The persistence of (HIV in) memory (T cells)

COMMENT

recently reported that VacA lowers the degradation capacity of late endosomes and lysosomes and, at the same time, partially neutralizes the luminal pH of these compartments3. In addition, VacA inhibits antigen processing by B cells4 and alters the permeability properties of epithelial cell monolayers (E. Papini et al., submitted); cytosolic expression of VacA is expected to help in elucidating the molecular basis of the

multiple cellular effects caused by this unusual bacterial protein toxin. Marina de Bernard Centro CNR Biomembrane, Dipartimento di Scienze Biomediche, Università di Padova, 35121 Padova, Italy Beatrice Aricò Istituto Richerche

Immunobiologiche Siena, Via Fiorentina 1, 53100 Siena, Italy References 1 Montecucco, C. et al. (1994) FEBS Lett. 346, 92–98 2 Telford, J.L. et al. (1997) Curr. Opin. Immunol. 9, 498–503 3 Satin, B. et al. (1997) J. Biol. Chem. 272, 25022–25028 4 Molinari, M. et al. (1998) J. Exp. Med. 187, 135–140

The persistence of (HIV in) memory (T cells) Donald M. Coen ‘Such a long, long time to be gone and a short time to be there’ ‘Box of Rain’ Grateful Dead (words by Robert Hunter)

S

everal of the most important developments in the management and understanding of HIV over the past few years derive from the use of new, potent antiviral drugs. Not only have these drugs prolonged and improved the lives of patients infected with HIV, but studies using these agents have also increased our understanding of HIV biology in vivo enormously. Three years ago, Wei et al.1 and Ho et al.2 published studies in which patients were treated with protease inhibitors. The results showed that HIV and HIV-infected cells are rapidly turned over, remaining in the blood of patients only for a short time. Subsequent findings published last year further demonstrated that treatment with combinations of protease and reverse transcriptase (RT) inhibitors reduces virus to undetectable levels in the blood of some patients and that these reductions can be sustained for more than two years3–5. This led to wellpublicized hopes that continuous therapy with such combinations

for a few years could cure patients of HIV. These hopes have been predicated on a scenario published in May of last year6. In this scenario, it is assumed that there are only two phases of turnover of HIV and HIVinfected cells; the rapid phase first noted following administration of protease inhibitors1,2 (with an estimated tg of ≤6 h and 1.6 d, respectively6), and a slower phase discerned following administration of combination chemotherapy (estimated tg of infected cells of 1– 4 weeks)3,6. However, in a less optimistic scenario6, a third, slower phase of decay, resulting, for example, from a long-lived population of latently infected cells, could not be excluded. In this case, HIV could be undetectable for a long time but could come storming back when antiviral drug therapy was stopped. Indeed, it was shown that the tg of HIV DNA in peripheral blood mononuclear cells

D.M. Coen is in the Dept of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 250 Longwood Avenue, Boston, MA 02115, USA. tel: +1 617 432 1691, fax: +1 617 432 3833, e-mail: [email protected]

Copyright © 1998 Elsevier Science Ltd. All rights reserved. 0966 842X/98/$19.00 TRENDS

IN

MICROBIOLOGY

129

(PBMCs) exceeds 14 weeks6. In this study, the PBMCs could not be activated to produce infectious virus, so it is possible that the detected DNA represented defective provirus. Naturally, the more optimistic scenario captured the public imagination. However, previous studies of patients with active infections had found that a very small fraction of purified cells (~7 in 106 cells) with the properties of resting CD4+ T cells could be induced to yield infectious HIV (Refs 7,8). Given this low frequency, it is difficult to be certain of the precise characteristics of the inducible cell. Nevertheless, resting CD4+ cells correspond to memory T cells. With apologies to Salvador Dali, the persistence of memory (T cells) is famous. Thus, the question arose: are cells that can be induced to yield infectious HIV present in patients following long-term therapy with drug combinations? The answer came quickly. In November of last year, three papers4,5,9 describing such patients appeared. HIV RNA was undetectable in the plasma of these patients, with limits of detection ranging from 50 (Ref. 4) to 500 copies per ml (Ref. 9). Activated CD4+ T cells from these patients did not contain PII: S0966-842X(98)01232-3

