Challenges in the therapy of HIV infection

TiPS -May 1993 [Vol. 141

Robert Yarchoan, Hiroaki Mitsuya and Samuel Broder inhibit human i~muno~eficienc~ virus U-W) replication have been shown to Irate clinical ufilify in patieFzfs with HIV j~fectiun. however, the immunological improvemenf inauced by available anfi-HIV therapies in patients with acquired immune deficiency syndrome (AIDS) is incomplete and transient. Explanations for this may include immunological barriers to compiete reco~zstjtufi~~~,low therapeutic indices of the available drugs, and the deve~opnteni of viral resistance. An undersfund~r~g of fhese processes, us discussed here by Robert Yarchoan and colleagues, may provide important leads for the developmenf of improved therapy for AIDS.

Dnzgs thaf

Since the discovery of human immunodeficiency virus (HIV) as the causative agent of AIDS, a number of compounds have been identified as having in vitro antiretroviral activity’. To date, the most clinical information exists for a class of compounds called dideoxynucfeosides2-5. Three of these compounds, zidovudine, 2’,3’-dideoxyinosine (dd1) and 2’,3’-dideoxycytidine (ddC) have undergone controlled multicenter testing and are approved in the USA and elsewhere for the treatment of HIV infection. With the available therapies, however, the immunological improvement attained is only partial and transient. Patients with advanced HIV infection who are administered zidovudine, for example, generally have CD4+ Tcell count increases of only 30-60 cellsmm-3 by week 6 to 10, after which they begin to decline3n6. Why does this occur? One reason is that the available drugs all have low therapeutic indices; at doses that are tolerable for long-term use, they induce only partial suppression of HIV replication. Another reason is the outgrowth of resistant HIV strains, although the relationship of this phenomenon to late falls in numbers of

CD4+ cells has not yet been formally proven. A third contributing factor is the toxicity of zidovudine and other drugs for immune cells. This is probabIy the reason that patients administered very high doses of zidovudine have an even more transient CD4” cell elevation than those receiving lower dose@. These concerns all indicate that the need for new anti-HIV strategies is as urgent as ever, and this issue will be discussed below. An unresolved question is whether inhibition of HIV replication in patients with advanced HIV infection will ever be sufficient in itself to induce complete and prolonged immunoreconstitution. The ability of HIV to destroy CD4+ lymphocytes in vitro initially suggested that the fall in

CD4 cell counts in HIV-infected patients occurred through their direct destruction in vivo. However, the observation that reiatively few (less than 1 in 10000) CD4 cells in the peripheral blood of patients contain actively replicating HIV as measured by in situ hybridization7, suggested that indirect mechanisms may be involved, and a number of potential mechanisms have since been defined’ (Table I). For example, HIV binding to CD4 cells may cause apoptosis, perhaps in part mediated by binding of the oral-associated major histocompatibility complex molecule, HLA DR, to the T-cell receptor or conceivably by HIV acting as a superantigen9,~0. Cells to which HIV gp120 binds may also be killed by anti-gpl20 antibodies, by antibody-dependent cellular cytotoxicity, or by cytotoxic T cells’l. Moreover, processes such as T-cell dysfunction or death caused by anti-CD4, anti-HLA DR or other autoantiantibodies, bodies might continue the process of immune destruction even in the face of complete viral suppression’“. Also, there is evidence that gp120 of HIV, or suppressive factors and cytokines released in HIVinfected patients, may impair the maturation and stimulation of immune elements8*13*‘4. Finally, there is some evidence that dysfunction of dendritic cells (or other antigen-presenting cells) as a result of HIV infection might contribute to immune dysiimction15*16. It should be noted that recent findings have indicated that more than 1 in 100 of the CD4+ cells in the lymph nodes of AIDS patients

TABLE I. Some possible indirect mechanisms of immune suppression in HIV infection Destruction of cells binding HIV or gpl20 by anti-HIV antibodies, cyfotoxic T-cell responses, antibody-dependent cellular cytotoxicity Abnormal T-ceil maturation induced by thymic damage or thymic HIV tnf~tion Destruction of the architecture oi iymph nodes and other lymphoid organs Autoantibodies to antigens on immune cells (for example to HLAclass If molecules or CD4) CD4 T-cell dysfunction caused by the binding of gp120 to CD4 Ove~r~uction

of suppressive factors or toxic cytokines

Programmed cell death (apoptosis) induced by the binding of gp120 to CD4, possibly in conjunction with Hf_A DR incorporated into HIV Effects of circulating immune complexes Suppression of bone marrow stem cells and other progenitor cells by direct HIV infection or by dest~ction of the bone marrow microenvironment

R. Yarchoan is Head, Retroviral Diseases

Section.

