D. Hardy, Jr., et al., Cancer Res. 36, 582 (1976);
L. J. Anderson, O. Jarret, H. M. Laird, J. Natl. Cancer
Inst. 47, 807 (1971).
R. C. Gallo et al., Cancer Res. 43, 3892 (1983);
F. Wong-Staal et al., Nature (London) 302, 626
(1983).
K. Nagy, P. Clapham, R. Cheinsong-Popov, R. A.
Weiss, Int. J. Cancer 32, 321 (1983).
M. Popovic et al., in preparation.
H. Mitsuya, H. G. Guo, M. Megson, C. D. Trainor, M.
S. Reitz, S. Broder, Science 223, 1293 (1984).
M. Essex et al., ibid. 220, 859 (1983).
J. Schüpbach, M. G. Sarngadharan, R. C. Gallo, ibid.,
in press; T. H. Lee et al., Proc. Natl. Acad. Sci. U.S.A.,
in press.
M. Robert-Guroff et al., in preparation.
D. A. Morgan, F. W. Ruscetti, R. C. Gallo, Science
193, 1007 (1976).
F. W. Ruscetti, D. A. Morgan, R. C. Gallo, J.
Immunol. 119, 131 (1977); B. J. Poiesz, F. W.
Ruscetti, J. W. Mier, A. M. Woods, R. C. Gallo, Proc.
Natl Acad. Sci. U.S.A. 77, 6134 (1980).
R. C. Gallo, F. W. Ruscetti, R. E. Gallagher, in
Hematopoietic Mechanisms, B. Clarkson, P. A.
Marks, J. Till, Eds. (Cold Spring Harbor Press, Cold
Spring Harbor, N.Y., 1978), vol. 5, p. 671.
J. Svoboda, Natl Cancer Inst. Monogr. 17, 277
(1964); J. Svoboda and R. Dourmashkin, J. Gen.
Virol. 4, 523 (1969); M. Popovic, J. Svoboda, J. Suni,
A. Vaheri, L. Ponten, Int. J. Cancer 19, 834 (1977);
M. Popovic, J. Svoboda, F. L. Kisselyov, K. Polakova,
Folia Biol. 26, 244 (1980).
R. C. Gallo et al., Science 224, 500 (1984).
J. Schüpbach, M. Popovic, R. V. Gilden, M. A. Gonda,
M. G. Sarngadharan, R. C. Gallo, ibid. 224, 503
(1984).
G. Shaw and F. Wong-Staal, unpublished data.
M. G. Sarngadharan, M. Popovic, L. Bruch, J.
Schüpbach, R. C. Gallo, Science 224, 506 (1984).
M. Popovic, M. Grofova, N. Valentova, D. Simkovic,
Neoplasma 18, 257 (1971).
H. F. Bach, B. J. Alter, B. M. Widmer, M. S. Segall, D.
Dunlap, Immunol Rev. 54, 5 (1981).
J. H. Monroe and P. M. Brandt, Appl. Microbi­ol. 20,
259 (1970).
F. Barré-Sinoussi et al., Science 220, 868 (1983).
L. Montagnier et al., in Human T-Cell Leuke­mia
Viruses, R. C. Gallo, M. Essex, L. Gross, Eds. (Cold
Spring Harbor Press, Cold Spring Harbor, N.Y., in
press).
S. Arya et al., in preparation.
We thank B. Kramarsky for help in electron
microscopic examination of HTLV-III infected cells,
E. Richardson and R. Zicht for technical help, and A.
Mazzuca for her editorial assist­ance.
30 March 1984; accepted 19 April 1984
Frequent Detection and Isolation of
Cytopathic Retroviruses (HTLV-III) from
Patients with AIDS and at Risk for AIDS
Robert C. Gallo, Syed Z. Salahuddin, Mikulas Popovic, Gene M. Shearer, Mark Kaplan, Barton F.
Haynes, Thomas J. Palker, Robert Redfield, James Oleske, Bijan Safai, Gilbert White, Paul Foster,
Phillip D. Markham
Peripheral blood lymphocytes from patients with the acquired immuno­deficiency syndrome
(AIDS) or with signs or symptoms that frequently precede AIDS (pre-AIDS) were grown in
vitro with added T-cell growth factor and assayed for the expression and release of human Tlymphotropic retroviruses (HTLV). Retroviruses belonging to the HTLV family and collectively
designated HTLV-III were isolated from a total of 48 subjects including 18 of 21 patients with
pre-AIDS, three of four clinically normal mothers of juveniles with AIDS, 26 of 72 adult and
juvenile pa­tients with AIDS, and from one of 22 normal male homosexual subjects. No HTLVIII was detected in or isolated from 115 normal heterosexual subjects. The number of HTLV-III
isolates reported here underestimates the true prevalence of the virus since many specimens
were received in unsatisfactory condition. Other data show that serum samples from a high
proportion of AIDS patients contain antibodies to HTLV-III. That these new isolates are members
of the HTLV family but differ from the previous isolates known as HTLV-I and HTLV-II is
indicated by their morphological, biological, and immunological characteristics. These results
and those reported elsewhere in [the 4 May 1984 issue of Science] suggest that HTLV-III may be
the primary cause of AIDS.
T
he acquired immunodeficiency syn­
drome known as AIDS was initially
rec­ognized as a separate disease entity in 1981 (1). Groups reported to be at risk
for AIDS include homosexual or bisexu­al
males (about 70 percent of reported cases),
intravenous drug users (about 17 percent of
cases), and Haitian immi­grants to the United
States (about 5 percent of cases). Also at risk
are hetero­sexual contacts of members of the
high­est risk group, hemophiliacs treated with
blood products pooled from donors, re­cipients
of multiple blood transfusions, and infants
born of parents belonging to the high-risk
groups (2). AIDS is diagnosed as a severe,
unexplained, immune deficiency that usually
involves a reduc­tion in the number of helper T
lympho­cytes and is accompanied by multiple
opportunistic infections or malignancies. A
number of other clinical manifesta­tions, when
occurring in members of a group at risk for
AIDS, are identified as its prodrome (preAIDS). These include unexplained chronic
lymphadenopathy or leukopenia involving a
reduction in the number of helper T lymphocytes (1, 2). The increasing incidence of this
disease, the types of patients affected, and
10
other epidemiological data suggest the exist­
ence of an infectious etiologic agent that can
be transmitted by intimate contact or by whole
blood or separated blood com­ponents (2). As
indicated by Popovic et al. (3), we and others
have suggested that specific human T-lymphotropic retrovir­uses (HTLV) cause AIDS (4, 5).
Many properties of HTLV are consistent with
this idea (6).
An association of members of the HTLV
family with T lymphocytes from some AIDS
or pre-AIDS patients was reported previously.
For example, the first subgroup of HTLV to be
character­ized, HTLV-I, was isolated recently
from T cells from about 10 percent of AIDS
patients, and a virus related to HTLV-II was
isolated from one AIDS patient (4). Another
HTLV isolate was obtained from the lymph
nodes of a patient with lymphadenopathy and
at risk for AIDS (7). This isolate has been difficult to grow in quantities sufficient to permit its characterization. HTLV provi­ral DNA
was detected in T lymphocytes from two additional AIDS patients (8) and HTLV-related
antigens were found in another two patients
(4). Studies in which disrupted HTLV-I or
the purified structural proteins (p24 or p19)
Fig. 1. Reverse tran­scriptase
activity from lymphocytes
es­tablished in cell cul­ture
from a patient with preAIDS. Via­ble cell number
and Mg2+-dependent RT
activity were deter­mined
by established procedures
(13). Sym­bols: º, viable
cell number in 1.5 ml of
growth medium; •, RT in 5
μl of fivefold concentrated
condi­tioned medium sam­
pled at the indicated time.
A sudden verti­cal drop in
the dashed curve indicates
the time of subculturing of
cells to the indicated cell
number. Arrow indicates the time of addition of rabbit antiserum to α-interferon to a portion of the
cultured cells (also see legend to Table 1).
were used to detect antibodies in serum sam­
ples from patients with AIDS and pre-AIDS
indicated that 10 to 15 percent of the patients
had been exposed to HTLV-I (9). Essex and
his co-workers, using HTLV-infected T-lymphocyte cultures to detect antibody in serum
samples, found that about 35 percent of patients with AIDS and pre-AIDS had been ex­
posed to HTLV (5). Further studies sug­gested
that at least some of the antigens detected in
this system were products of the genome of a
member of the HTLV family (10), but it was
not known wheth­er the antibodies were specifically against HTLV-I, HTLV-II, or a virus
of a different subgroup.
With the availability of large quantities of
HTLV-III (3), it became possible to develop
specific immunological reagents that would
facilitate its characterization. HTLV-III was
found to share many properties with other
HTLV isolates (6), but it was morphologically,
biologically, and antigenically distinguishable
(3, 11). Here we describe the detection and
iso­lation of HTLV-III from a large number of
patients with AIDS and pre-AIDS.
For these studies we used cell culture conditions previously developed in our laboratory for the establishment of T lymphocytes
in culture and for the detec­tion and isolation
of HTLV-I and HTLV-II from leukemic donors
(12). Evidence for the presence of HTLV-III
included: (i) viral reverse transcriptase (RT)
activi­ty (12) in supernatant fluids; (ii) trans­
mission of virus by coculturing T cells with ir-
radiated donor cells or with cell-free fluids (3,
13); (iii) observation of virus by electron microscopy (12, 13); and (iv) the expression of
viral antigens in indirect immune fluorescence
assays using serum from a patient positive for
antibodies to HTLV-III as described (5, 11), or
antisera prepared against puri­fied, whole disrupted HTLV-III (11). Cells and supernatant
fluids were also monitored for the expression
of HTLV-I and HTLV-II by using antibodies to
the viral structural proteins pl9 and p24 and
by indirect immune fluorescence and radioimmunoprecipitation procedures (14).
As summarized in Table 1, we found HTLVIII in 18 of 21 samples from pa­tients with preAIDS, from three of four clinically normal
mothers of juvenile AIDS patients, three of
eight juvenile AIDS patients, 13 of 43 adult
AIDS patients with Kaposi’s sarcoma, and 10
of 21 adult AIDS patients with opportu­nistic
infections. Virus was detected in only one of
22 samples from clinically normal, nonpromiscuous homosexual males believed to be at
only moderate risk for AIDS. It is interesting,
however, that 6 months after these tests were
conducted the one positive normal ho­mosexual
subject developed AIDS. In no instance, 0 of
115, was virus detected in or isolated from
cells of the normal vol­unteers. Samples from
15 of these were tested under rigorously controlled condi­tions, which included addition of
anti­body to a-interferon.
Primary cells from patients usually produce
virus for 2 to 3 weeks (Fig. 1). After this time
11
Fig. 2. Transmission electron micrographs of fixed, sectioned lym­phocytes from a patient with pre-AIDS.
(A) x 10,000; (B) x 30,000; (C and D) x 100,000.
Table 1. Detection and isolation of HTLV-III from patients with AIDS and pre-AIDS. Peripheral blood
leukocytes were banded in Ficoll-Hypaque, incubated in growth medium (RPMI 1640, 20 percent fetal
bovine serum, and 0.29 mg of glutamine per milliliter) containing phytohemagglutinin (PHA-P; 5 μg/ml)
for 48 hours at 37°C in a 5 percent C02 atmosphere. They were then refed with growth medium containing 10 percent purified T-cell growth factor (TCGF). Cells and conditioned media from these lymphocytes
were assayed for the presence of HTLV-III. Samples exhibiting more than one of the following were
considered positive: repeated detection of a Mg2+-dependent reverse transcriptase activity in supernatant fluids; virus observed by electron microscopy; intracellular expression of virus-related antigens
detected with antibodies from seropositive donors or with rabbit antiserum to HTLV-III; or transmission
of particles, detected by RT assays or by electron microscopic observation, to fresh human cord blood,
bone marrow, or peripheral blood T lymphocytes. All isolates are distinguishable from HTLV-I or HTLV-II
by several criteria and are classified as HTLV-III on the basis of similar morphological features observed
by electron microscopy (Fig. 1); similar cytopathic effects (3); antigenic cross-reactivity (11); and nucleic
acid analysis (16).
*With the exception of the normal heterosexual donors and some of the clinically normal mothers of
juvenile AIDS patients, all others belong to one of the groups of people identified as being at risk for
AIDS (homosexual males, intravenous drug users, Haitian immigrants, heterosexual contacts of members of a group at risk, hemophiliacs treated with pooled blood products, recipients of multiple blood
transfusions, and infants born of parents belonging to other groups at risk). Pre-AIDS includes patients
with unexplained chronic lymphadenopathy and leukopenia, with an inverted T4 (helper)/T8 (suppressor)
lymphocyte ratio. The clinically normal, nonpromiscuous, homosexual subjects are from Washington, D.C,
and are believed to be at moderate risk. The clinically normal heterosexual donors include both male and
female subjects believed not to be at risk for AIDS.
12
the production of virus declines even though
the culture may contain actively replicating
cells that can be maintained for long periods
in the presence of added T-cell growth factor
(TCGF). In some instances virus release can
be reinitiated by the addition of antibody to αinterferon (Fig. 1). The HTLV-III–producing
cell cultures were characterized by established
immunological procedures (13). They were
predom­inantly T lymphocytes (E rosette recep­
tor–positive, OKT3+ and Leul+) with a helperinducer phenotype (OKT4+ and Leu3+).
The fairly uniform morphological ap­
pearance of HTLV-III is shown in Fig. 2. The
diameter of the virus is 100 to 120 nm, and
it is produced in high numbers from infected
cells by budding from the cell membrane. A
possibly unique fea­ture of this virus is the cylindrical shaped core observed in many mature virions.
The incidence of virus isolation report­ed
here probably underestimates its true incidence
since many tissue specimens were not received
or handled under what we now recognize as
optimal conditions (15). This is particularly
so for the sam­ples received from late-stage
AIDS pa­tients. Such samples usually contain
many dying cells and very few viable T4
lymphocytes. However, a high propor­tion
of patients with AIDS and pre-AIDS have
circulating antibody to HTLV-III (11).
The HTLV-III produced by cultured T cells
from patients with AIDS and pre-AIDS is
highly infectious and can be readily transmitted to fresh umbilical cord blood and adult peripheral blood or bone marrow lymphocytes.
The produc­tion of HTLV-III by these cells
is transient, often declining to undetectable
levels by 2 to 3 weeks after infection (data
not shown). The transmission of HTLV-III to
an established T-cell line (3), however, now
makes possible its production in large quantities for de­tailed analyses and for development
of reagents for its detection (3, 11).
