Can HIV be eradicated from a human host? Can currently available
combination therapy approaches eliminate HIV from every last cell within a
reasonable amount of time (short of the lifetime of the host)? This
provocative question was first raised before a global audience by David Ho
at Vancouver in July 1996. After months of rumors and an accumulation of
presentations at St. Petersburg in June, at Istanbul and Toronto in
September, and at Hamburg in October, three papers published in Science
and the Proceedings of the National Academy of Sciences (U.S.A.) in
November 1997 definitively laid to rest the idea that HIV could be
eradicated within three years using currently available triple combination
antiretroviral therapy. Mark Harrington helps us make sense of it all with
this sobering and long feared year-end report.
The problem is that current combination therapies, no matter how potent,
only act against the virus when it is replicating. If there is a
significant number of cells in which the virus is resting dormantly --
integrated into the nucleus -- the drugs will be unable to target these
infected cells which, when activated, will be able to reignite the
infection -- no matter how long someone has suppressed new virus production
with combination therapy. Thus, the last sixteen months have seen an
intense search for a cell population -- the so-called "third compartment" --
in which HIV might persist in dormancy, unresponsive to combination therapy.
These experiments built on work first shown by David Ho at Vancouver in July
1996, and followed up by Ho and Los Alamos Alan Perelson at the Fourth
Retrovirus Conference in January 1997, which estimated the time it might
take to eradicate HIV from an infected human host. These were the first
rational, quantitative estimates based on in vivo data on viral and host
cell kinetics, and they assumed that there were just two cellular
compartments in which HIV dwelled:
- A primary compartment consisting of activated, HIV-infected CD4
T cells actively producing virus and rapidly being killed either by
HIV or by the immune response, which turn over rapidly in the body
with a half-life of a 1.4 days, and produce over 90% of the new virus
- A second compartment -- possibly consisting of latently infected CD4 T
lymphocytes or macrophages -- which, when activated, produce virus, turning
over with a slower half-life of one to four weeks, and producing less than
10% of the new virus population.
Regardless of the number of HIV-infected cells in the body, if these were
the only two compartments in which HIV dwelt, the time-to-eradication could
be estimated. If the half-life of the second compartment was four weeks,
the time-to-eradication would range from two to three years, depending on
the number of HIV-infected cells. The entire model, however, would break
down if there proved to be a third compartment. If one existed, it would
be important to measure whether it, like the first two compartments, was
reduced in size by triple combination antiretroviral therapy and, if so,
by how much, and how fast. Only then could one reliably estimate the
time-to-eradication using current therapies. Now, it turns out that there
is a third compartment. It consists of latently infected, resting,
previously activated memory CD4 T lymphocytes with integrated proviral HIV
DNA in their nucleus. These cells are a small but crucial reservoir for HIV
and, at least after two years of triple therapy, do not appear to diminish
In other words, the third compartment does not have a half-life of decay,
because it does not appear to decay at all. If this holds true, then we
can no longer expect the current therapeutic approaches by themselves to
lead to HIVs eradication -- which is really just a fancy word for cure.
New approaches, however, could be developed to target this third
compartment, possibly speeding up the turnover of these cells and leading
to their elimination. Such approaches remain speculative, but some are
rapidly moving towards preliminary clinical trials. In the meantime,
however, no one should begin antiretroviral therapy with the belief that
triple (or even quadruple) antiretroviral therapy is likely to eradicate
HIV infection. The best we can hope for, at this time, is chronic
suppression of HIV and thereby preventing disease progression, protecting
the immune system from further damage, and avoiding the development of drug
resistant HIV strains.
HIV Infection, Reverse Transcription, CD4 Cell Activation, DNA Integration
Most HIV in the body is in the form of RNA, either as free virus particles
circulating in the blood, trapped within lymph nodes or inside cells either
just after invading a cell (before it is converted into DNA) or when an
infected cells HIV DNA is making new viruses and new viral RNA. Each
infected cell may produce from 1-200 new virions. The reservoir which
accounts for the persistence of HIV infection is, however, not viral RNA
but proviral DNA which has already been integrated into the cell nucleus.
HIV DNA can be detected either in an unintegrated form within the cell
cytoplasm, or integrated within the cells DNA in the nucleus. Robert
Siliciano, of Johns Hopkins University, and colleagues developed an assay
earlier this year to distinguish the two types of HIV DNA. They found that
if a cell is infected while in a resting state, reverse transcription may
occur, but the new HIV DNA will not be transported into the nucleus for
integration. If the cell is kept in a resting state for six days, cellular
proteins will degrade the HIV DNA and integration will be aborted. Thus,
cellular activation is required for integration and productive infection of a cell.