H.

Mitsrcya is Hend, Experimental

RetrovirologySection in the Medicine Branch and S. Broder is Director,NationaJ Cancer hfibfe, N~fj~~~~ f~sfifI~fes of ~eu~f~, Zelkda, MD 20892. USA. 0

1” ‘. Asevier Science Pubhers

Ltd (UK)

Perturbation of T-cell repertoire induced by virally-encoded superantigens Dysfunction of dendritic cells and other antigen-presenting cells HIV, human immunodeficiency virus; HLADR, complex.

0165 - 6147/93/$06.00

a com~nent of the major histocompati~fity

TiPS - May 1993 [Vol. 141 may be infected with HIV, suggesting that such indirect mechanisms may not be necessary to explain the immunosuppression in AIDS17. The clinical importance of indirect mechanisms in the pathogenesis of AIDS remains unresolved. Immunological approaches What are the barriers to complete immunoreconstitution in AIDS? There is an increasing appreciation that one such factor may be the destruction of the architecture of immune organs’*. Follicular dendritic cells in lymphoid organs serve to trap antigens in the germinal center. In early HIV infection, such cells reduce viremia by trapping eirculating HIV particles. In the later stages of the disease, a degeneration of the network of follicular dendritic cells is believed to contribute to the increased viral burden in the circulation”. Destruction of the thymus may also be important in the induction and maintenance of immunosuppression in AIDS. Autopsy studies show that patients dying of AIDS have severe abnormalities in their thymus glands”. Moreover, thymic epithelial cells can be infected by HIV (as can dendritic cells, although this point requires more research) and there is evidence that human thymic tissue can be infected with HIV when implanted into immunodeficient (SCID) mice”. While there is still some controversy about the importance of thymic function in adults, it is possible that destruction of the thymus by HIV may reduce the capacity to replenish destroyed T cells. As such, improving thymic function may be therapeutic benefit. One of approach involves the use of growth hormone or insulin-like growth factor. There are data to suggest that growth hormone can increase thymic size in aged rodents and increase the number of CD4+ cells in SCID mice given human peripheral blood lymphocyteszl. Therefore several groups are testing whether these hormones can enhance immune function in patients with HIV infection. Preliminary results from one study in the National Cancer Institute, however, suggest that this therapy does not induce consistent CD4+ T-cell elevations

197 in patients with advanced disease (Nguyen, B. Y. et al., unpublished). Another potential barrier to immunoreconstitution complete in AIDS is the immunosuppressive effect that may be exerted by opportunistic infections with pathogens such as cytomegalovirus, human herpesvirus 6, Mycobacteria, or mycoplasma by direct or indirect mechanisms. Such pathogens may aIso serve to enhance HIV infection22,23. For example, human herpesvirus 6 may induce expression of CD4 on CD4T cells, and regulatory genes of certain herpesviruses or adenoviruses may activate HIV replication, Control of these infections may thus secondarily reduce the level of HIV replication. We need to learn more about the networks of cytokines in the body and how they interact with HIV. However, some therapeutic leads have already emerged from our current level of understanding. For example, there is recent interest in the concept that the progression of HIV infection is associated with a decline in Trill responses and an augmentation of Tn2 responsesz4. In the mouse, where these CD4+ T-cell subsets are best defined, TH1 cells produce interferon y (IFN-y) and interleukin 2 (IL-2) and enhance cellymediated immunity, whereas THY cells produce B-cell stimulatory cytokines such as IL-4, IL-6 and IL-lo. Some cytokines released by Tn2 cells suppress Trill cells, and an augmentation of Tn2 responses may thus contribute to the deficiency of cell-mediated immunity in AIDS. Also, overproduction of IL-6 may augment HIV replication and may contribute to the development of non-Hodgkin’s lymphoma1s~25*26.As such, there is an interest in exploring ways to suppress Tn2 responses (as with antiIL-4 or anti-IL-6 antibodies) or to enhance TH1 responses (as with low-dose vaccination), The possibility should be kept in mind, however, that certain Tn2 responses may be beneficial in the setting of HIV infection, and additional research is needed on the implications of this switch in T-cell subsets. Another cytokine that is believed to play an important role in HIV infection is tumour necrosis factor (x (TNF-ar). This cytokine