That the viruses we have named HTLV-III
belong to the HTLV family is indicated by
their T cell tropism, Mg2+-dependent RT of
high molecular weight, antigenic cross-reactivity with HTLV-I and -II (11), cytopathic effects on T lymphocytes (3), and their morphological appearance in the electron micro­graph.
HTLV-III also contains some structural proteins similar in size to those of other members
of the HTLV family (11).
These studies of HTLV-III isolates from
patients with AIDS and pre-AIDS and from
some healthy individuals at risk for AIDS provide strong evidence of a causative involvement of the virus in AIDS.
References and Notes
Schüpbach, R. C. Gallo, Science 224, 506 (1984); J.
Schüpbach et al., ibid., p. 503.
12. B. J. Poiesz et al., Proc. Natl. Acad. Sci. U.S.A. 77,
7415 (1980); B. J. Poiesz, F. W. Ruscetti, M. S. Reitz, V.
S. Kalyanaraman, R. C. Gallo, Nature (London) 294,
268 (1981); V. S. Kalyanaraman et al., Science 218,
571 (1982).
13. P. D. Markham et al., Int. J. Cancer 31, 413 (1983);
P. D. Markham, S. Z. Salahuddin, B. Macchi, M.
Robert-Guroff, R. C. Gallo, ibid. 35, 13 (1984); S. Z.
Salahuddin et al., Virology 129, 51 (1983).
14. M. Robert-Guroff and R. C. Gallo, Blut 47, 1 (1983);
V. S. Kalyanaraman, M. G. Sarngad­haran, B. J. Poiesz,
F. W. Ruscetti, R. C. Gallo, J. Virol. 81, 906 (1981); C.
Saxinger and R. C. Gallo, Lab. Invest. 49, 371 (1983).
15. For virus isolation, samples of freshly drawn,
heparinized peripheral blood or bone marrow,
yielding a minimum of 107 viable cells (greater than
90 percent), are needed. These samples must contain
the cells of interest, namely, OKT4+ T cells, which are
frequently depleted in AIDS patients.
16. S. Arya et al., in preparation.
17. We thank M. Gonda for electron microscopy and
A. Patel, S. Roberson, A. Fladager, and E. Reid for
technical assistance. We are also in­debted to many
clinical collaborators who pro­vided patient
materials.
1. M. S. Gottlieb et al., N. Eng. J. Med. 305, 1425
(1981); H. Masur et al., ibid., p. 1431; F. P. Siegal et
al., ibid., p. 1439.
2. Centers for Disease Control, Morbid. Mortal, Week.
Rpt. 32, 688 (1984); J. W. Curran et al., New Eng.
J. Med. 310, 69 (1984); G. B. Scott, B. E. Buck, J. G.
Letterman, F. L. Bloom, W. P. Parks, ibid., p. 76; J.
Oleske et al., J. Am. Med. Assoc. 249, 2345 (1983).
3. M. Popovic et al., Science 224, 497 (1984).
4. R. C. Gallo et al., ibid. 220, 865 (1983); M. Popovic
and R. C. Gallo, in preparation.
5. M. Essex et al., Science 220, 859 (1983); M. Essex et
al., in Human T-Cell Leukemia Viruses, R. C. Gallo, M.
Essex, L. Gross, Eds. (Cold Spring Harbor Press, Cold
Spring Harbor, N.Y., in press),
6. R. C. Gallo et al., Cancer Res. 43, 3892 (1983); in
Cancer Surveys, L. M. Franks et al., Eds. (Oxford Univ.
Press, Oxford, in press).
7. F. Barré-Sinoussi et al., Science 220, 868 (1983).
8. E. P. Gelmann et al., ibid., p. 862.
9. M. Robert-Guroff et al., in Human T-Cell Leu­kemia
Viruses, R. C. Gallo, M. Essex, L. Gross, Eds. (Cold
Spring Harbor Press, Cold Spring Harbor, N.Y., in
press).
10. T. H. Lee et al., personal communication; J.
Schüpbach, M. G. Sarngadharan, R. C. Gallo, in
preparation.
11. M. G. Sarngadharan, M. Popovic, L. Bruch, J.
30 March 1984; accepted 19 April 1984
13
Serological Analysis of a Subgroup
of Human T-Lymphotropic Retroviruses
(HTLV-III) Associated with AIDS
Jörg Schüpbach, Mikulas Popovic, Raymond V. Gilden, Matthew A. Gonda, M. G. Sarngadharan,
Robert C. Gallo
The two main subgroups of the family of human T-lymphotropic retroviruses (HTLV) that have
previously been characterized are known as HTLV-I and HTLV-II. Both are associated with certain
human leukemias and lymphomas. Cell surface antigens (p6I and p65) encoded by HTLV-I are
frequently recognized, at low titers, by antibodies in the serum of patients with acquired immunodeficiency syndrome (AIDS) or with signs or symptoms that precede AIDS (pre-AIDS). This
suggests an involvement of HTLV in these disorders. Another subgroup of HTLV, designated
HTLV-III, has now been isolated from many patients with AIDS and pre-AIDS. In the studies described in this report, virus-associated antigens in T-cell clones permanently producing HTLV-III
were subjected to biochemical and immu­nological analyses. Antigens of HTLV-III, specifically
detected by antibodies in serum from AIDS or pre-AIDS patients and revealed by the Western
blot technique, are similar in size to those found in other subgroups of HTLV. They include at
least three serologically unrelated antigenic groups, one of which is associated with groupspecific antigens (p55 and p24) and another with envelope-related (p65) proteins, while the
antigens in the third group are of unknown affiliation. The data show that HTLV-III is clearly
distinguishable from HTLV-I and HTLV-II but is also significantly related to both viruses. HTLV-III
is thus a true member of the HTLV family.
M
embers of the family of human lymphotropic retroviruses (HTLV) have
the following features in common: a
pro­nounced tropism for OKT4+ lympho­cytes
(1), a reverse transcriptase (RT) with a high
molecular weight (100,000) and a preference
for Mg2+ as the divalent cation for optimal enzymatic activity (2, 3), and the capacity to inhibit T cell function (4) or, in some cases, kill
T cells (5). Many HTLV also have the capacity
to transform infected T cells (1). The two major subgroups that have been charac­terized (6)
are HTLV-I, which is causatively linked to certain adult T-cell ma­lignancies (7), and HTLVII, which was first identified in a patient with
hairy cell leukemia (8).
Viruses of the HTLV family have been detected in some patients with the ac­quired immunodeficiency syndrome (AIDS) (9) or with
pre-AIDS, a condition frequently progressing
to AIDS (10). A high proportion of patients
with AIDS or pre-AIDS, as well as a significant num­ber of hemophiliacs, have antibodies
in their serum that recognize a cell surface
glycoprotein (gp61) that is present on certain
human T cells infected with HTLV-I (11).
Gp61 and p65, a slightly larger protein that is
14
a homolog of gp61 and occurs in another cell
line producing HTLV-I, were subsequently
shown to be related to the HTLV viral glycoprotein (12, 13). Studies of blood transfusion
recipients who later developed AIDS and of
their blood donors have revealed the presence,
in the blood of the donors, of antibodies to
a retrovirus of the HTLV family (14). These
findings sug­gest an involvement of viruses of
the HTLV family in the cause of AIDS and
pre-AIDS. An involvement of HTLV-I alone
appeared doubtful, however, be­cause antibody
titers to gp61 of HTLV-I in these patients are
generally very low and antibodies to the structural proteins of HTLV, notably p24 and pl9
(15), are not detectable in most AIDS patients
(16). Instead, it seemed likely that anoth­er
member of the HTLV family might be involved
in the etiology of AIDS. Here we describe our
studies of a group of cytopathic viruses (collectively designat­ed HTLV-III) isolated from
patients with AIDS or pre-AIDS. Isolation of
these viruses was achieved by means of a novel system permitting the continuous growth
of T-cell clones infected with the cytopathic
types of HTLV found in these disorders (17).
We show that antigens associated with human
Fig. 1 (top). Serologic detection of antigens in HTLV-III
producer cell clones. Strip
RIA were performed with human serum as described elsewhere in detail (21). Briefly,
lysates of HTLV-III producer
cell clones were subjected to
electrophoresis under reducing conditions on preparative sodium dodecyl sulfate
(SDS)–polyacrylamide
slab
gels, and electroblotted to
nitrocellulose sheets (18). The
sheets were cut into strips.
These were incubated with
human serum diluted 1:100.
After three thorough washings, bound antibodies of immunoglobulin G (IgG) and immunoglobulin M (IgM) classes
were made visible with radiolabeled, affinity-purified goat
antiserum to human IgG and
IgM (H-chain specific) and
autoradiography. (A) Analysis
with H4/HTLV-III cells. (Lanes
a, d, and g) U.S. patients with
AIDS; (lane b) a French heterosexual male who developed AIDS after receiving a blood transfusion in Haiti
(24); (lane c) an AIDS patient from Switzerland; (lane e) a normal heterosexual control; (lane f) a French
pre-AIDS patient (24); (lane h) a Swiss heterosexual drug addict; (lane i) a normal homosexual control.
(B) Analysis with H17/HTLV-III cells. (Lane a) An infant with AIDS whose mother is a prostitute; sera from
both are highly positive for antibodies to the HTLV membrane antigen (11, 25) and in our ELISA with
disrupted HTLV-III (19); (lane b) same serum as in (A), lane d; (lane c) normal heterosexual control; (lane
d) another Swiss AIDS patient; (lane e) a Swiss heterosexual male intravenous drug abuser with generalized lymphadenopathy and thrombocytopenic purpura (pre-AIDS). Fig. 2 (bottom). (A) Specificity of
the antigens recognized. Lysates of cloned cells before and after infection with HTLV-III were analyzed
by the Western blot technique (18) with a 1:500 dilution of the serum shown in Fig. IB, lane e. (Lane a)
The H17 clone before and (lane b) the same clone after infection (H17/HTLV-III); (lane c) the H4 clone
before and (lane d) the same clone after in­fection (H4/HTLV-III). All reactive antigens are virus-related
with the exception of that with a molecular weight of 80,000 in H17 cells; this antigen binds antibodies
from all human sera investigated. Normal human serum did not bind to any of the virus-related bands
(not shown). (B) Comparison of antigens in (lanes a) cells and (lanes b) virus. Lysates of H4/HTLV-III (250
µg per lane) or virus purified from the cell culture flu­ids (19) (5 µg per lane) were analyzed with 1:500
dilutions of human sera. (Panel I) Same serum as in Fig. 2A; (panel II) serum of a Swiss male ho­mosexual
with fatigue and generalized lymphadenopathy (pre-AIDS); (panel III) serum from same AIDS patient as
in Fig. IB, lane d. An antigen with a molecular weight of 110,000 and p41, p39, and p24 are enriched in
the virus preparation [see (20)]. The serum in panel III recognized a sub­set of the antigens recognized by
the sera used in panels I and II.
cells infected by HTLV-III are specifically recognized by antibodies in serum from AIDS
and pre-AIDS patients, and present a preliminary biochemical and immunological analysis
of these antigens.
Lysates of two immortalized and in­fected
human T-cell clones, H4/HTLV-III and H17/
HTLV-III (17), were tested with samples of
human serum in a strip radioimmunoassay
(RIA) based on the Western blot technique
(18). The sera were from patients with AIDS
or pre-AIDS, from contacts of such patients,
and from homo- or heterosexual male controls. Sera from the same patients were also
tested by the enzyme-linked immunosorbent
assay (ELISA) with pu­rified HTLV-III as part
15
Fig. 3. Relation between HTLV-III and HTLV-II.
Serum of an AIDS patient at a dilution of 1:500 was
tested in a competition RIA on strips (20) prepared
with H4/HTLV-III cells. (Lane a) The human serum
was added directly to the strip (uncompeted con­
trol); (lanes b to e) the serum was first ab­sorbed
for 3 hours at 37°C with 1 mg of cellular extract.
In (b) the absorption was with uninfected H4 cells
(not producing virus); in (c) the absorption was
with H4/HTLV-III cells producing HTLV-III (positive
control); in (d) the absorption was with C3/44
cells (26) producing HTLV-II; in (e) the absorption
was with HUT 102 cells producing HTLV-I (2).
of a larger, sys­tematic serologic study of the
prevalence of antibodies to HTLV-III in AIDS
and pre-AIDS patients (19).
Representative results are shown in Fig. 1.
Sera from patients with AIDS or pre-AIDS,
and from some homosexuals and heroin-addicts, recognized a number of specific antigens not detected by sera from heterosexual
subjects. The most prominent reactions were
with antigens of the following molecular
weights: 65,000, 60,000, 55,000, 41,000,
and 24,000. Antigens with molecular weights
of approximately 88,000, 80,000, 39,000,
32,000, 28,000, and 21,000 gave less prominent reactions. The reaction with the antigen
of 55,000 (p55) only occurred in sera that also
recognized p24, suggest­ing a relationship between the two anti­gens.
The specificity of these reactions was studied by comparing lysates of H4/HTLV-III and
H17/HTLV-III with ly­sates of the same cell
clones, H4 and H17, before viral infection
(Fig. 2A). No antigen from the uninfected
clones reacted with the sera, with the excep16
tion of a protein with a molecular weight of
80,000 in Hl7 which bound antibodies from
all of the human serum samples tested (see
Fig. 1B) but not from rabbit or goat serum.
Antigens newly expressed after viral infection and recognized by the human serum used
for this analysis in­cluded p65, p55, p41, p39,
p32, and p24. A large protein with a molecular
weight of approximately 130,000 and a protein of 48,000 were also detected. With this serum, p55 consistently appeared as a doublet of
bands of similar intensity. With normal human
serum, none of the antigens was detected (not
shown). These results show clearly that the
anti­gens detected after virus infection are either virus-coded proteins or cellular antigens
specifically induced by the in­fection.
The antigens of H4/HTLV-III were also
compared with antigens from virus purified
from the culture fluids of H4/HTLV-III (Fig.
2B). Extensive accumu­lation of p24 and
p41 [see (20)] occurred in the virus preparation (Fig. 2B, panels I and II). Protein stains
showed that these molecules are the major
components of the virus preparation (19). P24
and p41 may therefore be considered viral
struc­tural proteins. Furthermore, an antigen
with a molecular weight of approximate­ly
110,000 was detected in the virus preparation
but was below limit of detec­tion in the cells.
Also, p39 [see (20)] was present in the virus
preparation. It is interesting that p24 in the
virus prepara­tion consistently appeared as a
doublet (p24/p23), whereas in the cells it ap­
peared as p24 alone. The significance of this
is under investigation. P55 was not detected
in the virus; however, the inten­sity of the p55
band in the cells (Fig. 2B, lanes a) appeared
to correlate with the intensity of p24/p23 in
the virus preparation (Fig. 2B, lanes b), thus
again sug­gesting a relation between these
anti­gens. The p55 is probably a precursor of
p24, since a group-specific antigen of similar size (Pr 54gag) in HTLV-I–infected cells is
the precursor of p24 and the other gag-coded
proteins (21). Occasion­ally an additional set
of antigens was recognized by a serum (Fig.
