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Clinical significance of zidovudine-resistant human immunodeficiency viruses
4th FOR UM I N VIROLOG Y
134
Clinical significance of zidovudine-resistant human immunodeficiency viruses C . A . B . Boucher
Antiviral Treatment Laboratory~Department o f Medical Microbiology, Academic Medical Center, University o f Amsterdam, Meibergdreef 15, 1105 A Z Amsterdam
Introduction Treatment of individuals infected with the human immunodeficiency virus (HIV) with zidovudine (AZT) was started only 5 years ago. The first report on the emergence of AZT-resistant strains originates from 1989. Thus the available knowledge on the relation between the emergence of AZT-resistant isolates and clinical progression is limited. However, certain conclusions can be drawn from the available data.
The appearance of drug-resistant isolates In the first publication of zidovudine-resistant isolates by Larder et ak (1989) a distinction was made between partially resistant isolates and "high-level" or fully resistant isolates based on the in vitro sensitivity. Based on the 50 % inhibitory concentration (IC50), as determined in the HeLa CD4 + plaque assay, all isolates with an ICs0 ranging between 0.005 and 0.01 ~.M AZT (corresponding to a decrease in sensitivity of a maximum 20-fold) were defined as partially resistant. Isolates with an ICs0 above 0.01 gM AZT were said to have high-level resistance; they showed a decrease in sensiti~vity ranging from 20- to 100-fold. Sequence analysis of the genes encoding for the reverse transcriptase (RT) of these partially and highlevel resistant isolates showed that mutations at 5 codons (41, 67, 70, 215 and 219) were associated with a decrease in AZT sensitivity (Larder and Kemp, 1989). Introduction of these mutations in the RT gene of a molecular HIV clone proved that these mutations caused a decrease in AZT sensitivity. In addition, it was noted that a relation existed between the level of resistance and the number and combination of mutations. Mutations at either codon 70 or 215 alone (or in combination) caused partial resistance. For high-level resistance, 4 mutations are required. In addition to codons 70 and 215, in all cases a third mutation was encountered at codon 67; a fourth mutation can occur at either codon 41 or 219.
Therefore by analysing the composition of these 5 codons or a subset, one can make predictions about the level of resistance of a particular isolate. An approach was developed to determine the composition of a codon on RT by making use of the polymerase chain reaction (PCR). (Larder et ai., 1991). In the first step, a part of the RT gene containing the required codon is amplified, using primers which anneal~ to a conserved region. In the second (selective) step, a distinction is made between the wild-type and mutant codon. This is done by performing in parallel two separate PCR on the amplified fragment. In both reactions the same primer binding to the conserved region is added. In addition, to one reaction, a wtldtype primer annealing to the wild codon is added, and in the other reaction, the mutant primer is added. By analysing the products on a gel, it is possible to distinguish between a predominantly wild-type population, a predominantly mutant population or a mixture population.
Relationship between natural history and development of drug resistance To investigate the relationship between the development of disease progression and AZT resistance, we studied a group of 18 HIV-infected homosexual men. They were selected to enter a trial, from a previously described cohort, because they were considered to be at risk for rapid development of disease since they were HIV p24 antigenaemic and because the majority had less than 500 CD4 + cells per microlitre. All received, after an initial period of 2 months when different dosage schemes were used, 1,000 mg AZT per 24 hours (Boucher et al., 1990). During follow up, participants were seen each month and clinically evaluated, and peripheral blood mononuclear cells (PBMC) were stored. After a follow up period of two years, 6 of the 18 participants had progressed to AIDS and 12 were still asymptomatic, confirming results obtained in larger trials that, despite continuous treatment with AZT, disease progression can take place. This raised the question
RESISTANCE TO A N T I V I R A L DRUGS as to whether progression in this group was correlated with a difference in AZT sensitivity. To answer this question, virus was isolated through co-cultivation both with HIV-antibodynegative donor cells and ceils from the MT2 cell line. Three-time points were analysed: start, approximately 6 months later and either at 2 years for the nonprogressors or around the moment of AIDS diagnosis (for the progressors). At all moments, virus could be isolated using the donor PBMC and, as expected and described before, only a subgroup of the cocultivations with the MT2 cell line was positive. The latter group was tested in the HeLa CD4 ÷ plaque assay, 5 of the 6 subjects who had progressed to AIDS had developed AZT-resistant isolates. However, the level of resistance, as determined in the plaque assay, in all cases was below 1 I~M AZT. These partially resistant isolates were analysed by selective PCR for 4 codons (67, 70, 215 and 219) on RT (Boucher et al., 1992). All isolates lacked mutations at codon 219 and only