can upregulate HIV infection and may contribute to certain AIDSassociated symptoms such as cachexia and weight lossz7. The drug pentoxifylline has been found to inhibit the production of TNT-o, and clinical trials are under way to determine whether it might be of value in advanced HIV infection in combination with anti-HIV agentsz8. The activation of HIV replication induced by TNF-a: appears to be mediated through a reduction in intracellular glutathione levels and an activation of the transcription factor NF-KB, which interacts with the long terminal repeat (LTR) of HIV’7~29.There is evidence that cysteine precursors such as N-acetyl+cysteine (NAC) can prevent the reduction in intracellular glutathione levels and thus reduce the degree of TNF-rrinduced HIV activationz9. Clinical trials are now under way to test whether administration of NAC can increase intracelluiar glutathione levels in vivo and by this mechanism reduce H.V rephcation. As ~111 be ‘discussed below, gene therapy approaches to blocking LTR-driven HIV gene expression are also being explored. A number of immunological approaches designed to boost the immune system nonspeci 3cally in patients with HIV infection are also being explored. These include the use of stimulator-y factors (such as IL-2 or granulocytecolony-stimulating macrophage factor) or immunostimulato~ drugs. Additional areas of interest include bone marrow transplantation, extracorporeal expansion of certain immunological populations such as CD8+ cells, or expansion of uninfected stem cell populations in patients3’. While there are concerns that some bone marrow stem cells may be infected by HIV (and indeed, these may contribute to the immunodeficiency in this disease), there is evidence to suggest that at least a subset of stem ceils remains uninfected31. There is presently an interest in learning how to identify and expand such a population in vitro, possibly in conjunction with gene therapy3’. Another area of interest Lies in boosting the specific immune response to HIV. Preliminary studies therathat suggested have peutic vaccination of HIV-infected

TiPS - May 2993 [Vol. 141

~-Aztdo-~,~~i~o~thymidine (Zidovudine,AZT)

~.~5id~yd~-2’,~dideoxythymidine (Stavudine, D4T)

~-Ftuo~.~,~-dideoxythymidine (FLT) W

I

ti

Ii

~,~-Di&o~cytidine (Zatcitabine,ddC)

2’,3’-Dideoxyadenosine (ddA)

T,3’-Dideoxy-3’-thiacytidine (-) enantiomer (3TC)

2’,3’-Dideoxyinosine (Didsnosine,ddl)

fig. 1. ~tures of seven did~~nucf~sides that have entered ~inicaf testing f51 HIV infectiarf. Three of these, zidovudine, ddC and ddl, are now approved for use, and a fourth, LMT, is available in the USA undera paralleltrack mechanism. In oatients, ddA is rapidly converted to ddl by the ubiquitous &%!yme adenosine dearnina&

patients with viral glycoproteins or cells expressing these proteins can enhance the immune response to certain antigenic dete~inants33. These vaccines, as well as other vaccine approaches, are worthy of further study. At least one randomized trial of therapeutic vaccination is already under way, and others are planned. Inhibition of HIV replication HIV replication may be inhibited at early or late stages. Drugs that act at early stages So far, this discussion has focused on the means by which HIV disturbs the immune system and on immunological approaches to addressing this problem. This is certainly an important area of research and one that may lead to effective strategies in the not-too-

distant future. As noted above, however, the available anti-HIV agents shaw only limited ability to block HIV replication in viva and improved anti-HIV regimens are urgently needed. The widest clinical experience to date has been with the dideoxynucleoside drugs {Fig. 1). These agents have no apparent anti-HIV activity in their own right, but instead must be modified in target cells to their active moieties (believed to be the 5’-triphosphates)‘. While there are similarities among these drugs with respect to their mechanism of action, they are activated by different enzymes within the target cells and have different activity and toxicity profiles. As such, there continues to be an interest in exploring other members of this family beyond zidovudine, ddC and ddI. Indeed,