2B, panel III) but their relation to the antigens
described above is unclear.
Thus we have shown that viral or virusinduced antigens in cloned human T cells
infected with HTLV-III are spe­cifically recognized by antibodies in the serum of patients
Fig. 4. Electron microscopy of
thin sections of cells producing HTLV-I, -II, and -III. (Top)
HUT 102 cells producing
HTLV-I (2). (Middle) Cells from
an AIDS patient (J.P.) producing HTLV-II (24). (Bottom)
Cells from a patient [described in (27)] with pre-AIDS,
producing HTLV-III. (Panels a)
Virus particles budding from
the cell membrane. (Panels b)
Free particles have separated
from the membrane. (Panels
c) Free particles sectioned in
a different plane. Note the
dense, cylindrical core region
of HTLV-III.
with AIDS or pre-AIDS. The detection of p65
by many of the serum samples is of special
interest. We have tested these sera on strips pre­
pared from lysates of cells producing HTLV-I
or -II. Some of these cells pro­duce a p65 that
has been shown (13) to be coded for by the
env gene of HTLV-I and to be the homolog of
the gp61 de­scribed by others (11, 12). Many
of the sera recognizing p65 in HTLV-III–infected cells also recognized, though somewhat
faintly, p65 in cells producing HTLV-I or –II,
and some of them also recognized gag-related
antigens (data not shown).
In addition, the reaction of some hu­man sera
with virus-related antigens of HTLV-III–infected cells could be par­tially inhibited by large
amounts of ex­tracts of cells producing HTLVII (Fig. 3). When a human serum not recognizing p65 was used, the antigens for which
there was competition included p55, p48, p4l,
p39, and p24. These results were confirmed
by the demonstration that a rabbit antiserum
raised against purified HTLV-III showed some
reactivity with antigens of HTLV-II and, to a
lesser extent, with HTLV-I. In contrast, anti­
serum to HTLV-II recognized both HTLV-I
and -III antigens, and an antise­rum to HTLVI reacted well with HTLV-II, but only faintly
with HTLV-III (22). Moreover, nucleotide
sequences of HTLV-III have been found to be
related to HTLV-I and -II (23). Although the
morphology of HTLV-III particles appears to
be somewhat different from the morphology
of HTLV-I and -II (Fig. 4) , and although some
differences are also found in the protein patterns of purified virus preparations (19), these
immunological and nucleic acid data clearly
indicate that HTLV-III is a true member of the
HTLV family and that it is more closely related to HTLV-II than to HTLV-I.
References and Notes
1. M. Popovic, P. S. Sarin, M. Robert-Guroff, V. S.
Kalyanaraman, D. Mann, J. Minowada, R. C. Gallo,
Science 219, 856 (1983); P. D. Markham, S. Z.
Salahuddin, V. S. Kalyanaraman, M. Popovic, P.
Sarin, R. C Gallo, Int. J. Cancer 31, 413 (1983); S.
Z. Salahuddin, P. D. Mark­ham, F. Wong-Staal, G.
Franchini, V. S. Kal­yanaraman, R. C Gallo, Virology
129, 51 (1983).
2. B. J. Poiesz, F. W. Ruscetti, A. F. Gazdar, P. A. Bunn, J.
D. Minna, R. C. Gallo, Proc. Natl. Acad. Sci. U.S.A. 77,
7415 (1980).
3. H. M. Rho, B. Poiesz, F. W. Ruscetti, R. C Gallo, Virology
112, 355 (1981); M. Seiki, S. Hattori, Y. Hirayama, M.
Yoshida, Proc. Natl. Acad. Sci. U.S.A. 80, 3618 (1983).
4. M. Popovic et al., in preparation.
5. H. Mitsuya, H. G. Guo, M. Megson, C. O. Trainor, M. S.
Reitz, S. Broder, Science 223, 1293 (1984).
6. For a brief review, see M. G. Sarngadharan et al., in
Human Carcinogenesis, C. C. Harris and H. H. Autrup,
Eds. (Academic Press, New York, 1983), p. 679.
7. V. S. Kalyanaraman, M. G. Sarngadharan, Y. Nakao, Y.
Ito, T. Aoki, R. C. Gallo, Proc. Nail. Acad. Sci. U.S.A. 79,
1653 (1982); M. Robert-Guroff, Y. Nakao, K. Notake,
Y. Ito, A. H. Sliski, R. C. Gallo, Science 215, 925 (1982);
Y. Hinuma et al., Int. J. Cancer 29, 631 (1982); W. A.
Blattner et al., ibid. 30, 257 (1982); J. Schüp­bach,
V. S. Kalyanaraman, M. G. Sarngad­haran, Y. Nakao,
R. C. Gallo, ibid. 32, 583 (1983) ; J. Schüpbach, V. S.
Kalyanaraman, M. G. Sarngadharan, W. A. Blattner, R.
C. Gallo, Cancer Res. 43, 886 (1983).
8. V. S. Kalyanaraman et al., Science 218, 571 (1982); E.
P. Gelmann et al., Proc. Natl. Acad. Sci. U.S.A. 81, 993
(1984).
17
9. R. C Gallo et al., Science 220, 865 (1983); E. P.
Gelmann et al., ibid., p. 862.
10. F. Barré-Sinoussi et al, ibid., p. 868.
11. M. Essex et al., ibid., p. 859; M. Essex et al., ibid. 221,
1061 (1983).
12. T. H. Lee, J. E. Coligan, T. Homma, M. F. McLane, N.
Tachibana, M. Essex, Proc. Natl. Acad. Sci. U.S.A., in
press.
13. J. Schüpbach, M. G. Sarngadharan, R. C. Gallo, Science,
in press.
14. H. W. Jaffe et al., Science 223, 1309 (1984).
15. V. S. Kalyanaraman, M. G. Sarngadharan, P. A. Bunn,
J. D. Minna, R. C. Gallo, Nature (London) 294, 271
(1981); V. S. Kalyanaraman, M. Jarvis-Morar, M. G.
Sarngadharan, R. C Gallo, Virology 132, 61 (1984).
16. M. Robert-Guroff et al., in Cancer Cells, vol. 3, Human
T-Cell Leukemia Viruses, R. C Gallo and M. Essex, Eds.
(Cold Spring Harbor Labo­ratory, Cold Spring Harbor,
N.Y., in press).
17. M. Popovic, M. G. Sarngadharan, E. Read, R. C. Gallo,
Science 224, 497 (1984).
18. H. Towbin, T. Staehelin, J. Gordon, Proc. Natl. Acad.
Sci. U.S.A. 76, 4350 (1979).
19. M. G. Sarngadharan, M. Popovic, L. Bruch, J.
Schüpbach, R. C. Gallo, Science 224, 506 (1984).
20. Although in Fig. 2B the p41 in the virus prepara­tion
appears to be larger than the p41 in cells, the two
molecules are of the same size. During application
of the lysates to the gel in another experiment, a
small amount of cellular lysate was spilled into the
neighboring lanes and the cellular p41 moved with
the same velocity as the viral p41. A connecting
band was thus formed between the p41 in the lane
containing the cells and the p41 in the lane with the
virus. The same situation occurred with p39 in cells
and virus.
21. J. Schüpbach, V. S. Kalyanaraman, M. G. Sarn­gadharan,
R. C. Gallo, in preparation.
22. M. G. Sarngadharan et al., in preparation.
23. S. Arya et al., in preparation.
24. J. B. Brunet et al., Lancet 1983-I, 700 (1983).
25. M. Essex, personal communication.
26. M. Popovic, V. S. Kalyanaraman, D. L. Mann, E.
Richardson, P. S. Sarin, R. C. Gallo, in Cancer Cells,
vol. 3, Human T-Cell Leukemia Viruses, R. C. Gallo and
M. Essex, Eds. (Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y., in press).
27. R. C. Gallo et al., Science 224, 500 (1984).
28. We thank J. Ahmad for technical assistance and R.
Lüthy and M. Vogt, Division of Infectious Diseases,
Department of Medicine, University Hospital, Zurich,
and O. Haller, Institute for Immunology and Virology,
University of Zu­rich, Zurich, Switzerland, for making
some sera from AIDS and pre-AIDS patients available
and for providing clinical information. J.S. is a Fogarty
International Fellow of the National Can­cer Institute.
30 March 1984; accepted 19 April 1984
Antibodies Reactive with Human
T-Lymphotropic Retroviruses (HTLV-III)
in the Serum of Patients with AIDS
M. G. Sarngadharan, Mikulas Popovic, Lilian Bruch, Jörg Schüpbach, Robert C. Gallo
In cats, infection with T-lymphotropic retroviruses can cause T-cell proliferation and leukemia or
T-cell depletion and immunosuppression. In humans, some highly T4 tropic retroviruses called
HTLV-I can cause T-cell proliferation and leukemia. The subgroup HTLV-II also induces T-cell
proliferation in vitro, but its role in disease is unclear. Viruses of a third subgroup of human
T-lymphotropic retroviruses, collectively designated HTLV-III, have been isolated from cultured
cells of 48 patients with acquired immunodeficiency syndrome (AIDS). The biologi­cal properties of HTLV-III and immunological analyses of its proteins show that this virus is a member of
the HTLV family, and that it is more closely related to HTLV-II than to HTLV-I. Serum samples
from 88 percent of patients with AIDS and from 79 percent of homosexual men with signs and
symptoms that frequently precede AIDS, but from less than 1 percent of heterosexual subjects,
have antibodies reactive against antigens of HTLV-III. The major immune reactivity appears to be
directed against p41, the presumed envelope antigen of the virus.
T
he incidence of the acquired immunodeficiency syndrome (AIDS) in homosexual men with multiple sexual partners, intravenous drug abusers, he­mophiliacs,
blood transfusion recipients, and close heterosexual contacts of mem­bers of these high-risk
groups (1-7) strongly suggests that the disease
spreads by the transmission of an infec­tious
18
agent (8, 9). The agent’s primary targets within
the body appear to be specific subpopulations
of T cells. The severe immune deficiency of
AIDS pa­tients results from an unusually low
pro­portion of helper T lymphocytes (OKT4+)
and a resulting lack of many helper functions,
including production of antibodies by B cells
(1, 3).
Table 1. Antibodies to HTLV-III in serum samples from patients with AIDS and pre-AIDS and from control subjects. Wells of 96-well Immulon plates were coated overnight with a lysate of density-banded
HTLV-III (30) at 0.5 (µg protein per well in 100 µl 50 mM sodium bicarbonate buffer, pH 9.6. The wells
were washed with water and incubated for 20 minutes with 100 µl of 5 percent bovine serum albumin in
phosphate buffered saline (PBS). The wells were washed again in water, and then 100 µl1 of 20 percent
normal goat serum in PBS were added to each well, followed by 5 or 10 µl of the test sera. These were
allowed to react for 2 hours at room temperature. The wells were washed three times with 0.05 percent
Tween-20 in PBS and incubated for 1 hour at room temperature with peroxidase-labeled goat antiserum
to human immunoglobulin G at a dilution of 1:2000 in 1 percent normal goat serum in PBS. The wells
were successively washed four times with 0.05 percent Tween-20 in PBS and four times with PBS and
reacted with 100 µl of the substrate mixture containing 0.05 percent orthophenylene diamine and 0.005
percent hydrogen peroxide in phosphate-citrate buffer, pH 5.0. The reactions were stopped by the addition of 50 µl of 4N H2S04, and the color yield was measured with a Dynatech ELISA reader. Assays were
done in duplicate and absorbance reading greater than three times the average of four normal negative
control readings was taken as positive.
Retrovirus infections are known to lead to
depressed immune functions in animal systems. For example, in cats, a major result of
infection with feline leu­kemia virus (FeLV) is
loss of normal immune function. More FeLVinfected cats die from consequences of this
im­mune dysfunction than from the leuke­mia
itself (10). FeLV provides an exam­ple of a single T-cell tropic retrovirus that causes both target cell proliferation (leukemia) and depletion
(immunosup­pression). By analogy, a human
retrovi­rus with a tropism for T cells should
be considered a serious candidate in the etiology of human AIDS. Two subgroups of a
family of human T-lymphotropic retroviruses
(HTLV) have been isolated and characterized
(11). The first, HTLV-I, was isolated from a
black American with an aggressive form of
T-cell lymphoma (12) and has been etiologically linked to the pathogenesis of adult T-cell
leukemia-lymphoma (ATL) (13-15). In­fection
with HTLV-I in vitro can alter T-cell function
(16) and, in some cases, lead to T-cell death
(17). HTLV-II was isolated from a patient with
a T-cell variant of hairy cell leukemia (18).
Although there are distinct differences between HTLV-I and HTLV-II, they have the
following common features: a tropism for
OKT4+ lymphocytes (19); a Mg2+-dependent
reverse transcriptase (RT) of high molecular
weight (100,000) (20); some antigenic crossreactivity in their proteins (18); a novel set
of nucleo­tide sequences called pX at the 3'
end of the viral genome; a limited amount of
nucleic acid homology in their genomes (21);
and similar morphology. Both HTLV-I and
HTLV-II have been isolat­ed from cultured T
cells of patients with AIDS (22, 23). Another
retrovirus was isolated from a homosexual
patient with chronic generalized lymphadenopathy (24), a syndrome that often precedes
AIDS and is therefore referred to as preAIDS. Proviral DNA of HTLV-I was detected
in the cellular DNA of two AIDS patients (25),
19
Fig. 1. Comparison of the SDS–polyacrylamide
gel profile of HTLV-III with profiles of HTLV-I and
Rauscher murine leukemia virus (R-MuLV). Lane
1, HTLV-I; lane 2, HTLV-III; lane 3, R-MuLV; lane
4, molecular weight standards: phosphorylase b
(94,000), bovine serum albumin (68,000), ovalbumin (45,000), chymotrypsinogen (25,500), and
lysozyme (14,000).
and serum samples from some patients were
shown to react with antigens of HTLV-I (26).
A larger proportion of the sera reacted with a
cell membrane antigen specific to HTLV-I–infected cells (27). This antigen has since been
identified as a precursor of the envelope glycoprotein, gp46, of HTLV-I (28, 29). However,
the correlation be­tween AIDS and serum antibodies to HTLV-I protein (including the cell
mem­brane antigen, p61) is weak.