one had a mixed population of wild type and mutant at codon 67. This indicates that in the studied group of individuals with less than 500 CD4 ÷ per ~I cells who started AZT therapy while still asymptomatic, disease progression can occur in the presence of partially resistant isolates. The group of non-progressors, who stayed relatively stable with regards to their clinical situation as well as to the level of CD4 ÷ cells over time, harboured, for the greater majority during this period, isolates which did not grow in the MT2 cell line. In order to get an insight into their level of sensitivity, selective PCR was done for codons 70 and 215 (at 0, 6 and 24 months) because these mutations appear first. In addition, from the last isolate obtained after two years of treatment, codons 67 and 219 were also analysed. No significant differences were found between progressors and non-progressors in the prevalence of mutations at either codon 70 or 215, at any time. Moreover, after approximately 2 years of treatment, none of the isolates of the stable group had mutations at codon 67 or 219, indicating that these isolates are only partially resistant as well. Thus, the pattern of mutation development in asymptomatic treated individuals over time is not significantly different between the group which develop disease rapidly and those which stay healthy. This does not rule out the fact that resistant isolates are involved in progression to disease in asymptomatic individuals. During 2-year treatment, all individuals developed partially resistant isolates, and virus could be recovered from one million PBMC during treatment. This indicates that during treatment with 1,000 mg AZT, replication of HIV is not completely inhibited. Thus, it might be that partially resistant isolates are a prerequisite for progression. However, they cannot account for the different rates of disease progression in the cohort we analysed. Additional factors are in-
135
volved in modulating the length of the period between the start of treatment and the development of AIDS. Longitudinal studies have shown that in approximately 50 % of the untreated individuals, a switch towards more rapidly growing syncytium-inducing (SI) HIV isolates can be observed before AIDS development. Therefore, we analysed these viral characteristics in our treated cohort described above and included 6 more individuals who entered the trial later. Prior to this, the viral characteristics associated with progression of the disease were studied by cocultivation of patient PBMC with donor PBMC. During this co-cultivation, cultures were monitored for the development of a cytopathoiogical effect (the presence of syncytia). In addition, established isolates were tested for the capacity to grow in different T-cell lines. An alternative assay was used in our cohort, PBMC of patient were directly co-cultivated with cells of the MT2 cell line and screened for syncytia. It appeared that both approaches resulted in similar results. Thus, detection of syncytia with the MT2 assay is an easy and convenient assay for the determination of the virulence of clinic HIV isolates. After a follow-up of 3 years, the following results were obtained. Twelve individuals persistently harboured isolates which did not grow in the MT2 cell line; of this group, only one showed disease progression. Of 8 individuals who showed a change towards syncytium-inducing MT2-positive isolates, 4 developed disease at a mean of 75 weeks (72-77 weeks) after the detection of SI/MT2 + isolates. All 4 individuals who entered the trial with SI/MT2 + isolates developed disease after 94 weeks of treatment (range 60-134 weeks). Thus, these data clearly show that "in vitro virulence" is one of the factors determining the length of the asymptomatic period during treatment. Two groups of asymptomatic individuals emerge from these studies. Individuals who had or who developed SI/MT2 + isolates progressed to AIDS rapidly. The other group of individuals with persistently NSI/MT2- isolates did not show disease progression. In both groups, only partially resistant isolates were encountered. Thus, it can be concluded that, at least for individuals with SI isolates, partial resistance seems sufficient for disease progression. On the other hand, disease progression in treated individuals with MT2- isolates seems a rarer event. A follow-up of this group is particularly important to determine which is more important for progression: a switch in virulence of the virus or the development of high resistance to zidovudine. In other words: will disease develop in the presence of partially resistant, MT2-/NSI (non-SI) isolates or is the development of high resistance required for disease progression in the setting of MT2- viruses ? Alternatively, independent of the level of resistance,
136
4th F O R U M I N V I R O L O G Y
development of MT2-tropic isolates might be an absolute requirement.
In conclusion, at present all asymptomatic individuals develop partial AZT-resistant isolates during treatment. Partially resistant, MT2-tropic syncytium-inducing isolates seem to be sufficient for inducing rapid progression to disease. For individuals with non-MT2-tropic isolates, a longer follow-up is required. The question to be answered for this group is which event will herald progression, the development of high resistance or conversion to MT2-tropic viruses.