a variety ,lt other dideoxynucleosides have been identified with in vitro anti-HIV activity and several of these, including 2’,3’-did?hydro-2’,3’-dideoxythymidine (D4T), 3’-fluoro-3’-dideoxythymidine and the negative enantiomer of 2’,3’-dideoxy3’-thiacytidine, clinical now entered have testing*36 (Fig. 1). D4T is now available in the USA, produced by Bristol-Myers Squibb under a parallel track mechanism (established to make certain that investigational drugs for life-threatening diseases are available to patients who have no other therapeutic options). Another reason to continue to develop a variety of anti-HIV agents, even within the same class, relates to the issue of resistance. There is a body of evidence indicating that the outgrowth of resistant strains of HIV coniributes to the late fall in numbers of CD4 cells seen in patients on zidovudine therapysrt3s. Strains resistant to zidovudine generally have two or more mutations in the HIV pal gene (most frequently Met41+ Leu, Asp67+Asn, Lys’lO+Arg, Thr215-+Tyr or Phe, or Lys219+ Gin). Zidovudine-resistant strains preserve their sensitivity to most other dideoxynucleosides (including ddC and ddi). HIV strains with a decreased sensitivity to ddC or dd1 have also been reported in patients receiving those drugs, although the change in sensitivity is generally of a lesser magnitude than that seen with zido~d~e3’,~‘. Interestingiy, the most frequent mutation associated with a decreased sensitivity to ddI (Leu74+ Val) appears to enhance sensitivity to zidovudine in zidovudineresistant HIV isolates with Thr215+ Tyr. This observation provides one rationale for the use of these drugs in combination. Dideoxynucleosides are not the only group of compounds that block HIV replication by acting at the level of reverse transcriptase. In 1990, Pauwels et al. reported that certain benzodiazepine analogs and thione derivatives had potent and selective anti-HIV-l activity”. Several other chemicaliy diverse compounds, including BIRG587 (nevirapine) and L697661, were found to have a similar activity profile (in~ludin a lack of activity against HIV-2)4 d*43. These non-nucleoside, reverse transcrip-

TiPS - May 1993 1Vot. 141

199

TABLEII, Stages in the HIV life-cycle that may be targets for therapy Possible Intervention

Stage

Early Binding to target ceil

Antibodies to HIV or receptors; CD4 analogues Possibly polyanionic compounds

Fusion to target cell

Antibodies that block the gp41 fusogenic domain Antibodies to the V3 loop of gpl20

Entry, uncoating, and RNA release Bicyctams and hypericin may in part act

at this step

Transcription of RNA to doublestranded DNA

Dideoxynucleosides (for example, zidovudine, ddC, ddl), non-nucleoside reverse transcriptase inhibitors

RNA degradation by RNase H activity

Specific inhibitors of RNase H may be found

integration of HIV into host DNA

Specific inhibitors of pal-encoded integrase may be found

Late Efficient tran~~ption translation

and

Tat inhibitor; TAR decoys Inhibitors of Rev may be found Antisense constructs Ribozymes

Enhancement by cellular factors

Kacetyl-cysteine Pentoxi~lline

and analogues

Ribosomal frameshifting

Frameshift inhibitors may be found

Gag-Pol polyprotein cleavage

HIV protease inhibitors

Mydstoylatton and gly~sylation by cellular enzymes

Drugs (for exampte castanos~rmine

Dimerization, binding lysine tRNA

Inhibitors may be found

Packaging of the virus

Antisense inhibitors of the packaging sequence Zinc-ej~ting aromatic com~unds act in part at this step

Viral budding

lnterferons or interferon inducers Antibodies to viral antigens involved with viral release

and analogues)

