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The clinical significance of antiviral drug resistance G. Darby and B.A. Larder
Department o f Molecular Sciences, The Wellcome Research Laboratories, Beckenham, Kent BR3 3BS (UK)
In order to approach the question of the clinical significance of drug resistance, we need to define the terminology that we use in this area and to understand the underlying reasons for the appearance of drug-resistant variants. When we refer to drug resistance in relation to antivirals we are usually referring to a decrease in sensitivity to the drug measured in a tissue culture system. The value is usually quoted as an IC50, the concentration required to produce a 50 % inhibition of infectivity, and it may be measured in focal systems such as plaque reduction assays, or in yield reduction systems. In the latter assays, virus yield can itself be assessed in different ways including the use of surrogate markers of virus replication such as cytotoxicity, production of virus antigens or enzymes, or by estimating the extent of genome replication. It is important to appreciate that the actual values for ICs0 will vary widely according to the systems used to measure them, and they are therefore only meaningful if compared with values obtained for parental wild-type viruses. This comparison is not always possible, particularly when looking at clinical isolates, and judgement about whether or not an isolate is resistant will then have to be based on available base-line data derived using the same system with viruses that have never been exposed to the drug. The emergence of resistant strains, either in culture or in drug-treated patients, implies either that genetic variants initially present as minor components in the virus population have been selected out by the continued presence of the drug, or that such variants arise and are selected during drug treatment. The only phenotypic characteristic necessary to ensure their appearance is that they outgrow the parent strain in the presence of inhibitors. There is no requirement that they be more virulent, and so predicting their likely impact on the course of disease is not straightforward. In fact, drug-resistant variants which have been looked at so far in relation to virulence, mostly members of the herpes family, are less virulent than the
parent strains. Furthermore, the pattern of pathogenesis is also modulated so that the disease is less invasive (Field and Wildy, 1978; Larder and Darby, 1985 ; Field and Cohen, 1986; Larder et ai., 1986). It therefore cannot be stressed enough that there is no simple relationship between the emergence of drug-resistant strains and the future course of the disease process - - whether treatment is continued or not. This relationship can only be established with confidence by the collection of appropriate clinical and virological data. The term clinical resistance is used to describe a situation in which a virus disease which would be expected to respond to drug therapy responds less well or not at all. Clinical resistance may or may not be due to the evolution of resistant strains of virus, but in this discussion we will restrict ourselves to situations where resistance is the underlying cause. One other point which should be made is that a distinction must he drawn between those situations in which there is no response to treatment, i.e., the disease process continues as if no drug were present, and those where the response to treatment is less than optimal. Often these two situations are difficult to distinguish, but it would clearly be wrong to discontinue the drug treatment of a patient on the basis that there is clinical resistance unless it could be established that there would be no further benefit from that treatment. The phenotypic changes seen in the virus are the result of underlying mutations in the viral genome. Such mutations normally affect the interactions between drugs and their target enzymes and they constitute the genetic basis of resistance. We should now consider the state of knowledge in relation to the clinical implications of resistance, and the discussion will be restricted to the two systems which have been the focus of much of our own work over the past few years. These are: the treatment of herpes simplex infection of the immunocompromised with acyclovir, and the treatment of HIV infection with zidovudine.
tively infrequently, results in expression of enzymes with markedly reduced affinity for drug-related substrafes (acyclovir itself in the case of TK or acyclovir triphosphate in the case of DNA-pol), but little or no change in the affinity of the enzymes for their natural substrates. Unlike the severely attenuated TKdeficient viruses, "substrate specificity" mutants are very similar to wild-type strains in their interactions with animal hosts.
Resistance to acyclovir
Two enzymes encoded by herpes simplex virus (HSV) are involved in the mode of action of the nucleoside analogue acyclovir: thymidine kinase (TK), which is responsible for the first phosphorylation step, and DNA polymerase (DNA-pol), which is the ultimate target of the drug being inhibited by acyclovir triphosphate.
From a molecular genetic analysis of substrate specificity mutants, it is now clear that this phenotype normally results from single amino acid substitutions, usually in highly conserved sequence motifs. As more variants have been studied, it has become increasingly clear that such substitutions involve a restricted set of amino acid residues within these motifs, and it is likely that these are the residues which interact directly with the enzyme substrates. Thus an added bonus in the study of resistance is a clearer understanding of structure/function relationships of the enzymes themselves.