References Boucher, C.A.B., Tersmette, M., Lange, J.M.A., Kellam, P., de Goede, R.E.¥., Mulder, J.W., Darby, (3.,
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., Ramautarsing, C., Kellam, P., Darby, G., Lange, J.M.A., Goudsmit, J. & Larder, B.A. (1992), Ordered appearance of zidovudine (AZT) resistance mutations during treatment. J. infect. Dis., 165, 105-110. Larder, B.A., Darby, G. & Richman, D.D. (1989), HIV 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, 1155-1158. Larder, B.A., Kellam, P. & Kemp, S.D. (1991), Zidovudine resistance predicted by direct detection of mutations in DNA from HIV-infected lymphocytes. AIDS, 5, 137-144.
Molecular aspects of AZT resistance in HIV1 P. Levantis and J.S. O x f o r d
Department o f Academic Virology, Medical Microbiology, The London Hospital Medical College, Whitechapel, London E1 2AD (UK)
The problems of antiviral chemotherapy: the need for rational drug design The use of xenobiotic compounds in the treatment of viral infections has, up to the present, incurred two fundamental problems. 1) Selectivity ofaction. - - With the exception of acyclovir in the treatment of herpes simplex virus infection, most man-made chemotherapeutic agents arrest virus propagation by the inhibition of transcription or translation of viral genetic material, and may affect host cell/function in the same manner. Thus, antiviral treatments for communicable diseases may, of necessity, be a severe form of therapy for the individual, being more akin to the use of anti-cancer agents than the use of antibiotics for bacterial infection. 2) Development o f drug resistance. - - The emergence of resistance to antiviral agents has been well documented for viruses as diverse as influenza (Ox-
ford and Potter, 1973) and herpes simplex virus (Field et al., 1980), and has been regarded as concomitant with drug therapy. In consideration of these two effects of antiviral chemotherapy, detailed study of the interaction of drugs with their virus targets is necessary to monitor both their effects on the host and their role in the emergence of resistant strains in the presence of drug selection. At present, these considerations are of paramount importance in the treatment of AIDS with nucleoside analogues such as azidodeoxythymydine, AZT (Yarchoan et aL, 1986; Richman et ai., 1987 ; Fischl et al., 1987), dideoxyinosine (DDI) (Yarchoan et al., 1989) and dideoxycytidine (DDC) (Brandi et al., 1991). All three antiviral compounds act as DNAchain synthesis terminators in a reaction catalysed by the reverse transcriptase enzyme (RT) encoded by the pol gene of HIVI, the principal aetiologic agent causing AIDS and AIDS-related complex. Azidothymi-
134
Clinical significance of zidovudine-resistant human immunodeficiency viruses C . A . B . Boucher
Antiviral Treatment Laboratory~Department o f Medical Microbiology, Academic Medical Center, University o f Amsterdam, Meibergdreef 15, 1105 A Z Amsterdam
Introduction Treatment of individuals infected with the human immunodeficiency virus (HIV) with zidovudine (AZT) was started only 5 years ago. The first report on the emergence of AZT-resistant strains originates from 1989. Thus the available knowledge on the relation between the emergence of AZT-resistant isolates and clinical progression is limited. However, certain conclusions can be drawn from the available data.
The appearance of drug-resistant isolates In the first publication of zidovudine-resistant isolates by Larder et ak (1989) a distinction was made between partially resistant isolates and "high-level" or fully resistant isolates based on the in vitro sensitivity. Based on the 50 % inhibitory concentration (IC50), as determined in the HeLa CD4 + plaque assay, all isolates with an ICs0 ranging between 0.005 and 0.01 ~.M AZT (corresponding to a decrease in sensitivity of a maximum 20-fold) were defined as partially resistant. Isolates with an ICs0 above 0.01 gM AZT were said to have high-level resistance; they showed a decrease in sensiti~vity ranging from 20- to 100-fold. Sequence analysis of the genes encoding for the reverse transcriptase (RT) of these partially and highlevel resistant isolates showed that mutations at 5 codons (41, 67, 70, 215 and 219) were associated with a decrease in AZT sensitivity (Larder and Kemp, 1989). Introduction of these mutations in the RT gene of a molecular HIV clone proved that these mutations caused a decrease in AZT sensitivity. In addition, it was noted that a relation existed between the level of resistance and the number and combination of mutations. Mutations at either codon 70 or 215 alone (or in combination) caused partial resistance. For high-level resistance, 4 mutations are required. In addition to codons 70 and 215, in all cases a third mutation was encountered at codon 67; a fourth mutation can occur at either codon 41 or 219.