HIV,human immunodeficiency virus. tase inhibitors were found to operate by a mechanism that is non-competitive with respect to the primer, the template and tRNA. Subsequent studies showed that resistance to these compounds develops very rapidly, and this appears to blunt their clinical activity when used alone. These compounds, however, may be useful when combined with dideoxynucleosides. There is substantial {but not complete) crossresistance among the various members of this class, and this is an important factor linking them as a class of drugs42*43. The development of resistance to these compounds has been associated with several mutations in the pal gene (encoding reverse transcriptase), including Tyrl$l-+ Cys and Lysl03--Asn. One such mutation is sufficient to confer a substantial decrease in sensitivity to these drugs. A recent study of the structure of HIV reverse transcriptase by X-ray crystallography showed that BIRG587 binds in a pocket of ~66 on top of a p hairpin motif containing Asp185 and Asp186. This is located just under-

neath the active site for polymerization4*. The sidechains of Tyr181 and Tyr183 were also found to be in contact with this inhibitor. This may explain the ability of mutations at those sites to induce resistance. We are now able to identify critical portions of reverse transc~ptase in which mutations are associated with a loss of function. It is possible that in the future we will be able to design drugs that bind to those areas. It is hoped that any emerging resistance to such drugs would, if it developed, be associated with a decreased ability of HIV to replicate. Our understanding of the resistance induced by the dideoxynucleoside inhibitors is incomplete at present. The mutations associated with decreased sensitivity to zidovudine or dd1 do not lie close to the active site for polymerization. Kohlstaedt et RI. have suggested that these drugs might confer resistance by altering interactions between the protein and template strand44. They could affect the way the template is bound and in turn alter the

enzyme’s ability to discriminate between the usual physiological substrate and the nucleosideS’triphosphate inhibitor. We should also keep an open mind for the possibility that one or more unidentified intermediates of drugs such as zidovudine help bring about the inhibition of reverse transcriptase. Additional studies will be needed to sort out these issues. There continues to be an interest in developing drugs that act at other early stages. For example, a new class of drugs, bicyclams, has been reported to block the uncoating of HIV (Ref. 45). Many of the anti-HIV drugs up to this time have been discovered by screening and this process continues to identify active compoundP. However, we are now moving into an era where we can begin to rationally design inhibitors that act at specific stages in the HIV life-cycle (Table II). Among the first such drugs described were analogs of CD4 such as recombinant soluble CD4 (rCD4) and rCD4-immunoglobin G immunoadhesin. Such inhibitors of viral binding have potent activity against laboratory strains of HIV, although their activity against fresh isolates is substantially blunted47.

One of the more exciting areas of current research in AIDS therapy is the development of inhibitors to HIV aspartyl protease (Fig. 2). The Gag and Pal proteins of HIV are initially translated as a large Gag-PO1 fusion polyprotein. This polyprotein must then be cleaved by a specific viral protease (a product of the pal gene) in order to form infectious virions. The t!seedimensional structure of HIV protease, a 99-amino acid dimeric protein with a C-2 axis of SYIIImetry, has been determined by X-ray crystallography and several groups have produced specific inhibitors of this enzyme*&‘*. These inhibitors, which generally have activity against both HIV-l and HIV-Z act at a late step in HIV replication. As such, they can inhibit the production of infectious virus by chronically infected cells, including monocyte-derived cells, which produce HIV for long periods of time without themselves being destroyed by the

TiPS - May 1993 fVr31.141

Substrate sequence

leucine

i

asparagine

KM272

Fig. 2. Amino acid sequence of lhe physiological substrate for HIV-1 protease (top) and .sWXure of an ~~U~r~ease i~fbjfor, KN1272 (?.Wtom). KN1272, a ~ransi~o~ state mimetic proiease ~nb~b~f~~~ confains the f~~~~~~n St&t? moiety, ~ff~~b~n~f~~~~n~ lf2s3s)-3-arnino-2-hyd~~~~henylb~~~~ acfdj. in @ace of ilze Phe-Pro s&tile bond of the physfological substrate for HIV protease. me Phe 167~Fro 163 scissile bond is identified here by arrotvsj KNl272 exerts a potenf ant/vim/ acfivify against a wide spectrum of HIV strains in a variety of human fargefcells in vitro (see f?ef. 50).