Resistance to acyclovir has been extensively studied, and it is now well established that it can occur as a result of mutation in either target enzyme (for review see Collins and Darby, 1991). The most common phenotype is TK deficiency, i.e., insufficient TK is made to initiate activation of the drug. This can result from amino acid substitutions in the TK polypeptide, but more commonly it involves insertion or deletion of single nucleotides in the coding sequence of the gene. The latter usually results in translational frame-shifting and truncated nonfunctional products. The loss of TK function does not prevent virus growth in culture, but such variants are severely attenuated in animal model systems. This is most marked in their interactions with the nervous system where their virulence is reduced and where they appear to be defective in reactivation from latency.
The clinical picture appears to mirror very closely the picture built up from the tissue culture studies and genetic analyses. Resistance is seen mainly in the immunocompromised and it is generally due to the emergence of TK-deficient viruses. Substrate specificity mutants have rarely been described (Ellis et al., 1987; Parker et aL, 1987; Nugier et al., 1992). In the immunocompromised, the disease may be refractory to treatment and so we see clinical resistance. There is, so far, no evidence of the transmission of resistant strains, and furthermore, even in those in-
Resistance to acyclovir need not involve loss of enzyme function. It can arise through subtle alterations in the biochemical properties of either TK or DNA-pol resulting from an amino acid substitution in either gene. This phenotype, which is seen rela-
A 1 .I,
C D E .,. ~
I ~. . . .
Fig. 1. Mutations in HIV RT associated with zidovudine (Q) or ddI (O) resistance. The specific mutations are: residue 41, M -. L; 67, D -* N; 70, K -~ R; 215, L --* F or Y; 219, K -. Q and 74, L -, V (data from Larder and Kemp, 1989, St. Clair et al.. 1991, and KeUam et al., 1992). The boxes indicate conserved motifs in the polymerase domain of the RT polypeptide.
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dividuals where we see the emergence of such strains, subsequent reactivations involve only sensitive viruses (McLaren et al., 1983; Straus et ai., 1984; Parker et al., 1987). This is consistent with the attenuation of TK-deficient viruses and their failure to reactivate from latency.
Zidovudine resistance Whereas in the case of herpes simplex and acyclovir resistance was observed initially in tissue culture systems and the lessons learned in the laboratory were applied to the clinic, in the case of HIV and zidovudine this sequence has largely operated in reverse. The initial observation that resistant strains of HIV could emerge following selection with zidovudine was made not using laboratory isolates but using isolates from patients who had been exposed to the drug (Larder et al., 1989). Work by Larder and Kemp (1989) indicated that there were 4 mutations in the reverse transcriptase (RT) gene of the virus which were involved in the expression of the resistance phenotype, but that number has recently been increased with the identification of a fifth mutation (Kellam et al., 1992). These mutations are illustrated in figure 1. The accumulation of this set of mutations results in a shift in sensitivity of approximately 100-fold. Identification of the mutations responsible for the resistance phenotype was made difficult by the inherent genetic variability of HIV. In two-way comparisons of the sequences of RT genes derived from pre-therapy and resistant isolates, there were usually a large number of genetic differences. When several pairs (5) were analysed, a number of mutations were seen which were common to different pairs. This provided circumstantial evidence for the involvement of these mutations in resistance, but the final proof was provided by introducing these mutations, singly and in various combinations, by genetic manipulation into an infectious DNA clone of the wild-type virus (Larder and Kemp, 1989; Kellam et al., 1992). Using this approach it has been confirmed that each of the identified mutations has a role in the evolution of the resistance phenotype. In many clinical isolates which have been examined (Boucher et ai., 1990, 1992) the observed sensitivities to zidovudine could be explained by the presence or absence of these mutations. We cannot exclude the possibility that other mutations may be involved in the resistance phenotype. Indeed, it is likely that others will be identified. However, we should be cautious in ascribing a role in resistance to an observed mutation until we have rigorous proof of that role.
This picture is quite different from that seen with acyclovir and HSV, where resistance was generally due to single amino acid substitutions in either TK or DNA-pol, and it may explain the difficulty experienced in isolating resistant HIV variants in culture systems (Larder et al., 1991). The involvement of multiple mutations poses the question whether there is an ordered appearance of mutations as treatment progresses. The answer, although we do not yet have the whole story, appears to be that there is. This has emerged from study of sequential isolates from a cohort of "high-risk" asymptomatic infected homosexual men. The picture which has emerged so far is illustrated in figure 2. The first mutation to appear is that at residue 70, but this is transient and it disappears as the mutation at residue 215 appears. This may be followed by the appearance of the residue-41 mutation and the reappearance of the residue-70 mutation. Only when the infection progresses to AIDS do we see the appearance of the 67- and 219-mutations associated with the most resistant phenotypes (Boucher et al., 1992; Kellam et al., 1992).
67 70 • 0 ": .:
215 219 Q 0 ".. -
." I' ".