Most cells which experience activation and integration then undergo mitosis
(cell division), in the process of which HIV copies are made. The cell is
either killed by direct cytolytic effects of the virus or by antiviral
effector mechanisms of the immune system (cytotoxic T lymphocytes ("CTLs"),
antibodies, macrophages). However, a small minority of infected cells may
survive with integrated HIV DNA. Of these, only a small subset are
replication competent. Of one million white blood cells taken from an
infected individual, 1,000-100,000 cells may have detectable HIV DNA
provirus, but only 10-100 of these cells will have integrated provirus
and only 1-10 of these will be replication competent. (Other cells,
however, may remain targets for the immune mechanisms mentioned above
if any viral proteins, even defective ones, are made.)
Discovery of the Third Compartment
Resting memory (CD45RO+) cells are the major reservoir for integrated
HIV-1 provirus in resting CD4 T cells. Three studies (Finzi/Siliciano,
Spina/Richman and Chun/Fauci, 1997) show that there is no apparent decrease
in the number of latently-infected resting CD4 cells over two to 32 months
of potent antiretroviral therapy. Even among individuals with fewer than
200 RNA copies/mL, one to ten infectious units per million cells (IUPM)
can be detected after up to 32 months of treatment. The assay is imprecise
(with up to one log variation), but this suggests that there may be a very
slow half-life for decay of this infected cell population, confirming
estimates made several years ago by Angela McLean of Oxford University.
All three teams took a similar approach in searching for the resting,
latently infected CD4 cells. First they eliminated B cells, macrophages,
natural killer cells and CD8 cells. Activated CD4 cells were eliminated
to make sure the sample cells were truly resting. Then they stimulated
the resting T cells into activation thereby "turning on" any
replication-competent HIV DNA which may reside within this cell
population. They measured HIV within the activated, previously resting
cells in several ways: by looking for proviral DNA in the nucleus, or
unintegrated DNA in the cytoplasm; by amplifying proviral DNA with PCR;
and -- importantly -- by culturing the cells to see if replication-competent
HIV could be grown from the cells. It could.
In the largest study, led by Silicianos team, Finzi and colleagues
examined cells from 22 individuals treated with tripe therapy for up to
30 months. Subjects had to 1) be on highly active antiretroviral therapy
("HAART"); 2) be highly adherent; 3) rapidly have achieved "undetectable"
plasma HIV RNA levels (<200 RNA copies/mL by RT-PCR); and 4) have remained
"undetectable" for the duration of the study. Replicating virus was
detected in all 18 cases when sufficient resting CD4 cells were isolated
(they could not be isolated from four individuals with low CD4 counts).
Very few resting cells had integrated HIV DNA which could be stimulated
into active replication -- just 0.2 to 16.4 infectious units per million
resting cells. But what proved to be the most discouraging finding is that
when, in a cross-sectional analysis, the number of infectious cells in
individual patients were correlated with their time on HAART, there
appeared to be no relationship between time on HAART and number of
infectious cells -- and thus no apparent decay in the third compartment.
Serial samples were taken from only one patient -- whose level of
infectious cells actually rose slightly between months four and eight.
Thus, while the third compartment appears to be small, it also appears
to be very stable.
To see how old the provirus in these resting cells was -- and whether it
had evolved resistance to combination therapy -- the investigators sequenced
the protease genes. The good news is that (in patients with plasma RNA
levels continually suppressed below 200 copies/mL), the proviral HIV did
not display mutations associated with drug resistance. This indicates
that the cells had likely been infected prior to the initiation of triple
therapy, and that viral evolution appeared to have greatly slowed down --
if not stopped altogether -- after the initiation of triple therapy.
(Some individuals had mutations from previous experience with monotherapy.)
"Taken together," the authors explain, "these results
demonstrate that purified resting CD4+ T cell populations from
patients on HAART harbor replication-competent virus that in some cases
is cytopathic." (Given the inherent vagaries of translating results
from in vitro experiments to in vivo conditions, however, the question of
whether these latently infected T cells can be reactivated to produce
virus in treated individuals remains, for now, unanswerable.
GMHCs treatment wonk Dave Gilden, writes in a second exhaustive
analysis (Treatment Issues, November 97) of the 3 eradication papers,
"It is not clear, though, that any particular therapy is needed for
latent cells. It is possible that as the immune system recovers in the
nearly HIV-free environment, anti-HIV cytotoxic lymphocytes will be able to
quickly kill the few cells with active HIV infection and prevent spread of
the virus;" to which Diamonds Ho replies matter-of-factly, "I
wouldnt bet on it.") The Johns Hopkins investigators are
understandably wary to come down on either side of this issue and warn only
that "the existence of a small but relatively stable compartment of
latently infected cells should be considered in deciding whether treatment
should be stopped in patients with no other evidence of residual virus."
In the second paper, Joseph Wong, Doug Richman and colleagues conducted
a similar but smaller study at U.C. San Diego. They isolated resting CD4
T lymphocytes from six individuals who had achieved undetectable
(<50 copies/mL) viral load for up to two years on HAART. As in the
previous study, infrequent cells with integrated proviral HIV DNA
were found, their protease genes sequenced, and no genotypic evidence of
protease resistance identified, suggesting that the third compartment is
stable over up to two years of HAART and that HAART prevents viral
evolution and the development of drug resistance. In summary, there is
evidence for a third phase of viral persistence among latently infected
CD4 cells with integrated provirus. These cells are predominantly CD45RO+,
and their isolation requires CD8 cell depletion and CD4 cell activation.