virus. Also, such inhibitors have synergistic anti-HIV activity with dideoxynucleosides in vifru, There are substantial technical problems in the development of protease inhibitors that relate primarily to issues of solubility, plasma half-life, oral bioavailability and difficulty in large-scale synthesis. Efforts are underway to address these issues5’ and at least two protease inhibitors have now entered clinical trials. Other late targets for anti-HIV therapy are viral budding and RNA packaging. Interferon tr (IFN-a) has been reported to have anti-HIV activity in part by blocking viral budding. Also, zinc-ejecting aromatic C-nitroso cornpour, 3s have recently been reporteci to interfere with HIV replication in part by causing defective packaging of genomic viral RNA (resulting in non-infectious particles)5*. Efforts are being made to identify agents that block the activity of the adds-activating (Tat) protein of HIV. This 8damino acid pr >tein binds to a receptor called the ~ru~s-acti~~ response (TAR) element on the long-terminal re-

peat of HIV and promotes the efficient production of viral polyproteins. Several inhibitors of Tat have now been identified”. The side-effects of one such agent, Ro53335, include nephrotoxicity and yellow tissue discoloration, and an analog, Ro247429, has now entered chnical testing. Tat has afso been identified as a target that may be inhibited by gene therapy. Sullenger and co-workers reported that HIV expression was decreased in cells in which a tRNA transc~ption unit was utilized to overexpress TAR-containing sequences (TAR decoys)32. TAR decoys are believed to block viral replication by binding up free Tat protein within the cells. The general approach of rendering celts resistant to HIV infection through genetic manipuiatio~ (called ‘intracellular immunization’) is presently being studied in a number of laboratories, It could be applied to CD%+ cells, or alternatively to bone marrow stem cells that might then be able to repopulate the immune system. Another potential targeted approach to MIV therapy is the use

of anti-sense oligodeoxynucleosides, short segments of DNA (or modified DNA) that may hybridize to messenger RNA and inhibit translation53E54. A number of antisense constructs have been found to have anti-HIV activity in vitro. Some of these compoundsI such as phosphorothioat~ oligomers, have also been found to have sequenceindependent effects {by acting at an early stage of HIV replication). Self-cleaving RNAs (ribozymes) are also being explored in the laboratory as a potential therapeutic modality. The principal problems in the development of all of these compounds relate to issues of their differential activity against different strains of HIV, differential activity in various target cells, cost of production, oral biaavaiIabili~ and separating nonspecific from specific effects. While it is unlikely that any such approach will enter clinical practice soon, a preliminary study of an anti-sense compound directed at HIV is expected to start this year. In the future it may be possibi;e to use a modification of this approach to target HIV proviral DNA. Hanvey, Babiss and co-workers have recently reported that polyamide oligomers called peptide nucleic acids (PNAs} can invade duplex DNA strands in a sequence-specific manner, causing displacement of one DNA strand and the formation of a D100~~“. Such constructs can block transcription in vifro. This approach is highly experimental and nuclear microinjection is required to achieve an adequate concentration in cells. However, it is conceivable that a modification of this technique, perhaps in combination with enzymes that degrade DNA, might enable the selective destruction of proviral HIV and possibly even the consideration of curative approaches. It must be pointed out that no such approach is even near clinical development now, nor will be in the foreseeable future.

Cambinationstiategies So far, we have focused

on individual agents or therapeutic approaches. However, ii is likely that the most effective therapy for HIV will involve combinations of drugs and/or immunological approaches. Potential benefits of combining anti-HIV drugs, for ex-

TiPS -May

1993

[Vol. 141

201

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0~ CD4 Count Interval (Cells/mm3) Fig. 3. Hazard of death ~s~~~ as deaths per ~tie~tper muntb) in 55pa~ents totluw~ on Nationat Cancer tnstitute zidovudine-containing protocols, in retattonshtp to their absolute CD4 count. The error bars denote the 95% confidence intervals. Numbers above the bars show the number of patients dying within each CD4 count interval. Data adapted from Ref. 59.

ample, include the possibility of drug synergy, a reduction of drug toxicity, delayed development of resistance and the targeting of different cell targets. One reason for the interest in developing drugs that act at late stages of HIV replication is the possibility of combining them with dideoxynucleosides, which act at early stages. In fact, as noted above, even combinations of dideoxynucleosides can be beneficial. The combination of zidovudine and ddC was felt to induce greater and more sustained CD4 T-cell elevations than zidovudine used alone, and this combination is now approved in the FJSA’. Also, there are preliminary results from our group to indicate that CD4 T-cell elevations lasting for more than one year can be attained with zidovudine and ddI in combination, and that the simultaneous administration of these drugs raises numbers of CD4 T cells to