Therefore by analysing the composition of these 5 codons or a subset, one can make predictions about the level of resistance of a particular isolate. An approach was developed to determine the composition of a codon on RT by making use of the polymerase chain reaction (PCR). (Larder et ai., 1991). In the first step, a part of the RT gene containing the required codon is amplified, using primers which anneal~ to a conserved region. In the second (selective) step, a distinction is made between the wild-type and mutant codon. This is done by performing in parallel two separate PCR on the amplified fragment. In both reactions the same primer binding to the conserved region is added. In addition, to one reaction, a wtldtype primer annealing to the wild codon is added, and in the other reaction, the mutant primer is added. By analysing the products on a gel, it is possible to distinguish between a predominantly wild-type population, a predominantly mutant population or a mixture population.
Relationship between natural history and development of drug resistance To investigate the relationship between the development of disease progression and AZT resistance, we studied a group of 18 HIV-infected homosexual men. They were selected to enter a trial, from a previously described cohort, because they were considered to be at risk for rapid development of disease since they were HIV p24 antigenaemic and because the majority had less than 500 CD4 + cells per microlitre. All received, after an initial period of 2 months when different dosage schemes were used, 1,000 mg AZT per 24 hours (Boucher et al., 1990). During follow up, participants were seen each month and clinically evaluated, and peripheral blood mononuclear cells (PBMC) were stored. After a follow up period of two years, 6 of the 18 participants had progressed to AIDS and 12 were still asymptomatic, confirming results obtained in larger trials that, despite continuous treatment with AZT, disease progression can take place. This raised the question
RESISTANCE TO A N T I V I R A L DRUGS as to whether progression in this group was correlated with a difference in AZT sensitivity. To answer this question, virus was isolated through co-cultivation both with HIV-antibodynegative donor cells and ceils from the MT2 cell line. Three-time points were analysed: start, approximately 6 months later and either at 2 years for the nonprogressors or around the moment of AIDS diagnosis (for the progressors). At all moments, virus could be isolated using the donor PBMC and, as expected and described before, only a subgroup of the cocultivations with the MT2 cell line was positive. The latter group was tested in the HeLa CD4 ÷ plaque assay, 5 of the 6 subjects who had progressed to AIDS had developed AZT-resistant isolates. However, the level of resistance, as determined in the plaque assay, in all cases was below 1 I~M AZT. These partially resistant isolates were analysed by selective PCR for 4 codons (67, 70, 215 and 219) on RT (Boucher et al., 1992). All isolates lacked mutations at codon 219 and only one had a mixed population of wild type and mutant at codon 67. This indicates that in the studied group of individuals with less than 500 CD4 ÷ per ~I cells who started AZT therapy while still asymptomatic, disease progression can occur in the presence of partially resistant isolates. The group of non-progressors, who stayed relatively stable with regards to their clinical situation as well as to the level of CD4 ÷ cells over time, harboured, for the greater majority during this period, isolates which did not grow in the MT2 cell line. In order to get an insight into their level of sensitivity, selective PCR was done for codons 70 and 215 (at 0, 6 and 24 months) because these mutations appear first. In addition, from the last isolate obtained after two years of treatment, codons 67 and 219 were also analysed. No significant differences were found between progressors and non-progressors in the prevalence of mutations at either codon 70 or 215, at any time. Moreover, after approximately 2 years of treatment, none of the isolates of the stable group had mutations at codon 67 or 219, indicating that these isolates are only partially resistant as well. Thus, the pattern of mutation development in asymptomatic treated individuals over time is not significantly different between the group which develop disease rapidly and those which stay healthy. This does not rule out the fact that resistant isolates are involved in progression to disease in asymptomatic individuals. During 2-year treatment, all individuals developed partially resistant isolates, and virus could be recovered from one million PBMC during treatment. This indicates that during treatment with 1,000 mg AZT, replication of HIV is not completely inhibited. Thus, it might be that partially resistant isolates are a prerequisite for progression. However, they cannot account for the different rates of disease progression in the cohort we analysed. Additional factors are in-
135