These results are consistent with the idea
that the primary cause of AIDS is another
member of the HTLV family with limited
cross-reactivities with the known HTLV subgroups. Sera with high titers of antibodies to
the AIDS-specific virus might show a detectable reaction with antigens of HTLV-I and
HTLV-II, whereas the reaction of sera with
low titers might be too weak to recognize in
such a cross-reactive system. Our attempts to
isolate other retroviruses from AIDS patients
resulted in the identifica­tion of a number of
HTLV isolates that are similar to each other
20
but are distin­guishable from HTLV-I and
HTLV-II. These new isolates are designated
HTLV-III and are described in the accompanying reports (30-32). Here we describe the use
of HTLV-III in an im­munological screening of
serum samples from patients with AIDS and
pre-AIDS and from individuals at increased
risk for AIDS.
The virus was purified from supernatants of
cell cultures supporting the con­tinuous production of HTLV-III (30). The virus showed a
difference in the makeup of its protein components as revealed by sodium dodecyl sulfate
(SDS)-polyacrylamide gel electrophore­sis of
a sucrose density banded prepara­tion (Fig.
1, lane 2). Like HTLV-I (lane 1), and unlike
common mammalian re­troviruses (for example, Rauscher mu­rine leukemia virus, lane 3),
HTLV-III (lane 2) has a major group-specific
anti­gen (gag protein) with a molecular weight
of 24,000 (p24). It has a reverse transcriptase
with a molecular weight of about 100,000,
another protein with a molecular weight of
41,000 (presumably the envelope glycoprotein), and shows a tropism for OKT4+ lymphocytes. How­ever, it lacks the band separating at
a molecular weight of 19,000 (pl9). In­stead, it
has a smaller band that is miss­ing in HTLV-I.
Immunological studies presented in an accompanying report (32) also indicate that HTLV-III
is antigenically different from HTLV-I and -II,
but that it also shares a variety of antigenic determinants with them, especially with HTLVII. This relatedness has also been confirmed
by comparison of nucle­otide sequences of the
three types of HTLV (33).
Serum samples were obtained from patients
with clinically documented AIDS, Kaposi’s
sarcoma, sexual con­tacts of AIDS patients,
intravenous drug abusers, homosexual men,
and hetero­sexual subjects. These sera were
tested for their reactivity to HTLV-III by
means of the enzyme-linked immunosor­bent
assay (ELISA) (34). Lysates of su­crose density
banded HTLV-III were coated on 96-well microtiter plates. The test sera were diluted with
normal goat serum, added to the wells, and allowed to react for 2 hours or overnight at room
temperature. The primary immune com­plex
formed with the antibodies in the human sera
was detected by adding peroxidase-labeled
goat antiserum to human immunoglobulins
and assaying for a col­ored peroxidase reaction
product (34). The results are presented in Table
1. Of 49 clinically diagnosed AIDS patients,
43 (88 percent) showed serum reactivity in
this assay. Two of the subjects whose serum
reacted positively with the HTLV preparation had developed AIDS after receiving
blood transfusions, one in Hai­ti and the other
in Aruba. Of 14 homo­sexual men with preAIDS, 11 (79 per­cent) were positive. Of 17
homosexual men with no clinical symptoms
of AIDS, seven were positive. At least one
of these was known to be a long-time sexu­al
partner of a patient with clinically diagnosed
AIDS. Another had persistent fatigue and possibly other early symp­toms of AIDS. Because
these 17 men had been seeking medical assistance, they are not a representative sample
of the homosexual population, and the high
incidence of HTLV-III–specific antibod­ies in
their sera may not reflect the true incidence in
the homosexual population. One of the three
intravenous drug abus­ers that were positive
for serum antibod­ies to HTLV-III was also a
homosexual. Serum samples from only one
of 186 control subjects reacted positively
in this test. These control subjects included
three with hepatitis B virus infection, one
with rheumatoid arthritis, six with systemic
lupus erythematosus, four with acute mononucleosis, and eight with var­ious forms of
lymphatic leukemias and lymphomas, some
of whom were posi­tive for HTLV-I. The rest
were normal donors of unknown sexual preference including laboratory workers ranging
in age from 22 to 50.
To understand the molecular nature of the
antigens recognized by ELISA, we conducted
the following experiment. A lysate of HTLVIII was fractionated by SDS–polyacrylamide
gel electrophoresis and transferred to a nitrocellulose sheet by the eleetrophoretic blotting
(Western) technique of Towbin et al. (35).
The nitrocellulose sheet was cut into 0.5-cm
strips and reacted with samples of the human
sera. Antigen-antibody complex­es formed
were detected by autoradiography after incubation of the strips with 125I-labeled goat
antibody to human immunoglobulin. Figure
2 shows that the antigen most prominently
and commonly detected among all of the sera
from AIDS patients had a molecular weight of
41,000 (p41). This corresponds to one of the
major proteins of the virus (Fig. 1) and is pre-
Fig. 2. Identification of HTLV-III antigens recognized by sera of AIDS patients. HTLV-III was lysed
and fractionated by electropho­resis on a 12 percent polyacrylamide slab gel in the presence of
SDS. The protein bands on the gel were electrophoretically transferred to a nitrocellulose sheet
according to the proce­dure of Towbin et al. (35).
Strip solid-phase radioimmunoassays were then
performed as described (36). The sheet was incubated at 37°C for 2 hours with 5 percent bovine
serum albumin in 10 mM tris-HCl, pH 7.5 containing 0.9 percent NaCl and cut into 0.5-cm strips.
Each strip was incubated for 2 hours at 37°C and
2 hours at room temperature in a screw cap tube
containing 2.5 ml of buffer-1 (20 mM tris-HCl, pH
7.5, 1 mM EDTA, 0.2M NaCl, 0.3 percent Triton X100, and 2 mg of bovine serum albumin and 0.2
mg of human Fab per milliliter). Test sera (25 µl)
were then added to individual tubes containing the
strips and in­cubation was continued for 1 hour at
room temperature and overnight in the cold. The
strips were washed three times with a solution
containing 0.5 percent sodium deoxycholate, 0.1M
NaCl, 0.5 percent Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 10 mM sodium phosphate,
pH 7.5. The strips were incubated for 1 hour at
room temperature with 2.4 ml of buffer-1 and 0.1
ml of normal goat serum. Affinity-purified and 125Ilabeled goat antiserum to human immunoglobulin (µ chain and Fc fragment) (1.25 x 106 count/
min) were added to the reaction mixture and the
incuba­tion was continued for 30 minutes at room
temperature. The strips were washed as de­scribed,
dried, mounted, and exposed to x-ray film. Strip 1,
adult T-cell leukemia; strip 2, normal donor; strip 3,
mother of a child with AIDS; strips 4 and 6 to 10,
AIDS patients; and strip 5, patient with pre-AIDS.
21
sumably the envelope protein. Strip 7 shows
the result obtained with serum from an AIDS
patient that reacted negatively in the ELISA
but in this more sensitive strip assay it gave a
low, but definitely positive, result. Reactivity
to p24 of the virus was generally very weak
and was clear only in two cases (strips 4 and
5). This may be a reflection of the relative titer
toward different antigens. One would expect
the highest antibody titer against the envelope
of the infecting agent, especially if the infection causes a pronounced immune deficiency
and de­creased capacity to make antibodies in
response to subsequent antigenic chal­lenge.
Additional reactivities against antigens with
molecular weights of 66,000 and 51,000 were
seen in some sera. In strip 8 the serum reacted
with an additional antigen that has a molecular
weight of 31,000. These additional anti­gens
appear to be related to those detect­ed by sera
from the same patients in HTLV-III–producing cells (33). Strips 1 and 2 show that sera
from a patient with ATL who was positive for
HTLV-I and from a normal subject do not react with the antigens of HTLV-III.
Of particular interest is the finding that
among the serum samples that reacted positively with HTLV-III two were from young
children (ages 7 months and 2 years). These
children were free of known opportunistic infections including cytomegalovirus,
Epstein-Barr virus, Pneumocystis carinii,
and fungus. The mother of one of them was
positive in both tests described here. The
children presumably acquired the infection
in utero, by their mother’s milk, or by another route.
Among the positive serum samples from
AIDS patients there appears to be a wide
variation in antibody titer to HTLV-III.
Generally, the titers in sera from patients
with advanced AIDS are significantly lower
than those in sera from newly diagnosed patients and pa­tients with pre-AIDS. This is
consistent with the idea that HTLV-III infection causes an initial lymphoid proliferation
but eventually causes death of the target lymphocytes (OKT4+) leading to the ab­normal
T4+/T8+ ratios and loss of helper T-cell
functions including antibody pro­duction by
B cells. Therefore, the low or negative result
in the ELISA of sera from some cases of advanced AIDS may be a consequence of the
22
natural course of the disease. To prove this
it will be necessary to study antibody titers
in sera obtained at intervals from subjects at
risk for the disease. The serum of one AIDS
patient showed a low positive ti­ter, but serum from his homosexual part­ner with no
symptoms of AIDS had a significantly higher antibody titer. It is interesting that the
serum of one AIDS patient that was negative in the ELISA did show a definite but
low positive reaction with p41 in the more
sensitive Western blot assay (Fig. 2, strip 7).
The ELISA with purified p41 might prove to
be even more sensitive. It is significant that
although HTLV proviral sequences were
clearly detected in DNA from cell samples
obtained from two AIDS pa­tients early in
the course of their disease, these sequences
could not be detected in cells obtained after
1 year in one case and 2 months in the second case (25). It is conceivable that the subset of T lym­phocytes that forms the target
of the provirus had been depleted before the
second samples were obtained in each case.
In conclusion, we have shown a high incidence of specific antibodies to HTLV-III in
patients with AIDS and pre-AIDS. Among
the antibody-positive cases reported here a
few are of particu­lar importance with respect to the trans­mission of the disease. For
example, the mother of the baby with AIDS
was posi­tive for HTLV-III as was a long-term
sexual partner of a homosexual with AIDS.
Recipients of blood products orig­inating
from individuals at risk for AIDS were also
positive for HTLV-III and, as described in
an accompanying report (31), the virus has
been isolated from several children with
AIDS as well as from their mothers. The
data presented here and in the accompanying reports (30-32) suggest that HTLV-III is
the primary cause of AIDS.
7. J. W. Curran, D. N. Lawrence, H. Jaffe, J. E. Kaplan, L.
D. Zyla, M. Chamberland, R. Weinstein, K.-J. Lui, L. B.
Schonberger, T. J. Spira, W. J. Alexander, G. Swinger, A.
Ammann, S. Solomon, D. Auerback, R. Stoneberger, J.
M. Mason, H. W. Haverkos, B. L. Evatt, N. Engl. J. Med.
310, 69 (1984).
8. Centers for Disease Control, Task Force on Kaposi’s
Sarcoma and Opportunistic Infections, ibid. 306, 248
(1982).
9. J. P. Hanrahan, G. P. Wormser, C. P. Maguire, L. J. De
Lorenzo, G. Davis, ibid. 307, 498 (1982).
10. M. Essex, W. D. Hardy, Jr., S. M. Cotter, R. M. Jakowski,
A. Sliski, Infect. Immun. 11, 470 (1975); W. D. Hardy,
Jr., P. W. Hess, E. G. MacEwen, A. J. McClelland, E. E.
Zuckerman, M. Essex, S. M. Cotter, Cancer Res. 36,
582 (1976) ; L. J. Anderson, O. Jarrett, H. M. Laird, J.
Natl. Cancer Inst. 47, 807 (1971).
11. R. C. Gallo, in Cancer Surveys, L. M. Franks et al., Eds.
(Oxford Univ. Press, Oxford, in press).
12. B. J. Poiesz, F. W. Ruscetti, A. F. Gazdar, P. A. Bunn, J.
D. Minna, R. C. Gallo, Proc. Natl. Acad. Sci. U.S.A. 77,
7415 (1980).
13. V. S. Kalyanaraman, M. G. Sarngadharan, Y. Nakao, Y.
Ito, T. Aoki, R. C. Gallo, ibid. 79, 1653 (1982).
14. W. A. Blattner, V. S. Kalyanaraman, M. Robert-Guroff,
T. A. Lister, D. A. G. Galton, P. Sarin, M. H. Crawford,
D. Catovsky, M. Greaves, R. C. Gallo, Int. J. Cancer 30,
257 (1982).
15. R. C. Gallo et al., Cancer Res. 43, 3892 (1983).
16. M. Popovic, N. Flomberg, D. Volkman, D. Mann, A. S.
Fauci, B. DuPont, R. C. Gallo, in preparation.
17. H. Mitsuya, H. G. Guo, M. Megson, C. D. Trainor, M. S.
Reitz, S. Broder, Science 223, 1293 (1984).
18. V. S. Kalyanaraman, M. G. Sarngadharan, M. RobertGuroff, I. Miyoshi, D. Blayney, D. Golde, R. C. Gallo,
ibid. 218, 571 (1982).
19. M. Popovic, unpublished observation.
20. H. M. Rho, B. J. Poiesz, F. W. Ruscetti, R. C. Gallo,
Virology 112, 355 (1981).
21. E. P. Gelmann, G. Franchini, V. Manzari, F. Wong-Staal,
R. C. Gallo, Proc. Natl. Acad. Sci. U.S.A. 81, 993
(1984).
22. R. C. Gallo et al., Science 220, 865 (1983).
23. M. Popovic et al., in preparation.
24. F. Barré-Sinoussi et al., Science 220, 868 (1983).
25. E. P. Gelmann, M. Popovic, D. Blayney, H. Mazur, G.
Sidhu, R. E. Stahl, R. C. Gallo, ibid. p. 862.
26. M. Robert-Guroff et al., unpublished results.
27. M. Essex, M. F. McLane, T. H. Lee, L. Falk, C. W. S. Howe,
J. I. Mullins, C. Cabradilla, D. P. Francis, Science 220,
859 (1983).
28. J. Schüpbach, M. G. Sarngadharan, R. C. Gallo, ibid., in
press.
29. T. H. Lee, J. E. Coligan, T. Homma, M. F. McLane, M.
Tochipana, M. Essex, Proc. Natl. Acad. Sci. U.S.A., in
press.
30. M. Popovic, M. G. Sarngadharan, E. Read, R. C. Gallo,
Science 224, 497 (1984).
31. R. C. Gallo et al., ibid., p. 500.
32. J. Schüpbach, M. Popovic, R. V. Gilden, M. A. Donda, M.
G. Sarngadharan, R. C. Gallo, ibid. p. 503.
33. S. Arya et al., in preparation.
34. C. Saxinger and R. C. Gallo. Lab. Invest. 49, 371
(1983).
35. H. Towbin, T. Staehelin, J. Gordon, Proc. Natl. Acad.
Sci. U.S.A. 76, 4350 (1979).