higher levels than does an alternating regimen (Yarchoan, R. and Broder, S., unpublished). One reason for this may lie in the preferential targeting of different cell populations by these two drugs; there are data to suggest that zidovudine is preferentially phosphorylated in actively dividing cells, while ddl has relatively better phosphorylation in resting cells56,57. There may be evolutionary limitations in the abifity of reverse transcriptase to develop resistance to multiple drugs simultaneously. Chow et al. have recently reported that at least one combination of mutations conferring resistance to zidovudine, ddI and a nonnucleoside reverse transcriptase inhibitor was incompatible with viral replication58. While it is still possible that other combinations of mutations will permit viral replication in the presence of the this result has three drugs, heightened interest in the study of

multidrug resistance by HIV. Clinical trials are now planned to test whether this approach, called convergent combination therapy, can prevent or substantially delay the development of HIV resistance in patients. The development of antiretroviral dideoxynucleosides has demonstrated that anti-HIV therapy can beneficially affect the clinical course of patients with AIDS. At the same time, however, it has become apparent that HIV is a formidable foe. There is a continued need for anti-HIV agents that target various cell po~ulations (including those within the central nervous system) at various stages of the HIV life-cycle and have different toxicity profiles. In addition to anti-HIV therapy, approaches to reconstitute the immune system and to specifically boost the response to HIV need to be explored, both individually and in combination. Ultimately, our goal in patients with AIDS is to keep viral replication in check and restore the immune system to normal. However, there are data to suggest that substantial benefits can be attained even with more modest immunological improvement. Early in the course of the AIDS epidemic, it was not unusual for patients to develop life-threatening opportunistic infections with CD4 cell counts as high as 200 cellsmm-3. With the development of effective prophylaxis, along with therapies for such conditions, the profile of many of these infections has changed and patients now rarely die of AIDS in most centers in the USA until their CD4 cell counts fall below 50 cellsmmw3 (Refs 59, 60) (Fig. 3). Accordingly, maintaining the counts above that level may reduce the probability of death and offer substantial redustions in morbidi~ and increases in survival. Thus, to the extent that therapeutic regimens can effect even modest improvements in immunological parameters, a substantial clinical benefit may be possible. References 1 Mitsuya, H., Yarchoan, R. and Broder, S. W?901Science 249. 1533-1544 2 Yakhok, R., Pluda, J. M., Perno, C. F., Mitsuya, H. and Ekoder, S. (1991) Blood 78,859-884 3 Fischl, M. A. ef Ql. (1987) New .&?I$. J. Med. 317,1&191 4 Kahn, J. 0. et R!. (1992) New Eq$. 1. Med.

TiPS - May 2993 [Vol. 141

302

5 b

.i-jigI) I

;

Harper,hl. E.. Mnrselle. L. MI.. Gallo.

(2. C. and Wang-Staal, F. (19S6) Pror. S11f1.iCJ‘f. Sd. USA 83, 772-776 5. M.. Foil, G., 8 Fauci, A. S , Schnittman, Koenig, 5. and Pantaleo, C. (1992) A~I. I,trtw. Med. 114, 678-693 185, 9 Laurent, C. A. rl RI. (1991) Vriology 829-839 L. 0. ct III. (1992) S&w 258, 10 Arthur, 1935-1938 R. F. et RI. (1988) Cell 54, 11 Siliciano, 561-575 R. B. 1-t nl. (1987) N~trrw 327, I2 Striker, xc-713 K. j. ct RI.(1989) /. 11wtnrwl. 13 W&hold, 142,3091-3097 14 Clerici, M., Roilides, E., Via, C. S., Pizza, P. A. and Shearer, G. (1992) Proc. Nat! Asnd. Sri. USA 89, 8424-8428 15 Langhoff, E. cf al. (1991) Proc. Nntl Acnd. Ser. USA 88,7998-8002 16 Macatonia, S. E.. Patterson, S. and Knight, S. C. (1989) I~~r~w~zolo~~/67, 285-289 17 Schnittman, S. et nf. (1990) Anwfs ftrf. Med. 113, 43P-443 18 Pantaleo, G., Grariosi, C. and Fauci, A. S. (1993) Nczr Et@. /. Med. 328, 327-335 19 Schuurman, H. J. et nl. (1989) Am. /. Patkol. 134, 1329-1338 20 McCune, J. M.. Namikawa, R., Shih, C-C., Rabin, L. and Kaneshima, H. (1990) Ssir~ce 247, 564-566 21 Murphy. W. J.. Durum, S. K. and Longo. D. L. (1992) Proc. N&l Acnd. Sri. USA 89, 4481-4485 22 Lusso. l’. et nl. (1991) Nature 349, 533-535 23 Nabel, G. J., Rice, S. A., Knipe, D. M. and Baltimore, D. (1988) Scimce 239, 1299-1302 24 Clerici, M. and Shearer, G. M. (1993) rmJa2r~lol. Today 14, 109-111 25 Birx, D. L. et RI. (1990) Blood 76, 2303-2310 26 Pluda, J. M. et al. J. Cfin. Oncol. (in