Fig. 2. Acquisition of mutation in RT with zidovudine treatment. Possible sequence of appearance of zidovudine resistance mutations deduced from studies of a cohort of infected homosexual men treated over extended periods with drug (based on data from Boucher et ai., 1992, and Kellam etal., 1992). • = mutant residue; (3 = wild-type.
TO A N T I V I R A L
Several very important questions remain unanswered and provide us with the challenges for tomorrow. We do not yet know whether resistance will develop at the same rate in all patient groups. Early indications are that it may develop more slowly in asymptomatic individuals, presumably a reflection o f the low level o f virus replication in these individuals. Perhaps more importantly, we need to know whether use of drug combinations will slow or prevent the development of resistance. In a recent study o f clinical isolates from patients who had been taken o f f zidovudine treatment and switched to ddl (St. Clair et al., 1991) it was shown that resistance to that drug also develops. Interestingly, the mutation responsible for ddl resistance (a mutation at amino acid residue 74 o f the RT gene) appears to suppress the phenotype of the 215-mutation involved in zidovudine resistance, causing the isolates to revert to zidovudine sensitivity (see fig. l). This observations clearly has important implications for the way we manage drug combinations. We do not yet know the full clinical implications o f resistance. For example, we do not know whether an individual who has developed partial drug resistance will benefit from further treatment with the drug. The fact that additional mutations accumulate with further treatment implies that the drug has a suppressive effect on the replication of partially resistant virus, and we need to know whether this correlates with clinical benefit. Furthermore, although it appears that full resistance may be associated with clinical deterioration, we do not know whether that deterioration is less rapid than it would be in the absence o f treatment. There are currently several trials underway in which drug combinations are being evaluated and it will be interesting to see whether such combinations result in a reduction in the incidence of resistance and in improved clinical outcome.
There are clearly many outstanding questions in relation to antiviral drug resistance, but it is clear that effective drugs elicit a response from viruses which encourages the evolution o f less sensitive variants. These variants particularly emerge during extended periods o f treatment such as those used in the treatment of HIV infection or in herpes simplex infection o f the immunocompromised. Although great strides have been taken in the past decade or so, the area o f antivirals generally still suffers from a lack o f effective drugs. Clinical experience with those that exist tells us that we need more. Only with a comprehensive arsenal o f effective antivirals will we be able to build on the early successes.
Boucher, C.A.B., Tersmette, M., Lange, J.M.A., Kellam, P., deGoede, R.E.Y., Mulder, J.W., Darby, G., Goudsmit, J. & Larder, B.A. (1990), Zidovudine sensitivity of human immunodeficiency viruses from high-risk, symptom-free individuals during therapy. Lancet, I, 585-590. Boucher, C.A.B., O'Sullivan, E., Mulder, J.W., Ramautarring, C., Kellam, P., Darby, G., Lange, J.M.A., Goudsmit, J. & Larder, B.A. (1992), Ordered appearance of zidovudine (AZT) resistant mutations during treatment. J. infect. Dis. (in press). Collins, P. & Darby, G. (1991), Laboratory studies of herpes simplex virus strains resistant to acyclovir. Rev. Med. Virol., l, 19-28. Ellis, N.M., Keller, P.M., Fyfe, J.A., Martin, J.L., Rooney, J.F., Straus, S.E., Nusinoff-Lehrman, S. & Barry, D.W. (1987), Clinical isolates of herpes simplex virus type 2 that induces a thymidine kinase with altered substrate specificity. Antimicrob. Agents a. Chemother., 31, 1117-1125. Field, H.J. & Wiidy, P. (1978), The pathogenicity of thymidine kinase deficient mutants of herpes simplex virus in mice. J. Hyg., 81,267-277. Field, H.J. & Coen, D.M. (1986), Pathogenicity of herpes simplex virus mutants containing drug resistance mutations in the viral DNA polymerase gene. J. Virol., 60, 286-289. Kellam, P., Boucher, C.A.B. & Larder, B.A. (1992), A novel fifth mutation in HIV-I reverse transcriptase contributes to the development of high-level resistance to zidovudine. Proc. nat. Acad. Sci. (Wash.) (in press). Larder, B.A., Coates, K.E. & Kemp, S.D. (1991), Zidovudine (AZT) resistant human immunodeficiency virus selected by passage in cell culture. J. Virol., 65, 5232-5236. Larder, B.A. & Darby, G. (1985), Selection and characterisation of acyclovir-resistant herpes simplex virus type l mutants inducing altered DNA polymerase