The half-life of this third compartment is likely to be many months to
years, but the virus isolated from these cells taken from patients with
very low HIV RNA levels (<50/mL) after two years of treatment showed no
evidence of viral evolution or the emergence of drug resistance. Thus,
hopes for short term eradication of HIV from an individual within three
to six years are "clearly unrealistic," according to Richman, unless we
can figure out a way to shorten the third phase, perhaps by stimulating
the latent infection out of the CD4 cells. "Conceivably," he continued,
"one could eradicate in ten years or more." Richman joked that inducing
toxic shock might be one way of shortening the third phase and concluded
that, "It would be wrong to be discouraged about anything but the prospect
for short term eradication; we have achieved suppression of viral
evolution, which bodes well for maintenance."
The third paper, from Faucis lab published by Chun et al. in P.N.A.S.,
also found persistent low level RNA production despite triple therapy.
Eighteen HIV-infected individuals on various antiretroviral regimens had
leukopheresis (blood extraction) at the NIH, and their cells were analyzed
for HIV RNA at baseline and, in thirteen patients, after a median of ten
months of "HAART." They were able to isolate resting memory CD4 cells with
integrated, replication-competent HIV provirus at low frequencies from 11
of the 13 patients on HAART. Some of these viruses (in cell culture) could
induce syncytia, indicating virulent cytopathicity. They also found low
levels of unintegrated DNA in some cells, indicating recent infection and
reverse transcription. The amount of unintegrated DNA was higher in
patients whose viral load was still detectable. In contrast to the
Siliciano and Richman paper, the NIAID study "suggests persistent active
virus replication in vivo," but this might simply reflect the fact that
viral suppression in the NIAID study was less stringent ("undetectable" in
the NIAID study was defined as <500 copies/ml) than in the other two
studies (Siliciano, <200; Richman, <50/mL).
In addition, the NIAID team found that "levels of integrated HIV-1 DNA
within resting CD4+ T cells in HAART naïve patients was not significantly
higher... than in treated patients... [suggesting] that resting CD4+ T
cells with integrated HIV-1 DNA do not decay rapidly in patients receiving
HAART and thus represent a stable reservoir of HIV-1 DNA." In other words,
the third compartment appears to lack a measurable decay curve despite
potent combination antiretroviral therapy. Chun et al. concluded their
paper by noting that "the time required for virus eradication -- if indeed
this is possible -- will be considerably longer than previously predicted,"
and they call for the development of "strategies for eradicating those
minor populations of infected cells that serve as reservoirs of inducible
and replication-competent HIV."
What Can Be Done to Eliminate the Third Compartment?
Several strategies are currently being considered to eliminate the third
compartment. One way might be to activate the latently-infected resting
cells so that potent antiretroviral combination therapy would interrupt
further cycles of virus replication. There are several obvious dangers to
this approach. First, the amount of virus that might be released might be
enough to overcome therapeutic barriers against the development of drug
resistance. Second, the activation of so many resting CD4 cells might
cause excess inflammation, fever or even shock.
ACTG 387 is a protocol in development which aims to determine more exactly
the kinetics of viral decay with the three current classes of
antiretroviral agents (nucleoside and non-nucleoside RTIs and protease
inhibitors) by measuring their pharmacokinetics and viral kinetics in the
first 72 hours of therapy. Participants will then begin four drug therapy
with AZT, 3TC, indinavir and nevirapine. After six days they will be
randomized to receive immune modulation with interleukin-12 (IL-12),
GM-CSF, or nothing. IL-12 is a Th1-type cytokine which induces production
of gamma interferon, and may inhibit CD4 cell apoptosis. Immune modulation
will continue for fourteen weeks and quadruple antiretroviral therapy will
continue for 48 weeks. The hypothesis here is that IL-12 will accelerate
the clearance of HIV from long-lived CD4 cells and that GM-CSF will do the
same for macrophages. This study may help to clarify whether it is
possible to use immune stimulatory cytokines to speed up clearance of the
It seems clear that, with maximal antiretroviral control of active HIV
replication, weve gotten as far as we can with "Its the virus, stupid."
Many of the most pertinent questions now relate to the immune system and
its ability (or inability) to contain HIV infection:
Some of us posed the last question six years ago. We still dont have the
- Can memory cells revert to naïve cells in humans? How frequent is this?
- Can latently infected cells undergo mitosis into infected daughter
cells without turning on viral replication?
- Do memory cells that revert to naïve cells retain their capacity for
further T cell receptor gene reshuffling?
- Is there new thymic emigration in HIV-infected adults?
- Is there extrathymic maturation of new naïve or memory CD4 cells?
- As the likelihood of eradication recedes, does the argument for
treatment of newly infected or asymptomatic individuals weaken?
- What destroys the lymphoid tissue late in disease?