p-w 27 Poli, G. et al. (1990) Proc. Nat1 Acnd. Sci. USA 87. 782-785 28 Fazely, F., Dezube, B. J.. Allen, R. J., Pardee. A. 8. and Ruprecht, R. M. (1991) Blood 77, 1653-1656 29 Staal. F. J. T., Roederer, M.. Herzenberg, L. A. and Herzenberg, L. A. (1990) Proc. Nntl Acnd. Sri. USA

87, 9943-9947

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Ro53335: 7-chloro-5-(2-pyrryl)-3H-1,4benzodiazepin-2H-one L697661: 3-( [(4,7-dichloro-1,3-benzoxazol2-yl)methyl]-amino)-5-ethyl-6-methylpyridin-2(lH)-one BIRG 587: 11-cyclopropyl-5,11-dihydro-4methvl-6H-dipvridol3,2-b:2’,3’-e111.4jdiazepin-6-one.

Drew M. Pardoll 1993 represents the 100th anniversary of William Coley’s first report of tumour regressions induced by immune system activation in response to bacterial toxins. While many subsequent cancer vaccine trials have yielded tantalizing results, active immunotherapy has not yet become an established modality of cancer therapy. Drew Pardoll reviews newer molecular vaccine approaches based on rational immunological principles that have resulted in improved systemic antitumour effects in animal models. Ultimately the genetic definition of tumour-specific antigens will allow the development of targeted antigen-specific vaccines for cancer therapy. The classic concept of a vaccine derives from the area of viral vaccines. For viral vaccines, individuals are immunized against viral antigens before encountering the pathogenic virus. Such a strategy is feasible with viruses because viral genomes are relatively simple, possessing a limited number of defined antigens. Such is not the case for most tumours, whose potential universe of antigens can be virtually limitless (see below). Therefore, when we talk about tumour vaccines, the most common clinical setting is one in which induction of a systemic immune response by the vaccine must occur subsequent to, rather

36, 202-205

37 Larder, B. A., Darby, G. and Richman, D. D. (1989) Sclcxce 243, 1731-1734 38 Larder, B. A. and Kemp, S. D. (1989) Science 246, 1155-1158 39 S. Clair. M. H. et ~1. (1991) Science 253, 1557-1559

54

59

34, 3402-3405

E. S.. Li. X. L.. Moudrril. T. and Ho, b. D. (1990) Pr&. Nntl”A;nd. Sci. USA

1799-1802 53 Matsukura,

D. M. Pnrdoll is Associnfc Professor irr the Depnrtrwrrts of Otrcolog!y, Medicine, nnd Molecrrlnr Biolppy nud Getwtics, Jolrr~s Hopkirls U?rioersify Sclrool of Mcdicitle, Bnltimore, 0

MD

1993. Elscvm

21205, Sclrnrc

USA.

Publishers

Ltd (UK)

than before, the antigenic insult. In the past, and for the near future, the major tumour-specific antigens upon which we can focus the immune system have not been molecularly defined. We must therefore produce the vaccine by using the patient’s own tumour cells as the source of antigen. In considering how to enhance most effectively immune responses against tumour antigens, it is important to appreciate the emerging principle that T-cell responses, rather than antibody responses, are the primary target of effective antitumour immunization strategies. The importance of T cells in vaccine-based tumour immunotherapy has a number of critical implications. First, since r cells can recognize peptide antigens brought to the cell surface in conjunction with major histocompatibility complex (MHC) proteins,

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