36. J. Schüpbach, V. S. Kalyanaraman, M. G. Sarn­gadharan,
R. C. Gallo, in preparation.
37. We thank K. L. Arnett for technical assistance and R.
Lüthy and M. Vogt, Division of Infec­tious Diseases,
Departme nt of Medicine, Uni­versity Hospital,
Zurich, and O. Haller, Insti­tute for Immunology and
Virology, University of Zurich, Zurich, Switzerland,
for providing us with sera from AIDS and pre-AIDS
patients and for clinical information. J.S. is a Fogarty
Inter­national Fellow of the National Cancer Institute.
30 March 1984; accepted 19 April 1984
References and Notes
1. M. S. Gottlieb, R. Schroff, H. M. Schanker, I. D.
Weisman, P. T. Fan, R. A. Wolf, A. Saxon, N. Engl. J.
Med. 305, 1425 (1981).
2. H. Masur, M. A. Michelis, J. B. Greene, ibid., p. 1431.
3. F. P. Siegal et al., ibid., p. 1439.
4. M. Poon, A. Landay, E. F. Prasthofer, S. Stagno, Ann. Int.
Med. 98, 287 (1983).
5. Morbid. Mortal. Weekly Rep. 31, 697 (1983).
6. B. Moll, E. E. Emerson, C. B. Small, G. H. Friedland, R. S.
Klein, I. Spigland, Clin. Immu­nol. Immunopathol. 25,
417 (1982).
23
The National Cancer Institute’s Role
in AIDS and Cancer
therapy (HAART) regimen that has
considerably extended the lives of
many HIV patients.
Robert Wiltrout and John Niederhuber
National Cancer Institute, National Institutes of Health
Though HAART has been a great
success, it is far from a cure.
Recently, NCI investigators,
working with industry, helped
develop another FDA-approved
drug (darunavir), a novel protease
inhibitor for treatment of drug
resistant HIV (9). Resistance
mechanisms are also under
investigation in monkey models,
thanks to the development by Dr.
Vineet KewalRamani in the HIV
Drug Resistance Program (DRP) of
an HIV-SIV hybrid which can cause
disease in monkeys, yet retains the
drug sensitivity characteristics of
HIV. The Laboratories of Molecular
Pharmacology and Medicinal
Chemistry are collaborating to
develop drugs that inhibit HIV
integrase, a target currently not
exploited in HAART regimens.
Bevirimat, an HIV maturation
inhibitor developed in the DRP, has
been shown to be effective against
drug resistant strains of HIV that
lack a mutation in the Gag capsid
protein targeted by the compound
in a Phase 2 clinical trial (1). Finally,
investigators are exploring several
vaccine strategies designed to
elicit cellular and humoral immune
responses necessary for effective
prophylaxis against HIV. Work
continues on DNA vaccines,
neutralizing antibodies, whole virus
vaccines based on inactivated
virions, and live vector vaccines
that utilize poxvirus or adenovirus.
I
n early 1981, a young man
presented to physicians at
NCI’s Metabolism Branch
with severe lymphopenia and
fever. A bone marrow biopsy
revealed extensive mycobacterial
infiltration. Though the etiology of
his immunodeficiency confounded
his physicians, his presenting
symptoms had recently been
described in a Centers for Disease
Control and Prevention Morbidity
and Mortality Weekly Report
addressing the appearance of
immunodeficiency and opportunistic infections among young
gay men (1). Similar cases were
soon being referred to NCI’s
Immunology Branch and reported,
in increasing numbers, in large
cities across the country.
NCI’s leaders quickly recognized
the emergence of a new epidemic
and the need to act swiftly. In
September 1981, NCI’s Division of
Cancer Treatment convened the
first national meeting to uncover
the causative factor(s) of the
new immunodeficiency disease.
HTLV-I, the first known human
retrovirus, had been identified as
the causative agent in adult T-cell
lymphoma just one year prior by a
group of investigators, led by Dr.
Robert Gallo, in NCI’s Laboratory
of Tumor Cell Biology (2). Because
HTLV-I caused immunodeficiency
and cancer, it was suggested
24
that it, or a similar virus, was
a possible etiologic agent for
acquired immunodeficiency syndrome (AIDS). This avenue paid
dividends when Dr. Gallo’s group
and Dr. Luc Montagnier in France
co-discovered HIV (3, 4). These
studies led to the development, by
Dr. Gallo’s group at NCI, of the first
diagnostic antibody test for AIDS,
thus ensuring the safety of the
blood supply.
NCI aggressively pursued the
characterization of the virus, in
an effort to develop an effective
treatment. By 1985, a trio of NCI
investigators, Samuel Broder,
Hiroaki Mitsuya, and Robert
Yarchoan, had demonstrated
in vitro the antiviral activity of
certain nucleoside analogs. This
work led to the development of
zidovudine (AZT) (5), the first
US FDA–approved drug in the
treatment of AIDS. Didanosine
(6) and zalcitabine (7) followed
shortly. Subsequent work by NCI
Frederick’s structural biology
group yielded the sequence and
structural conformation of the HIV
protease (8). This paved the way for
the development of a new class of
antiviral agents: protease inhibitors.
Today, along with nonnucleoside
reverse transcriptase inhibitors,
nucleoside analogs and protease
inhibitors are the foundation of
the highly active antiretroviral
Anti-HIV strategies are only one part
of the story. The catalyst for NCI’s
involvement in AIDS was the high
prevalence of cancer in the infected
population. Due to the HIV-induced
reduction in host resistance, many
AIDS-related malignancies have
viral etiologies. KSHV (Kaposi’s
sarcoma-associated herpesvirus),
EBV (Epstein-Barr virus) and
HPV (human papillomavirus) are
implicated in Kaposi’s sarcoma,
non Hodgkin lymphoma, and
cervical cancer—all of which are
more prevalent in the HIV-infected
population. Cancer has emerged
as the leading cause of death in
HIV-infected patients now that the
HAART regimen has led to the
decline of opportunistic infections
and advanced AIDS. Therefore,
NCI has devoted considerable
attention to the treatment of AIDSrelated malignancies. Dr. Robert
Yarchoan and Dr. Richard Little
have defined a therapeutic regimen
for Kaposi’s sarcoma; Dr. Little
and Dr. Wyndam Wilson defined
EPOCH (etoposide, vincristine,
doxorubicin, cyclophosphamide,
and prednisone) therapy for AIDSrelated lymphoma. Studies are
ongoing by the Yarchoan group
investigating the development of
treatment strategies for Kaposi’s
sarcoma using prodrugs activated
by viral enzymes that specifically
target KSHV-infected cells. Other
investigators have succeeded
in targeting the underlying viral
etiology in HIV-related cancer. In
2006, the FDA approved a vaccine
for cervical cancer prophylaxis
(11). The vaccine, developed in
part by Drs. Douglas Lowy and
John Schiller in NCI’s Laboratory of
Cellular Oncology, targets HPV 16
and 18, which are implicated in up
to 70 percent of cervical cancers.
25
Success in the development of this
vaccine has relevance far beyond
the HIV-infected population, as the
vast majority of cervical cancer
cases occur in the absence of HIV
coinfection. Similarly, EBV infection
in parts of Africa is endemic and
independent of HIV. It is estimated
that approximately 18 percent of
human cancers are caused by infection, both viral and nonviral (12).
Additional viruses have been found
to be associated with cancers,
though no causal link has been
established (13). As our ability to
detect infectious agents in association with cancer improves and
more studies are done to establish
causation, it is likely that the current
figure will rise. The expertise NCI
has gained in identifying, treating,
and preventing infectious causes
of cancer is likely to pay enormous
dividends in the near future.
NCI’s investment in and commitment to HIV-related research, ever
since that first patient in 1981, has
improved the lives of countless
patients with HIV, with further
advances to come. The work of
NCI scientists has also placed
the institute in an ideal position
to undertake the challenge of
uncovering new infectious causes
of cancer and building on the
intervention strategies that we
have developed in our HIV work to
successfully prevent or treat these
cancers at the earliest stages of
development.
References
1. Morbid. Mortal. Wkly. Rep. 3(25): 305-8,
1981.
2. B. Poiesz, F. W. Ruscetti, A. F. Gazdar et al.,
Proc. Natl. Acad. Sci. U.S.A. 77: 7415-19,
1980.
3. M. Popovic, M. G. Sarngadharan, E. Read, R.
C. Gallo Science 224: 497-500, 1984.
4. F. Barré-Sinoussi, J. C. Chermann, F. Rey,
M. T. Nugeyre, S. Chamaret, J. Gruest, C.
Dauguet, C. Axler-Blin, F. Vézinet-Brun, C.
Rouzioux, W. Rozenbaum, L. Montagnier
Science 220: 868-71, 1983.
5. H. Mitsuya, K. J. Weinhold, P. A. Furman et
al., Proc Natl Acad Sci U.S.A. 82(20): 7096100, 1985.
6. R. Yarchoan, H. Mitsuya, R. V. Thomas et al.,
Science. 245: 412-5, 1989.
7. H. Mitsuya, R. Yarchoan, S. Broder Science
249: 1533-44, 1990.
8. A. Wlodawer, J. Vondrasek Annual Review
of Biophysics and Biomolecular Structure 27:
249-284, 1998.
9. Y. Koh, H. Nakata, K. Maeda et al.,
Antimicrob. Agents Chemother. 47(10): 31239, 2003.
10. J. Lalezari, S. McCallister, M. Gigliotti
et al., 48th International Conference on
Antimicrobial Agents and Chemotherapy
(ICAAC 2008). Washington, D.C., October
25-28, 2008. Abstract H-89.
11. C. Carillo-Infante, G. Abbadessa, L. Bagella,
A. Giordano International Journal of
Oncology 30: 1521-28, 2007.
12. D. M. Parkin Int. J. Cancer 118: 3030–44,
2006.
13. M. E. McLaughlin-Drubin, K. Munger
Biochemia et Biophysica Acta 1782: 127-50,
2008.
Understanding Causation
Leads to Treatment
Samuel Broder, M.D.
Director, National Cancer Institute, Retired
Chief Medical Officer, Celera Corporation, Rockville, Maryland 20850
his year we mark the 25th anniversary of the four classic
Science publications by the
Gallo group, which identified the
pathogenic retrovirus HIV.
T
tients with AIDS. Within my group,
Hiroaki Mitsuya spearheaded the
lab-based discovery effort while
Robert Yarchoan did the same for
clinical investigation.
These discoveries overturned
entrenched beliefs including: (1)
active retroviruses did not exist
in human beings; or (2) if they
did, they were not associated
with human diseases; or (3) if
they were involved in disease,
such agents played a relatively
minor or anecdotal role in public
health. These beliefs were a barrier
to progress in the prevention,
diagnosis, and treatment of
HIV/AIDS.
We discovered the antiretroviral
features of a series of
dideoxynucleosides, the most
prominent of which was AZT (now
known as zidovudine), and took
them into clinical trials (1-6), a
process facilitated by a fortuitous
NIH tradition of locating research
labs adjacent to its Clinical Center.
These agents held promise,
partly because we found a huge
differential between in vitro drug
concentrations needed to inhibit
HIV replication, and the level
required to induce toxicity for
human host cells, inhibition of
antigen, or mitogen-driven T-cell
activation and immunoglobulin
production by indicator B cells.
The Gallo discoveries affected
many aspects of public health,
including the development of
effective antiviral agents and
targeting the proven causative
agent of the AIDS pandemic,
not secondary or opportunistic
infections, nor bystander microbes.
Such antiretroviral agents have
reduced the death rate of AIDS in
this country and other parts of
the world.
The publication of this work, and
none other, made it possible for my
laboratory, starting in May 1984,
to develop the first FDA-approved
antiretroviral agents for treating pa-
26
Approximately one year after the
key Gallo Science papers, the first
patient received AZT at the NIH
Clinical Center during the initial
Phase 1 clinical trial. We published
a series of papers summarizing this
drug family’s broad antiretroviral
activity and, more important by
far, evidence for clinical activity
including discoveries related to
pharmacology on both cellular
and clinical levels. Several of the
27
drugs studied were advanced into
randomized, multicenter clinical
trials sponsored by the National
Institute of Allergy and Infectious
Diseases (NIAID), which in turn led
to rapid approval by the US Food
and Drug Administration and by
health ministries in other countries.
Such agents are generally still used
in combination regimens. Several
of the agents were integrated
into later combinations with viral
protease inhibitors and other drugs
when it became apparent that no
single drug could be expected to
achieve durable viral suppression
and clinical benefits. Therefore,
it was often not necessary to
abandon already approved drugs,
but rather retain their clinical legacy
by inclusion in combinations with
newer agents. What ultimately
became known as HAART—highly
active antiretroviral therapy—
proved up to the task. At a
minimum, these events removed
the lingering doubt that pathogenic
retroviruses were inherently
untreatable, which had, until our
work, a significant impact on both
basic and clinical research.
What might have been considered
the most refractory feature of HIV/
AIDS, i.e., dementias associated
with infection in the central nervous
system, responded to therapy in
some cases, with improved neurologic signs and reversal of brain
metabolic abnormalities. Dideoxynucleosides like AZT, alone and in
combination, were shown quickly to
improve neurodevelopmental function and growth velocity in pediatric
patients with AIDS.
28
The public health consequences
are noteworthy. Proof that HIV
could be treated using clinically
active antiretroviral agents led, by
around 1995, to a precipitous drop
in the annual age-specific death
rate from HIV/AIDS in the United
States, reversing the inexorable rise
in statistics reported to the Centers
for Disease Control and Prevention
(http://www.cdc.gov/hiv/topics/
surveillance/resources/slides/
mortality). Moreover, the actuarial
statistics for people surviving after
a diagnosis of HIV/AIDS have
improved yearly since then.
Similarly, the availability of such
agents has led to a major change
in the outcomes and destinies of
HIV/AIDS patients, their families,
and communities in the developing
world. Low-cost versions of such
therapies have saved many lives
among the developing world’s
adults and children. Antiretroviral
therapies provide a practical way to
reduce maternal-child transmission
associated with pregnancy and
breast-feeding, the latter being
essential for infant survival in
many developing countries, but
unfortunately, also a route for
postpartum viral transmission.
While we mark the 25th anniversary
of the Gallo discoveries, there
remains unfinished business. This
is certainly true in HIV vaccine
development, but there is reason
for cautious optimism on this front
as well, a topic that will be covered
in more detail in other portions of
this booklet.
That said, a sense of complacency
based on progress from prior
work might reverse gains in
the future. There are worrisome
signs that unusual subtypes
or chimeric strains of HIV are
emerging, potentially affecting viral
load monitoring and the activity
of antiretroviral therapies. In
particular, non-B subtypes may
pose special challenges if they
manifest unique patterns of drug
susceptibility and resistance. This
makes several of the HIV molecular
epidemiology and surveillance
programs now carried out at
the Institute of Human Virology
and other centers all the more
important. Indeed, all of this
makes expanded support for
AIDS research at NIH a crucial
national priority.