activities. Virology, 146, 262-271. Larder, B.A., Lisle, J.J. & Darby, G. (1986), Restoration of wild-type pathogenicity to an attenuated DNA polymerase mutant of herpes simplex virus type I. J. gen. Virol., 67, 2501-2506. Larder, B.A., Darby, G. & Richman, D.D. (1989), H I e with reduced sensitivity to zidovudine (AZT) isolated during prolonged therapy. Science, 243, 1731-1734. Larder, B.A. & Kemp, S.D. (1989), Multiple mutations in HIV-I reverse transcriptase confer high-level resistance to zidovudine (AZT). Science, 246, I155-I158. McLaren, C., Core),, L., Dekker, C. & Barry, D.W. (1983), In vitro sensitivity to acyclovir in genital herpes simplex viruses from acyclovir-treated patients. J. infect. Dis., 148, 868-875. Nugier, F., Collins, P., Larder, B.A., Langlois, M., Aymard, M. & Darby, G. (1992), Herpes simplex virus isolates from an immunocompromised patient who failed to respond to acyclovir treatment express thymidine kinase with altered substrate specificity. Ant. Chem. Chemother. (in press). Parker, A.C., Craig, J.I.O., Collins, P., Oliver, N.M. & Smith, I.W. (1987), Acyclovir-resistant herpes sim-
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plex virus infection due to altered DNA polyrnerase. Lancet, II, 1461. St.Clair, M.H., Martin, J.L., Tudor-Williams, G., Bach, M.C., Vavro, C.L., King, D.M., Kellam, P., Kemp, S.D. & Larder, B.A. (1991), Resistance to ddI and sensitivity to AZT induced by a mutation in HIV reverse transcriptase. Science, 253, 1557-1559.
Straus, S.E., Takiff, H.E., Seidlin, M., Bachrach, S., Lininger, L., DiGiovanna, J.J., Western, K.A., Smith, H.A., Nusinoff-Lehrman, S., Creah-Kirk, T. & Ailington, D.W. (1984), Suppression of frequently recurring genital herpes. A placebo-controlled double-blind trial of oral acyclovir. New Engl. J. Med., 310, 1545-1550.
The pathogenicity of drug-resistant variants of herpes simplex virus H . J . Field and S.E. G o l d t h o r p e
Department o f Clinical Veterinary Medicine, University o f Cambridge, Madingley Road, Cambridge CB3 0ES (UK)
Historical perspective - - resistance first encountered in H S V
Herpes simplex virus (HSV) infections were among the very first to be widely treated by means of antiviral agents. Nucleoside analogues were introduced in the early 1960's and it was noted that resistance to idoxuridine (IUdR) could be obtained by selection in tissue culture in the presence of the drug (Smith, 1963). During the 1970's 3 nucleoside analogues, IUdR, adenine arabinoside (Ara-A) and trifluorothymidine (TFT) were widely employed as topical agents for treating the ocular manifestation of HSV infection, herpes keratitis. Resistance was widely held to be a problem by the ophthalmologists who regarded this as a cause of "treatment failure" and it was believed that changing from one drug to another sometimes resulted in clinical benefit for this reason. Furthermore, virus strains resistant to IUdR were detected in clinical isolates from such cases (White et al., 1968; Jawetz et al., 1970; Hirano et al., 1979; Isobe et al., 1982). Resistance was also detected in culture to Ara-A (Guari, 1979; Coen et al., 1982) and eventually TFT (Guari, 1979; Field et al., 1981). It must be emphasized, however, that the observations of clinical resistance were largely anecdotal, and formal evidence was rarely, if ever, obtained to prove an essential role of the resistant virus in disease. Indeed, it was reportedly difficult to reconcile
variations in clinical response with the biochemical properties of viruses which were isolated from the clinical cases (Coleman et al., 1968; Jawetz et al., 1970). These difficulties may, in part, have been due to the lack of knowledge of the biochemical nature of resistance and the shortcomings of the tissue culture systems available at the time, which were used to define the relative sensitivity of the virus isolates.
Acyclovir and other second generation nucleoside analogues - - resistance predicted
With the advent of acyclovir (ACV), which began to be used in the early 1980's, the biochemical knowledge of both the virus itself and the mode of action of nucleoside analogues had advanced significantly. Thus, before the drug entered clinical trials in man, the molecular sites for resistance in the virus had been predicted and resistant mutants had been defined (Coen and Schaffer, 1980; Schnipper and Crumpacker, 1980). Indeed, such resistance mutants, which were isolated in tissue culture, have been invaluable tools for the elucidation of the mechanism of action of ACV and other nucleoside and pyrophosphate analogue inhibitors of HSV. These studies confirmed that the two crucial virus-induced enzymes which are involved in the mode of action of many nucleoside analogues are the thymidine kinase (TK) and DNA polymerase (DNA-poI). It was