VOL. 6 NO. 4 APRIL 1998

COMMENT

detectable infectious virus5. Nevertheless, these patients harbored cells that could be induced to yield replication-competent virus. Detection of these inducible cells appears to require elaborate purification and induction protocols. The frequencies of these cells in the purified fractions were even lower than those previously observed in patients with active infections8, but did not decrease appreciably over the time of the study5. The amount of integrated HIV DNA per 106 purified cells was the same in patients with no detectable HIV as in those with active HIV infections9. In combination, these results suggest that the inducible cells are indeed long-lived, which is consistent with the persistence of memory cells. Taken at face value, the data argue that the cells were infected months/years ago. Although it is not known whether these cells could ever be induced to produce HIV in vivo, suffice it to say that none of the papers recommend discontinuing antiviral therapy after a few years to see if a cure has been achieved. An important question addressed by two of the papers4,5 is whether the virus contained within the inducible cells is drug-resistant. Sequencing of the RT- and proteasecoding sequences (genotypic assays) from induced virus isolates has revealed none of the mutations known to confer resistance to the RT inhibitors used in therapy and only a few of the mutations known to confer resistance to protease inhibitors. In these cases, the patients had undergone ritonavir monotherapy before the study, so the mutant viruses might not have arisen during combination therapy. However, none of the isolates was examined for mutations in cleavage sites for the protease, which can be determinants of resistance to indinavir10, or for drug resistance (phenotypic assays), so it remains possible that mutations at other positions in the viruses confer resistance to the drugs. Undoubtedly, efforts are under way to characterize the induced virus isolates more thoroughly. It is also possible that the viruses in these cells evaded drug therapy because the cellular milieu mitigated drug activity. For example, all of the drug combi-

TRENDS

IN

nations included either zidovudine (AZT) or stavudine (d4T), which are activated by cellular thymidine kinase. There is very little of this enzyme in resting cells; thus, if thymidine kinase levels were low when the cells were infected by HIV, AZT or d4T might have had little effect. Clinically, the patients are latently infected with HIV. Virologically, a fundamental question is whether the infection is truly latent or whether there is low-level viral replication. Herpesvirologists are painfully aware of how difficult it is to answer this question definitively. In the present studies4,5,9, the purified cell populations came from patients in whom no viral RNA was detected, and virus was only detected following induction protocols. However, the viral RNA assay is not sensitive enough to detect tens of molecules per ml and is performed on smaller volumes of blood than are used to purify the cells. Assays to detect infectious HIV are notoriously even less sensitive. The failure to detect drug-resistance mutations is consistent with a lack of viral replication. However, interpretation of this result is tempered by the issues raised above. Comparisons of partial sequences of HIV isolated from patients before combination therapy with virus isolates induced from purified cells two years later show a mean of nine changes per 1000 base positions4. This relatively low rate of viral evolution, together with other data, is consistent with low viral replication, although it is not definite that no viral replication at all occurred in the cells that are thought to be ‘latently’ infected. Indeed, one of the three papers9 suggests that persistent replication is occurring in these patients. This suggestion derives from quantitative PCR measurements that indicate 28 times less integrated DNA than unintegrated DNA (measured by subtracting integrated DNA from total DNA) in the purified cell populations. The authors argue9 that the unintegrated HIV DNA might arise from ongoing viral replication, as it is thought to be unstable. The patients studied had plasma HIV RNA levels of <500 copies per ml and had undergone a maximum of

MICROBIOLOGY

130

13 months of combination therapy. It would be interesting to follow the ratios of unintegrated to integrated DNA in these patients for longer periods, coupled with more-sensitive assays of HIV RNA. In any event, it is not yet known whether the infections in the inducible cells are truly latent. Many other questions remain: for example, what blocks HIV production in the inducible cells, and what signals induce virus production? There is an enormous literature on mechanisms of HIV latency in vitro11; ideas from this literature can be tested with the inducible cells from treated patients. Questions that address the possibility of curing HIV infections include whether drug-resistant virus can evolve in the inducible cells and what the halflife of the inducible cell population is. Might it make sense to try to induce these cells in vivo in the presence of combinations of antiviral drugs to eliminate reservoirs of virus that might otherwise be there for a long, long time? ‘One step done and another begun in I wonder how many miles’ ‘New Speedway Boogie’ Grateful Dead (words by Robert Hunter)

Acknowledgements I thank D. Marvel for helpful comments, and numerous basic and clinical investigators for informative conversations regarding antiviral chemotherapy, drug resistance and viral latency. Grant support from the NIH is gratefully acknowledged. References 1 Wei, X. et al. (1995) Nature 373, 117–122 2 Ho, D.D. et al. (1995) Nature 373, 123–126 3 Gulick, R.M. et al. (1997) New Engl. J. Med. 337, 734–739 4 Wong, J.K. et al. (1997) Science 278, 1291–1295 5 Finzi, D. et al. (1997) Science 278, 1295–1300 6 Perelson, A.S. et al. (1997) Nature 387, 188–191 7 Chun, T-W. et al. (1995) Nat. Med. 1, 1284–1290 8 Chun, T-W. et al. (1997) Nature 387, 183–188 9 Chun, T-W. et al. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 13193–13197 10 Zhang, Y-M. et al. (1997) J. Virol. 71, 6662–6670 11 McCune, J.M. (1995) Cell 82, 183–188

VOL. 6 NO. 4 APRIL 1998