Yet, this is also a moment to reflect
on the intellect, creativity, and
tenacity of the Gallo group, leading
to the discovery of HIV as the
causative agent of a terrible disease
without precedent in modern times
and to the discovery of antiretroviral
therapy with proven clinical utility.
These discoveries changed the
trajectory of a pandemic.
References
1. H. Mitsuya, S. Broder, Proc. Natl. Acad. Sci.
U.S.A. 83, 1911 (Mar. 1986).
2. H. Mitsuya, S. Broder, Nature 325, 773 (Feb.
26-Mar. 4, 1987).
3. H. Mitsuya et al., Proc. Natl. Acad. Sci. U.S.A.
82, 7096 (Oct. 1985).
4. H. Mitsuya, R. Yarchoan, S. Broder, Science
249, 1533 (Sep. 28, 1990).
5. R. Yarchoan et al., Lancet 1, 575 (Mar. 15,
1986).
6. R. Yarchoan, H. Mitsuya, C. E. Myers, S.
Broder, New Engl. J. Med. 321, 726 (Sep. 14,
1989).
Disclosure: The author is a former director of
the National Cancer Institute, and currently an
employee of the Celera Corporation, which has
commercial programs in molecular diagnostics for
HIV. He is also a co-inventor on government HIV
therapy patents and has received a royalty under
the Federal Technology Transfer Act.
On the Discovery of Human Retroviruses:
Interleukin-2; HTLV-I and HTLV-II; Isolation of HIV-1
Anders Vahlne
Karolinska Institutet, Stockholm, Sweden
T
o isolate T-cell lymphotropic
viruses one needs to culture
T lymphocytes. Working
with conditioned medium to grow
lymphocytes, Gallo together with
two of his postdoctoral fellows,
Doris Morgan and Frank Ruscetti,
discovered T-cell growth factor
(TCGF) later named interleukin 2
(IL-2). The first report of IL-2/TCGF
was published in 1976 (1) and
signaled a new capacity for in vitro
growth and analysis of human T
cells and, consequently, viruses
that target these cells.
Reverse transcriptase activity was
detected by Gallo’s group in a Tcell line established (using IL-2)
from a patient diagnosed originally
with mycosis fungoides in 1979. To
show that this was a novel human
retrovirus, i.e., not any of the known
animal retroviruses, Gallo and
29
co-workers demonstrated that:
virus could be isolated from tissue
samples of the same patient by
culturing primary T cells with IL-2, it
could infect human T cells in vitro,
specific antibodies to the virus were
found in serum from the patient,
proviral DNA was integrated in cells
from which the virus was isolated,
and specific antibodies were
documented in multiple patients.
Most or all these results were obtained by the time (August 1980)
Gallo submitted the first paper on
isolation of a human retrovirus (2)
which appeared that December.
Gallo designated the virus human
T-cell leukemia virus (HTLV). Gallo
and his group next isolated HTLV-I
from cells of a patient with Sézary
T-cell leukemia, submitting their results in May 1981 (3). Then, in June
and July, six and seven months,
respectively, after the first Gallo
paper on HTLV was published and
more than a year after it was submitted, Yorio Hinuma and his group
submitted two papers showing that
patients with adult T-cell leukemia
(ATL) or malignant T lymphoma
had antibodies against the MT-1
cell line, and that extracellular type
C virus particles were detected
in MT-1 cultures (4). Furthermore,
on transmission of virus from MT-1
cells (female) to cord blood cells of
a male infant, the latter cells were
transformed (immortalized) (5);
they designated the virus adult Tcell leukemia virus (ATLV).
In October of 1981, Gallo’s group
submitted a report (6) showing
that five of six tested ATL patients
in Japan had antibodies to HTLVI (the only HTLV known at the
30
time). Characterization of ATLV
as a retrovirus was published first
in March 1982. In this paper (7)
submitted in November 1981, it
was also reported that proviral DNA
was detected in fresh peripheral
lymphocytes from five patients with
ATL but not in healthy adults.
It should be mentioned that the
connection between HTLV-I and
ATL was facilitated greatly by
Kiyoshi Takatsuki’s earlier work
that revealed a distinct subset
of highly endemic leukemia in
southern Japan. The recognition
that HTLV-I causes ATL and that
breast-feeding readily transmits
virus led to a drastic reduction in
ATL incidence for Japan.
In November 1982, Gallo’s group
together with Japanese colleagues
Nakao, Miyoshi, Minowada,
Yoshida, and Ito reported that
HTLV-I and ATLV were the
same virus (8) and selected the
designation HTLV-I, recognizing
that Gallo’s group isolated the first
known human retrovirus. By 1982,
Gallo and co-workers reported a
second human retrovirus, HTLV-II,
in a patient with hairy cell leukemia
(9). However, no malignancy or
other disease has yet been linked
clearly to this virus.
The first report of a new human
retrovirus that was distinct morphologically from HTLV-I and HTLV-II
was an oral presentation by Luc
Montagnier at the Cold Spring Harbor meeting in September 1983 [a
proceedings was published September 1984] (10). The first publication in a peer-reviewed journal
showed a new retrovirus isolated
from siblings with hemophilia B,
one of whom had acquired immunodeficiency syndrome (AIDS)
(11). Immunological and molecular
characterizations of the isolated
virus did not discriminate convincingly the new virus from HTLV-I,
save for an electron micrograph
that clearly depicts virus with a
cylindrical or conical core, different
from the spherical cores in HTLV-I
and HTLV-II and consistent with
lentivirus morphology. The paper,
however, did not establish the new
virus as a causative agent for AIDS.
It has been claimed that the first
report describing a new human
retrovirus distinct from HTLV-I and
HTLV-II was the paper from Montagnier’s group that appeared in a
May issue of Science in 1983 (12).
However, I would argue that this
paper fails to prove a new virus.
First, the virus described in the
1983 paper was claimed to have
a type C morphology, not the
lentivirus morphology of HIV.
Second, the immunofluorescence
and immune precipitation data
lacked proper positive controls for
claiming the virus was distinct from
HTLV-I and no convincing evidence
was presented distinguishing it
from HTLV-II. Specifically, sera
from HTLV-I– and HTLV-II–infected
patients was not tested. The paper
was written apparently in haste
as acknowledged by Montagnier,
and contains numerous errors and
omissions from the figure legends.
Montagnier got the idea of a retrovirus as the cause of AIDS indirectly from Robert Gallo and Myron
Essex. The French group used
protocols for virus isolation and RT
detection that were developed by
Gallo and many of the reagents
they used were also obtained from
Gallo. Robert Gallo was not only
indispensible for the French group
to isolate HIV-1 (HTLV-III), his was
the first group to show convincingly
that this new human retrovirus was
the causative agent of AIDS (13
and see page 10). Gallo’s group
also provided the first blood test
to screen donors for the presence
of HIV infection (14, 15 and see
pages 18 and 4). The rapid implementation of the latter in the United
States and Europe probably saved
hundreds of thousands of lives.
References
1. D. A. Morgan, F. W. Ruscetti, R. Gallo, Science
193, 1007 (Sep. 10, 1976).
2. B. J. Poiesz et al., Proc. Natl. Acad. Sci. U.S.A.
77, 7415 (Dec. 1980).
3. B. J. Poiesz, F. W. Ruscetti, M. S. Reitz, V. S.
Kalyanaraman, R. C. Gallo, Nature 294, 268
(Nov. 19, 1981).
4. Y. Hinuma et al., Proc. Natl. Acad. Sci. U.S.A.
78, 6476 (Oct. 1981).
5. I. Miyoshi et al., Nature 294, 770 (Dec. 24,
1981).
6. M. Robert-Guroff et al., Virology 122, 297 (Oct.
30, 1982).
7. M. Yoshida, I. Miyoshi, Y. Hinuma, Proc. Natl.
Acad. Sci. U.S.A. 79, 2031 (Mar. 1982).
8. M. Popovic et al., Nature 300, 63 (Nov. 4, 1982).
9. V. S. Kalyanaraman et al., Science 218, 571
(Nov. 5, 1982).
10. L. Montagnier, J. C. Chermann, F. Barré-Sinoussi,
S. Chamaret, J. Gruest, M. Nugeyre, F. Rey,
C. Dauguet, C. Axler-Blin, F. Vézinet-Brun, C.
Rouzioux, G. A. Saimot, W. Rozenbaum, J. C.
Gluckman, D. Klatzman, E. Vilmer, C. Griscelli, C.
Foyer-Gazengel, and J. B. Brunet, A new human
T-lymphotropic retrovirus: Characterization and
possible role in lymphadenopathy and acquired
immune deficiency syndromes. R. C. Gallo, M.
Essex, L. Gross, Eds., Human T-Cell Leukemia/
Lymphoma Virus (Cold Spring Harbor Laboratory
New York, 1984), pp. 363-379.
11. E. Vilmer et al., Lancet 1, 753 (Apr. 7, 1984).
12. F. Barré-Sinoussi et al., Science 220, 868 (May
20, 1983).
13. R. C. Gallo et al., Science 224, 500 (May 4,
1984).
14. M. G. Sarngadharan, M. Popovic, L. Bruch, J.
Schüpbach, R. C. Gallo, Science 224, 506 (May
4, 1984).
15. M. Popovic, M. G. Sarngadharan, E. Read, R.
C. Gallo, Science 224, 497 (May 4, 1984).
31
An HIV Vaccine—When and How?
José Esparza,* Francine E. McCutchan,* George K. Lewis,**
and Robert C. Gallo**
*Bill & Melinda Gates Foundation, Seattle, WA 98102 , U.S.A.
**Institute of Human Virology, University of Maryland School of Medicine,
Baltimore, MD 21201, U.S.A.
S
oon after HIV was defined as
the etiological agent of AIDS
in 1983-1984 (1-6 and see
pages 4, 10, 14, and 18) there were
expectations that an effective HIV
vaccine would soon be developed.
However, scientific complexities
including the apparent inability
of natural immune responses to
control HIV replication, permanent
HIV proviral DNA integration, HIV
genome variation with nine genetic
subtypes plus many recombinants,
and rapid immune impairment
make it evident that developing
an HIV vaccine is one of the most
difficult challenges confronting the
biomedical community (7, 8).
Most successful viral vaccines are
based on live-attenuated or wholeinactivated viruses, options that
are not available for HIV because
of obvious safety concerns and/or
lack of impressive experimental
results. Developing HIV vaccines
requires more challenging
strategies like the use of subunits
as proteins or genes, to induce
humoral and/or cell-mediated
immune responses against
protective virus epitopes.
Clearly needed is a vaccine that
prevents infection but, so far, none
does. Some may prevent disease,
at least for a certain period of time,
but even this type of vaccine is
32
challenging because HIV quickly
causes immune system damage
and subverts the vaccine response.
In the first weeks of HIV infection,
there is near-complete destruction
of gut-associated lymphoid tissue
which harbors a majority of the
body’s immune cells. Moreover,
CD4+ T cells, which coordinate and
sustain the immune response to
pathogens, are the very cells that
HIV prefers to infect, especially
those that are activated and in the
process of combating infections.
A current strategy for HIV vaccine
development is to identify key correlates of protection from natural
infection studies and apply these
as criteria for the performance of
candidate vaccines in animal models and clinical trials. Several studies already pointed to human immune responses that are correlated
with protection from HIV infection.
High spontaneous ß-chemokine
production among HIV-negative
participants in the MACS study
(9) or hemophiliacs that remained
uninfected despite exposure to HIV
(10) established the production of
ß-chemokines as a correlate of HIV
protection. Neutralizing antibodies
(11, 12) were associated with longterm nonprogression among HIV+
individuals and effective CTL responses were associated with elite
controller status (13). Until we know
how to sustain high level production of ß-chemokines or neutralizing
antibodies, the way forward will be
empirical evaluation of candidate
vaccines in animal models in which
a relevant challenge can be employed with protection from infection or blockade of disease as the
outcome. The SIV and SHIV infection of rhesus macaques are the
only current models, but the limited
range of virus isolates that can be
used for challenge, differences in
the MHC Class I alleles that present antigens to T cells, and the very
few vaccine candidates that elicit
antibodies which neutralize challenge strains are symptomatic of
the model’s limitations.
Three overlapping “waves” of vaccines have been studied (14, 15).
The first was based on a concept
that neutralizing antibodies would
confer protection against HIV infection or development of AIDS. This
concept received early support
from chimpanzee protection experiments and from recent passive
transfer studies in macaques that
were protected from challenge.
These vaccines were based on the
envelope glycoproteins of HIV, especially gp120, and they induced
neutral-izing antibodies directed
against laboratory strains of HIV
with weak or no neutralizing activity
against clinically relevant primary
HIV isolates. Efficacy trials in people showed conclusively that these
candidate vaccines failed to protect against HIV infection and had
no impact on viral load in subjects
who became infected with HIV after
vaccination. It is evident that far
better envelope vaccines must
be developed.
The second wave started in the
mid 1990s with recognition of
the importance for CD8+ T-cell
responses in the control of HIV
infection. This led to DNA vaccines
and live recombinant virus
vectors, especially poxvirus and
adenovirus, capable of delivering
HIV-1 antigens that are presented
by the MHC Class I pathway.
These candidate vaccines
elicit CTL in some proportion of
vaccinees and induce crossreactive responses against multiple
HIV subtypes in some volunteers,
but even in these cases the key
question is whether the responses
will be durable in the face of HIV’s
ability to escape the CTL response.
There are obvious reasons to
suspect that the control of HIV
variants will not be permanent. The
first efficacy trial of a second-wave
candidate vaccine was initiated in
2003, priming with a canarypoxHIV vector combined with a gp120
boost, and results are not expected
until late 2009. Encouraging results
from preclinical and Phase 1
trials of adenovirus type 5 (Ad5)
vectored vaccines, that were
developed by Merck & Co. and
supported by NIH, led to a 2005
“proof-of-concept” trial with Ad5
containing HIV-1 gag/pol/nef
genes that was conducted in the
United States, Latin America, and
the Caribbean (the STEP trial).
A parallel trial was conducted
in South Africa (the Phambili
trial). Both were halted in 2007
when a startling fact emerged: a
subset of vaccine recipients with
higher levels of preexisting Ad5
antibodies had increased HIV
acquisition (16). The failure of the
STEP trial caused a turning point
33
in the field, because that vaccine
was considered by many as the
most promising candidate. Some
experts objected to this kind of
trial because it was based solely
on the induction of cell-mediated
immunity (17).