volved in modulating the length of the period between the start of treatment and the development of AIDS. Longitudinal studies have shown that in approximately 50 % of the untreated individuals, a switch towards more rapidly growing syncytium-inducing (SI) HIV isolates can be observed before AIDS development. Therefore, we analysed these viral characteristics in our treated cohort described above and included 6 more individuals who entered the trial later. Prior to this, the viral characteristics associated with progression of the disease were studied by cocultivation of patient PBMC with donor PBMC. During this co-cultivation, cultures were monitored for the development of a cytopathoiogical effect (the presence of syncytia). In addition, established isolates were tested for the capacity to grow in different T-cell lines. An alternative assay was used in our cohort, PBMC of patient were directly co-cultivated with cells of the MT2 cell line and screened for syncytia. It appeared that both approaches resulted in similar results. Thus, detection of syncytia with the MT2 assay is an easy and convenient assay for the determination of the virulence of clinic HIV isolates. After a follow-up of 3 years, the following results were obtained. Twelve individuals persistently harboured isolates which did not grow in the MT2 cell line; of this group, only one showed disease progression. Of 8 individuals who showed a change towards syncytium-inducing MT2-positive isolates, 4 developed disease at a mean of 75 weeks (72-77 weeks) after the detection of SI/MT2 + isolates. All 4 individuals who entered the trial with SI/MT2 + isolates developed disease after 94 weeks of treatment (range 60-134 weeks). Thus, these data clearly show that "in vitro virulence" is one of the factors determining the length of the asymptomatic period during treatment. Two groups of asymptomatic individuals emerge from these studies. Individuals who had or who developed SI/MT2 + isolates progressed to AIDS rapidly. The other group of individuals with persistently NSI/MT2- isolates did not show disease progression. In both groups, only partially resistant isolates were encountered. Thus, it can be concluded that, at least for individuals with SI isolates, partial resistance seems sufficient for disease progression. On the other hand, disease progression in treated individuals with MT2- isolates seems a rarer event. A follow-up of this group is particularly important to determine which is more important for progression: a switch in virulence of the virus or the development of high resistance to zidovudine. In other words: will disease develop in the presence of partially resistant, MT2-/NSI (non-SI) isolates or is the development of high resistance required for disease progression in the setting of MT2- viruses ? Alternatively, independent of the level of resistance,
136
4th F O R U M I N V I R O L O G Y
development of MT2-tropic isolates might be an absolute requirement.
In conclusion, at present all asymptomatic individuals develop partial AZT-resistant isolates during treatment. Partially resistant, MT2-tropic syncytium-inducing isolates seem to be sufficient for inducing rapid progression to disease. For individuals with non-MT2-tropic isolates, a longer follow-up is required. The question to be answered for this group is which event will herald progression, the development of high resistance or conversion to MT2-tropic viruses.
References Boucher, C.A.B., Tersmette, M., Lange, J.M.A., Kellam, P., de Goede, R.E.¥., Mulder, J.W., Darby, (3.,
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., Ramautarsing, C., Kellam, P., Darby, G., Lange, J.M.A., Goudsmit, J. & Larder, B.A. (1992), Ordered appearance of zidovudine (AZT) resistance mutations during treatment. J. infect. Dis., 165, 105-110. Larder, B.A., Darby, G. & Richman, D.D. (1989), HIV 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, 1155-1158. Larder, B.A., Kellam, P. & Kemp, S.D. (1991), Zidovudine resistance predicted by direct detection of mutations in DNA from HIV-infected lymphocytes. AIDS, 5, 137-144.
Molecular aspects of AZT resistance in HIV1 P. Levantis and J.S. O x f o r d
Department o f Academic Virology, Medical Microbiology, The London Hospital Medical College, Whitechapel, London E1 2AD (UK)
The problems of antiviral chemotherapy: the need for rational drug design The use of xenobiotic compounds in the treatment of viral infections has, up to the present, incurred two fundamental problems. 1) Selectivity ofaction. - - With the exception of acyclovir in the treatment of herpes simplex virus infection, most man-made chemotherapeutic agents arrest virus propagation by the inhibition of transcription or translation of viral genetic material, and may affect host cell/function in the same manner. Thus, antiviral treatments for communicable diseases may, of necessity, be a severe form of therapy for the individual, being more akin to the use of anti-cancer agents than the use of antibiotics for bacterial infection. 2) Development o f drug resistance. - - The emergence of resistance to antiviral agents has been well documented for viruses as diverse as influenza (Ox-
ford and Potter, 1973) and herpes simplex virus (Field et al., 1980), and has been regarded as concomitant with drug therapy. In consideration of these two effects of antiviral chemotherapy, detailed study of the interaction of drugs with their virus targets is necessary to monitor both their effects on the host and their role in the emergence of resistant strains in the presence of drug selection. At present, these considerations are of paramount importance in the treatment of AIDS with nucleoside analogues such as azidodeoxythymydine, AZT (Yarchoan et aL, 1986; Richman et ai., 1987 ; Fischl et al., 1987), dideoxyinosine (DDI) (Yarchoan et al., 1989) and dideoxycytidine (DDC) (Brandi et al., 1991). All three antiviral compounds act as DNAchain synthesis terminators in a reaction catalysed by the reverse transcriptase enzyme (RT) encoded by the pol gene of HIVI, the principal aetiologic agent causing AIDS and AIDS-related complex. Azidothymi-