The third wave of HIV vaccines
is starting now and involves
optimizing immune responses by
existing or soon-to-be-developed
candidate vaccines. The goals are
to develop candidate vaccines
that induce antibodies capable of
neutralizing clinical strains from
a wide variety of HIV-1 subtypes
and/or rapid antibody-dependent
cell cytotoxicity (ADCC), joined
with vaccines inducing high levels
of long-lasting, cross-reactive CTL
against different structural and
regulatory proteins of HIV.
Understanding these developments
helps explain why the conduct of
HIV vaccine trials is controversial.
Candidate vaccines are based on
the science available at the time,
but the pace of discovery in the
field of HIV biology is rapid and
soon the rationale for a candidate
vaccine is superseded by new
knowledge. This situation may lead
to calls for stopping the conduct of
trials with “old” candidate vaccines,
but the field needs the discipline
to ensure that appropriate vaccine
concepts are tested to completion,
incorporating new knowledge into
the analysis as trials proceed.
However, much more attention
must be given to the selection of
rational candidates.
Since the ultimate goal is to
prevent infection, the development
34
of envelope-based candidate
vaccines capable of inducing the
appropriate type of neutralizing
antibodies and/or ADCC is a
high priority. Though a major
challenge, it is one now being
approached by sophisticated
interactions of structural biology,
peptide chemistry, immunology,
and genetic engineering. Multiple
approaches are being explored
to direct or redirect the immune
response to neutralization-relevant
epitopes of gp120 or gp41, some
of which are normally hidden in
the envelope glycoprotein or only
transiently expressed during virus
binding to CD4 (18, 19).
In conclusion, vaccines are part of
the long-term strategy to prevent
expansion of the HIV epidemic.
In all probability, we will not
have a safe, effective preventive
HIV vaccine for at least another
decade. Moreover, developing a
vaccine that can halt the epidemic
will likely happen in stages, with
the first generation of vaccines
protecting some and improving
over time to protect everyone.
Because an estimated 95 percent
of all new HIV infections occur in
developing countries, we would
hope to see vaccines that are long
lasting, require one administration,
do not need refrigeration, and are
easily administered.
For the medium term, we
understand the importance of
developing and combining other
tools to expand options for slowing
the spread of HIV. For example,
broadening anti-HIV therapy to
reduce virus levels in infected
persons may in turn help prevent
transmission. However, it is clear
that the most powerful ultimate
prevention will be by an effective
vaccine, and that we have no
choice but to persevere until
success is finally achieved.
References
1. F. Barré-Sinoussi et al., Science 220, 868
(May 20, 1983).
2. R. C. Gallo et al., Science 224, 500 (May 4,
1984).
3. M. Popovic, M. G. Sarngadharan, E. Read, R.
C. Gallo, Science 224, 497 (May 4, 1984).
4. B. Safai et al., Lancet 1, 1438 (Jun 30, 1984).
5. M. G. Sarngadharan, M. Popovic, L. Bruch,
J. Schupbach, R. C. Gallo, Science 224, 506
(May 4, 1984).
6. J. Schüpbach, M. G. Sarngadharan, R. C.
Gallo, Science 224, 607 (May 11, 1984).
7. D. H. Barouch, Nature 455, 613 (Oct. 2,
2008).
8. F. E. McCutchan, J. Med. Virol. 78 Suppl 1, S7
(2006).
9. A. Garzino-Demo et al., Proc. Natl. Acad. Sci.
U.S.A. 96, 11986 (Oct. 12, 1999).
10. D. Zagury et al., Proc. Natl. Acad. Sci. U.S.A.
95, 3857 (Mar. 31, 1998).
11. P. Carotenuto, D. Looij, L. Keldermans, F. de
Wolf, J. Goudsmit, AIDS 12, 1591 (Sep. 10,
1998).
12. A. K. Pilgrim et al., J. Infect. Dis. 176, 924
(Oct. 1997).
13. F. Pereyra et al., J. Infect. Dis. 197, 563 (Feb.
15, 2008).
14. J. Esparza, S. Osmanov, Curr. Mol. Med. 3,
183 (May, 2003).
15. J. Esparza, N. D. Russell, in AIDS Vaccine
Development: Challenges and Opportunities
W. C. Koff, P. Kahn, I. D. Gust, Eds.
(Academic Press, Norfolk, 2007) pp. 77-86.
16. J. Cohen, Science 318, 1048 (Nov. 16, 2007).
17. R. C. Gallo, Lancet 366, 1894 (Nov. 26, 2005).
18. J. M. Gershoni, G. Denisova, D. Raviv, N. I.
Smorodinsky, D. Buyaner, FASEB J. 7, 1185
(Sep. 1993).
19. Q. J. Sattentau, J. P. Moore, J. Exp. Med. 174,
407 (Aug. 1, 1991).
New Directions in HIV Therapy and
Prevention—The Next 25 Years
Robert R. Redfield, M.D., and William A. Blattner, M.D.
Institute of Human Virology, University of Maryland School of Medicine
T
he first 25 years of discovery
in the field of HIV/AIDS
have been unprecedented.
The systematic application of
new knowledge gained from
basic science has revolutionized
HIV medicine. What was almost
universally fatal in the 1980s has
now become a treatable and
commonly survivable condition
when appropriate care is
available. Unfortunately, flawed
public policies, failed investment,
ignorance, stigma, and denial
allowed HIV to become pandemic,
with over 33 million persons
worldwide estimated to be living
with the virus. The vast majority
of infected individuals reside
in countries least equipped to
manage the destabilizing impact
of a still fatal condition that
exacerbates other communicable
diseases such as tuberculosis (1).
Goals for therapy were limited
initially to prevent opportunistic
infections. With the addition of first
generation antiretroviral agents (S.
Broder, page 27) medication was
used to delay the rate of HIV-induced immune damage by controlling, but rarely suppressing, viral
replication. Building on the early
work of Broder and others at NCI
(2, 3), the biomedical community
exploited key knowledge of the HIV
life cycle and expanded drug de35
velopment efforts targeting key HIV
viral targets (HIV reverse transcriptase, intregrase, protease, and fusion proteins of the virus). Twentyfive years after the historical publications in Science by Gallo and
his colleagues (4-7 and see pages
4, 10, 14, and 18) more than 20
unique chemical compositions in
routine clinical practice are providing durable therapeutic outcomes
of sustained viral suppression, CD4
immune reconstitution, and new
expectations of a natural life span.
Therapy during the peripartum period has virtually eliminated motherto-child transmission in resource
rich countries and is impacting
transmission in resource limited
environments when the intervention
is available, showing that treatment
also benefits prevention (8).
As the field of HIV treatment enters
the next 25 years, basic science
holds more promise for therapeutic
advances. As obligatory parasites,
human retroviruses exploit key host
pathways for successful viral replication. The recent application of
siRNA techniques identified more
than 250 potential host genes that
impact HIV replication, far greater
than the number defined previously (9). CCR5 is required for cell
attachment of most viral strains;
receptor antagonists are the only
agents directed at host targets that
have been successfully introduced
as a primary HIV therapeutic in
clinical practice. Drugs targeting
host pathways critical to HIV replication will eventually become the
back-bone of therapy and a paradigm for treating chronic human
viral pathogens.
36
Treatment approaches may also
target the immune system itself
since HIV infection causes progressive destruction of CD4+ cells with
significant immune dysregulation,
and immune activation associated
with disease progression (10).
Thus, two primary therapeutic targets are emerging: HIV itself and
immune activation. HIV exploits immune activation on multiple fronts.
Cellular activation increases the
efficiency of viral reverse transcription, provides opportunity for viral
host chromosomal integration,
increases target cell susceptibility
for HIV entry by increasing receptor and coreceptor expression, and
modifies optimal response of host
immunity to induce novel immune
responses. There is increasing
awareness of the potential for the
host to naturally control and suppress HIV as seen in the investigation of natural viral suppressors
(NVS)/elite controllers, where host
specific factors including immune
effectors are responsible for and/or
capable of controlling and even
potentially clearing HIV replication
and infection (11, 12). This is a key
area for research and offers the
potential of elucidating additional
therapy targets.
Among the greatest public health
initiatives in world history, the multilateral Global Fund to Fight AIDS,
Tuberculosis, and Malaria, and the
US-funded President’s Emergency
Plan for AIDS Relief (PEPFAR)
promote global HIV therapy and
redress previous public policy failures. Prior to 2003, few individuals
benefited from antiretroviral therapy
outside of the United States, Europe, Australia, and Canada, but
with global scale-up more than 2
million people living with HIV/AIDS
in sub-Saharan Africa are now
receiving antiretroviral therapy.
Reauthorization of PEPFAR through
the Tom Lantos and Henry J. Hyde
United States Global Leadership
Against HIV/AIDS, Tuberculosis,
and Malaria Reauthorization Act of
2008 was vital to sustaining progress in global health equity, and
even in the troubled world economy
of today, the health and economic
impacts will extend far beyond the
program’s 25-year horizon.
Antiretroviral treatment to prevent
mother-to-child transmission
demonstrated the efficacy of
preventative regimens. Now,
the role for HIV treatment as a
prevention approach has been
given added urgency in the face
of recent failed trials for HIV
vaccines and microbicides. Given
unequivocal evidence for the
impact of treatment on survival,
initiation of treatment at the earliest
phases of infection is emerging as
standard practice with the added
benefit that treatment-induced viral
load reduction lowers the likelihood
of HIV transmission. Recent public
health policy recommendations
are designed to optimize early
diagnosis of HIV infection through
routine opt-out testing. We now
have an opportunity to determine
if early diagnosis combined with
early effective therapy in broad
populations of HIV infected
individuals will prove an effective
strategy for preventing HIV
spread (13).
Since the proof that HIV infection
causes AIDS in seminal papers
from Gallo’s group published in
May 1984, basic science has
translated into public health impact
by making HIV a chronic, treatable disease. The failure of public
health policy to contain the epidemic in its earliest phase created
a public health disaster that now
demands further advances. Even
if an effective vaccine were to be
developed against HIV, the tens of
millions already infected will destabilize health care systems and
erode control of other public health
threats such as tuberculosis. Innovative therapies that are suitable
for public health scale-up, require
infrequent dosing, avoid viral drug
resistance, and do not require sophisticated medical infrastructures
will come from fundamental discovery of the pathogenic lynchpin at
the virus-host interface.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
References
Report on the global AIDS epidemic Executive summary. http://data.unaids.
org/pub/GlobalReport/2008/JC1511_
GR08_ExecutiveSummary_en.pdf
H. Mitsuya et al., Proc. Natl. Acad. Sci. U.S.A. 82, 7096 (Oct. 1985).
R. Yarchoan, H. Mitsuya, C. E. Myers,
S. Broder, New Engl. J. Med. 321, 726 (Sep. 14, 1989).
R. C. Gallo et al., Science 224, 500 (May 4, 1984).
M. Popovic, M. G. Sarngadharan, E. Read, R. C. Gallo, Science 224, 497 (May 4, 1984).
M. G. Sarngadharan, M. Popovic, L. Bruch, J. Schüpbach, R. C. Gallo, Science 224, 506 (May 4, 1984).
J. Schüpbach, M. G. Sarngadharan, R. C. Gallo, Science 224, 607 (May 11, 1984).
E. R. Cooper et al., J. Acquir. Immune Defic. Syndr. 29, 484 (Apr. 15, 2002).
A. L. Brass et al., Science 319, 921 (Feb. 15, 2008).
W. A. Blattner et al., J. Infect. Dis. 189, 1793 (May 15, 2004).
F. Pereyra et al., J. Infect. Dis. 197, 563
(Feb. 15, 2008).
M. M. Sajadi et al., J. Acquir. Immune Defic. Syndr. (Feb. 12, 2009).
C. F. Gilks et al., Lancet 368, 505 (Aug. 5,
2006).
37
Cancers and HIV Infection:
An Evolving Story
Robert Yarchoan and C. David Pauza*
HIV and AIDS Malignancy Branch, Center for Cancer Research,
National Cancer Institute, NIH and the *Institute of Human Virology,
University of Maryland School of Medicine
T
he appearance of Kaposi’s
sarcoma (KS), a hitherto rare
tumor in the United States,
in young, never-married men in
New York and San Francisco in
1981 was an early indication of
the global AIDS pandemic. It soon
became apparent that AIDS was
associated with a pronounced
increase in tumors, including KS
and aggressive non-Hodgkin
lymphoma (NHL). The discovery of
HIV and demonstration that it was
the cause of AIDS (1-3 and pages
10 and 4) laid the foundation for
understanding this association.
HIV is not a direct agent for AIDSrelated tumors, but rather acts as a
potent cofactor, largely by causing
profound T-cell immunodeficiency.
Curiously, this effect is directed
at a relatively small number of
tumors. For example, untreated
patients with HIV infection before
1989 had a greater than 50,000fold enhanced risk of KS (4, 5)
but no increased risk of breast or
colon cancer. With the discovery
of Kaposi’s sarcoma–associated
herpesvirus (KSHV or HHV-8),
it became apparent that most
AIDS-associated tumors are
linked to oncogenic DNA viruses
(6, 7). Immunologic control of
these viruses or their products
is diminished in AIDS patients,
allowing tumorigenesis to
38
occur. By contrast, otherwise
common cancers such as breast
or colon express relatively few
neoantigens, and the impact of
declining immune capacity was
correspondingly less dramatic.
Some cases of AIDS-NHL are not
associated with known oncogenic
viruses. HIV-induced B-cell
dysregulation is believed to play
a major role in their pathogenesis,
although new oncogenic viruses
may still be identified. Another
mechanism by which HIV may
contribute to oncogenesis is
through the HIV Tat protein, which
can stimulate tumor cell growth and
enhance entry of oncogenic viruses
into cells (8, 9). Studies of tumor
incidence among patients who
have natural control of HIV (“elite
or natural controllers”) may provide
important clues into mechanisms
for tumor susceptibility.
The identification of HIV as
the cause of AIDS enabled
rapid development of the first
antiretroviral drugs (10, 11
and reviewed by Broder on
page 27). Clinical testing soon
showed that these drugs could
induce immunologic and clinical
improvement, and prolong
the life of AIDS patients. The
subsequent development of HIV
protease inhibitors enabled the
consideration of highly active
combination antiretroviral therapy
(cART, also called HAART) that
almost completely blocked HIV
replication. Use of cART led to
a substantial decrease in AIDSrelated malignancies, especially
those associated with very
low CD4 counts, and helped
improve survival of patients with
malignancies (4). These advances
were made possible by long-term
commitments of the NIH, and the
NCI in particular, to research on the
interrelationship between viruses,
immunodeficiency, and cancer.
For a short period in the mid 1990s
there was optimism that cART
would be curative and a perception
that AIDS was disappearing as a
problem. We now know this is not
the case—development of cART
was more like the development
of insulin for Type 1 diabetes, in
that a rapidly fatal disease was
transformed into a chronic illness
with serious late complications and
mortality. The yearly number of new
adult HIV infections in the United
States has been relatively constant
since the introduction of cART
and with patients living longer, the
number of patients with AIDS has
more than doubled since 1996 (9).
As patients live longer, we are also
seeing a shift toward non-AIDS–
defining tumors. Epidemiologic
studies show that cancer is now the
most common cause of mortality in
HIV-infected patients, accounting
for about a third of deaths (4, 12).
More than half of cancer deaths
are from non-AIDS–defining
malignancies.
These changes are associated with
a shift of HIV infection to a longterm illness with subtle immune
defects. However, factors affecting
trends for individual tumors
are incompletely understood.
For example, the incidence of
Hodgkin lymphoma in HIV-infected
patients is increasing (4). Lung
cancer has become the most
common non-AIDS–defining fatal
malignancy, partly because people
at risk for HIV have high rates
of cigarette smoking, although
immunodeficiency may also play
a role. There is growing evidence
that some cases of head and neck
cancer arising in HIV-infected
patients are caused by human
papillomavirus (HPV) infection and
that these cancers comprise a
distinct disease subtype (13).
It is unclear how these trends will
play out. We now appreciate that
some patients on cART develop
KS and other AIDS-related tumors,
even in the face of good viral
control and apparent immune
reconstitution. As patients live
longer, their cumulative risk of
developing tumors will almost
certainly increase. KS and other
AIDS-related malignancies
continue to be major causes of
morbidity and mortality in Africa
and other developing regions. Thus
it is important that we consider
how best to prevent and treat
these tumors.
The recent development of
vaccines against HPV have
demonstrated that tumors can be
prevented by effective vaccination
against the causative viruses (14).
Vaccines against other key viruses
39
such as KSHV and EBV would
hold promise for preventing a large
fraction of AIDS-related tumors.
Viruses may also provide unique
targets by which to attack viralinduced tumors; understanding
viral biology and tumorigenesis
may yield novel and effective
therapies. It will be important to
study if and how the treatment of
various cancers should be modified
in the setting of HIV infection. To
facilitate this effort, the NCI has
recently undertaken an initiative
to reduce barriers to the entry of
HIV-infected patients with common
cancers into clinical trials. Finally,
there is a need to clarify how HIV
affects natural tumor surveillance
and consider approaches that
correct these defects.
Thus, while cancers are emerging
as an increasingly common
cause of death and morbidity in
HIV-infected patients, basic and
translational research holds the
potential to reduce this burden in
patients with HIV disease.
The Interface of Cancer and HIV/AIDS
Vaccines: Both Preventive and Therapeutic
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
Jeffrey Schlom, Douglas Lowy, and John Schiller
Center for Cancer Research, National Cancer Institute,
National Institutes of Health, Bethesda, Maryland
References
F. Barré-Sinoussi et al., Science 220, 868 (May 20, 1983).
R. C. Gallo et al., Science 224, 500 (May 4,
1984).
M. Popovic, M. G. Sarngadharan, E. Read, R. C. Gallo, Science 224, 497 (May 4, 1984).
E. A. Engels et al., AIDS 20, 1645 (Aug. 1, 2006).
J. J. Goedert et al., Lancet 351, 1833 (Jun. 20, 1998).
Y. Chang et al., Science 266, 1865 (Dec. 16, 1994).
R. Yarchoan, G. Tosato, R. F. Little, Nat. Clin. Pract. Oncol. 2, 406 (Aug. 2005).
B. Ensoli et al., Nature 345, 84 (1990).
Y. Aoki et al. Blood 104, 810 (2004).
H. Mitsuya et al., Proc. Natl. Acad. Sci. U.S.A. 82, 7096 (1985).
R. Yarchoan et al., Lancet 1, 575 (1986).
F. Bonnet et al., Clin. Infect. Dis. 48, 633 (Mar. 1, 2009).
R. Zelkowitz, Science 323, 580 (Jan. 30, 2009).
D. R. Lowy, J. T. Schiller, J. Clin. Invest. 116, 1167 (May 2006).
This work was supported in part by the
Intramural Research Program of the NIH, NCI
(R.Y.) and PHS grant CA113261 (C.D.P.).
T
o date, the most successful
vaccines targeting cancer
have been those that
prevent disease induced by
hepatitis B virus (HBV) and human
papillomavirus (HPV). The induction
of neutralizing antibodies appears
to be the key protective activity for
both vaccines. Liver disease from
HBV involves successive rounds
of viral replication, offering multiple
opportunities for the vaccineinduced systemic neutralizing
antibodies to act. By contrast,
genital HPV causes a local
infection whose individual lesions,
once established, do not appear
to be altered by neutralizing
antibodies (1).
Like HBV, HIV induces chronic
systemic infection, and most
efforts to develop preventive
HIV vaccines have focused on
reducing systemic infection of T
cells (2). The success of the HPV
vaccine suggests that preventing
initial anogenital mucosal infection
may be an attractive alternative
vaccination strategy. The HPV
virus-like particle vaccine trials
demonstrated potent sterilizing
immunity in the genital tract, thus
overcoming justified skepticism
concerning the prospects for a
successful prophylactic vaccine
against a local genital mucosal
infection (3).
40
Sexual acquisition of HIV usually
results from a single infectious
event, and HIV has low rates
of heterosexual transmission,
estimated to be 1 per 200-2000
exposures (4, 5). Inefficient
trafficking to its initial target cell,
due to slow rates of diffusion
through mucus, might be a
major contributing factor to low
transmission rates (6). Further
decreasing virion diffusion rate, via
vaccine-induced antibody/virion
complexes, might further reduce
the likelihood of transmission,
especially as HIV is a rapidly
inactivated enveloped virus. The
antibodies would need to have
reasonable affinity, but need not
prevent virus infection of cultured
cells. It should be considerably
easier to design immunogens that
generate this less restricted class
of virus-binding antibodies. Also,
virus escape through mutation
would be difficult if, as expected,
a broad spectrum of virion binding
antibodies were able to retard
diffusion. Vaccines that could
induce secretory IgA (sIgA) may
be more potent, since sIgA, but not
IgG, appears to specifically bind
mucus (7). Incremental challenge
dose-escalation trials in monkeys
may be required to fully reveal
the activity of this type of vaccine.
They might appear ineffective using
the high challenge doses required
41
to ensure infection of all control
animals after a single exposure.
Most recent attempts to develop
HIV vaccines have focused on
preventing what the virus does
best, destroy T lymphocytes.
Perhaps more attention should
be focused on what it does most
inefficiently, initiate infection. It
is encouraging that an antibodymediated vaccine can be so
effective against HPV, a virus that
appears to be more efficient at
sexual transmission than HIV.
As yet, no therapeutic vaccines
for cancer or HIV/AIDS have been
approved by the FDA. There
have been many more cancer
vaccine clinical trials thus far
carried out and consequently a
larger accumulation of knowledge
concerning therapeutic vaccines.
The differences in these two
diseases are obvious and
numerous, but there are some
lessons learned in the field of
cancer therapeutic vaccine
development that may have
implications in the development
of an AIDS vaccine. Evidence of
patient benefit in the use of cancer
vaccine trials has not yet been
translated into FDA approval; the
reasons include: (a) several Phase
3 trials were conducted with earlier
generation, less potent vaccines;
(b) they employed patient
populations with advanced disease
and often prior therapies that
damaged their immune system;
(c) endpoints were shrinkage
of visible tumor lesions. There
is now evidence that vaccines
may readily slow progression of
indolent disease or low burden
disease, leading to increased
42
survival. Recent studies show that
continuous boosting is possible
and appears to be beneficial to
keep the immune responses from
succumbing to down regulation.
Thus a paradigm shift in cancer
vaccine development has occurred
(8), particularly regarding patient
populations and clinical endpoints.
Since most tumor-associated
antigens (TAAs) are self-antigens,
which are non- or weakly
immunogenic, the major thrust of
cancer vaccine development has
been to improve the potency of
the immune response to the TAA.
Strategies employed to this end
have been: (a) the use of dendritic
cell vaccines, vector-based
vaccines containing multiple T-cell
costimulatory molecules, and the
addition of cytokines to the vaccine
regimen, (b) altering specific
amino acids of the TAA to produce
enhancer agonist epitopes, and
(c) diversified prime and boost
regimens. These more potent
vaccines not only induce higher
levels of T-cell responses, but also
greater T-cell avidity.
Cancer is a spectrum of diversified
cell types and within each cancer
type multiple TAAs have been
identified. Perhaps HIV/AIDS
vaccines should not focus entirely
on viral antigens, but evaluate
the HIV-infected cell as the
“target.” Recent advances in
immunotherapy of cancer suggest
that virally induced self-antigens
may also serve as targets for T-cell
mediated attack.
References
1.
2.
3.
4.
5.
6.
7.
8.
I. H. Frazer, D. R. Lowy, J. T. Schiller, Eur. J. Immunol. 37 Suppl 1, S148 (Nov. 2007).
B. D. Walker, D. R. Burton, Science 320, 760 (May 9, 2008).
J. T. Schiller, X. Castellsague, L. L. Villa, A. Hildesheim, Vaccine 26 Suppl 10, K53 (Aug. 19, 2008).
F. Hladik, T. J. Hope, Curr. HIV/AIDS Rep. 6, 20 (Feb. 2009).
R. E. Haaland et al., PLoS Pathog. 5, e1000274 (Jan. 2009).
A. Phalipon et al., Immunity 17, 107 (Jul. 2002).
J. Schlom, P. M. Arlen, J. L. Gulley, Clin. Cancer Res. 13, 3776 (Jul. 1, 2007).
A. Hoos et al., J. Immunother. 30, 1 (Jan.2007).
Identifying of patient populations
most likely to respond effectively
to vaccine therapy is critical. Prior
to vaccination, the number of
effector cell populations should be
analyzed, along with their ability to
respond to relevant antigens; the
number and function of regulatory
T-cell populations should also be
evaluated. Similarly, analysis of
serum cytokines and chemokines
has shown to be of some predictive
value in defining appropriate target
patient populations.
43
May 9-11, 2009
University of Maryland
School of Medicine and the
National Cancer Institute
PRESENT THE SYMPOSIUM
25 Years After
DISCOVERING HIV AS
THE CAUSE OF AIDS
Baltimore Marriott
Inner Harbor at Camden Yards
Baltimore, Maryland
May 9, 2009
AN EVENING HONORING
Robert C. Gallo, MD
Hilton Hotel
Baltimore, Maryland
For more information:
Call 410-706-8503, or visit
www.Gallo25.umaryland.edu
Rwanda
A country devastated by genocide and a crippling AIDS epidemic. Together with the Rwandan
Ministry of Education, AAAS is working to ensure that local children gain skills in science,
technology, math, and engineering. And this is just one of the ways that AAAS is committed to
advancing science to support a healthy and prosperous world.
Join us. Together we can make a difference. aaas.org/plusyou
May 9-11, 2009
Honoring the Accomplishments of Robert C. Gallo at the
National Cancer Institute and the Institute of Human Virology
JOIN THE SYMPOSIUM
25 Years After
Co-Sponsored by the University of Maryland School of Medicine,
E. Albert Reece, MD, PhD, MBA, Dean, and the
National Cancer Institute, John E. Niederhuber, MD, Director
DISCOVERING HIV AS
THE CAUSE OF AIDS
Organizing Committee Honorary Chairs:
Myron Essex, DVM, PhD, and William Haseltine, PhD
Saturday, May 9, 2009
Welcome
E. Albert Reece, MD, PhD, MBA, Dean,
University of Maryland School of Medicine
Introductory Remarks
John Niederhuber, MD, Director,
National Cancer Institute
Opening Remarks
Bernadine Healy, U.S. News and World Report,
Former Director, National Institutes of Health
Scientific Session
Introduction to the 25th Anniversary of
Discovering HIV as the Cause of AIDS
Max Essex, DVM, PhD
Tools that laid the foundation for
discovering human retroviruses
Chairs: Arsene Burny and Yiming Shao, PhD
Tom Waldmann, MD
The Common Gamma Chain Cytokines
Induced by HTLV-I Tax are of value
in the treatment of Cancer and in
Molecular Vaccines for Cancer and AIDS
Jean Claude Chermann, PhD
Origins of Human Retrovirology
The first human retroviruses and
cancers they cause — HTLV1 and HTLV2
Chairs: William Blattner, MD, and
Mitsuyaki Yoshida, PhD
Lee Ratner, PhD
HTLV Tax, Transactivation, T-Cell Activation,
and Tumorigenesis
Mitsuyaki Yoshida, PhD
Kuan-Teh Jeang, MD, PhD
Three decades of HTLV-1 research:
Insights into Adult T cell leukemia and
cellular transformation mechanisms
Lunch Break
Retrovirus research enabling the
identification of human oncogenes
Chairs: Luigi Chieco-Bianchi, MD, and
Isaac Witz, PhD
Carlo Croce, MD
Oncogenes, Retroviruses and Human Cancer
Stuart Aaronson, MD
Translating Gene Discovery to the Clinic
Discovering HIV and Proving the
Association with AIDS
Chairs: John Niederhuber, MD, and
Bernadine Healy, MD
Harold Jaffe, MD
The Early HIV / AIDS Epidemic
in the United States:
Views from Hollywood and Atlanta
Luc Montagnier, MD
New Prospects for Treatment and
Prevention of HIV Infection
Anders Vahlne, MD, PhD
On The Discovery of Human Retroviruses
Robert C. Gallo, MD
Lessons of the past and
applications for the future
Clinical science, HIV pathogenesis and
the emerging retroviral syndrome
Chairs: Robert R. Redfield, MD, and
John Bartlett, MD
Sam Broder, MD
Therapy of Pathogenic Retroviruses:
Past, Present, Future
Anthony Fauci, MD
HIV / AIDS — 1985 thru 2009:
The Evolution of the Science
Reflections on 25 Years of AIDS Advocacy —
A Tribute to Martin Delaney and the
Work He Inspired
Chairs: Robert C. Gallo, MD, and
John Moore, PhD
Brenda Lein, Director, Project Inform
Martin Delaney and AIDS Advocacy
Mark Harrington, Executive Director,
Treatment Action Group
Reflections on 25 Years of AIDS Advocacy —
A Tribute to Martin Delaney and the
Work He Inspired
Closing Remarks
William Haseltine, PhD,
William Haseltine Foundation
For the full Symposium schedule, please visit our website at:
http://www.gallo25.umaryland.edu/program.doc