3rd International Workshop on HIV Drug Resistance Workshop

Kauai, Hawaii — 2–5 August 1994

P1	INTRODUCTORY TALK: MODELLING HIV GENETIC VARIATION Int Wkshop HIV Drug Res 1994 Aug 2-5;3:1 John M. Coffin Most analyses (such as the in situ PCR) provide only snapshots of the dynamic processes. However, the rapid and dramatic effects of non-nucleoside RT inhibitors on HIV populations in vivo may well provide the key information necessary to resolve this problem, and approaches to this kind of analysis will be presented.
*Session 1: IN VITRO* RESISTANCE TO PROTEASE INHIBITORS** Co-chairs: Emilio Emini and Mike Otto Abstracts 1 thru 10, Pages 2 to 11
1	STRUCTURAL BASIS OF DRUG RESISTANCE TO HIV-1 PROTEASE INHIBITORS Int Wkshop HIV Drug Res 1994 Aug 2-5;3:2 (abstract 1 J.W. Erickson, E.T. Baldwin, T.N. Bhat, S. Gulnik, B. Liu, B. Yu Comparison of the wild type and mutant enzyme complexes revealed asymmetric structural effects on enzyme/inhibitor interactions. Main chain shifts in the Sl subsite residues 79-84 resulted in a repacking of mutant enzyme and inhibitor atoms in an unexpected fashion. These structural results suggest strategies for the re-design of drugs that will specifically target subsite mutants of HIV PR.
2	BIOCHEMICAL BASIS AND CONSEQUENCES OF ACTIVE SITE MUTATIONS IN THE HIV-l ASPARTYL PROTEASE Int Wkshop HIV Drug Res 1994 Aug 2-5;3:3 (abstract 2) D.J. Livingston, S. Pazhanisamy, J.A. Partaledis, C. Stuver, A. Cullinan, O. Futer, S.P. Chambers, J.R. Fulghum and R.D. Tung The changes in kinetic constants, as well as their activation energy, are dependent on the peptide substrate used in the assay, where each peptide substrate corresponds to a different gag-pol cleavage sequence. Finally, the effect of alterations on protease catalytic efficiency on HIV-l viral infectivity and replication rate have been examined in collaboration with Dr R. Byrn at the New England Deaconess Hospital, Boston, MA, USA.
3	IN VITRO SELECTION AND CHARACTERIZATION OF HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 (HIV-1) VARIANTS WITH DECREASED SENSITIVITY TO HYDROXYETHYLUREA ISOSTERE CONTAINING PROTEASE INHIBITORS Int Wkshop HIV Drug Res 1994 Aug 2-5;3:4 (abstract 3) Karen E. Potts¹, Mary L. Smidt¹, William C. Stallings Jr.¹, Michael Clare², Deenan Pillay³, Douglas D. Richman³ and Martin L. Bryant¹ Subtle perturbations of essential H-bon interactions among Asn88, Thr 31, Thr 74 and Asp29 could in turn influence the S2/S2´ inhibitor binding subsites. Other amino acid changes (positions 10, 46, 53 and 71) were found in some, but not all, selected resistant variants. The significance of the observed changes and their ability to confer cross resistance to other asymmetric protease inhibitors is currently under investigation.
4	DEFINED MUTATIONS IN THE HIV PROTEASE GENE CONFER REDUCED SUSCEPTIBILITY TO STRUCTURALLY DISSIMILAR HIV PROTEASE INHIBITORS Int Wkshop HIV Drug Res 1994 Aug 2-5;3:5 (abstract no. 4) D.L. Winslow, E.D. Anton, R.A. Horlick, R.J. Zagursky, R.J. Tritch, H. Scarnati, K. Ackerman, R. Buckery, and L.T. Bacheler These data show potentially significant cross-resistance between cyclic urea, linear diol, asymmetric mono-ol, and hydroxyethylene isostere protease inhibitors.
5	MULTIPLE SEQUENCE CHANGES WITHIN THE HIV-1 PROTEASE CONFER REDUCED SENSITIVITY TO A SYMMETRIC PROTEASE INHIBITOR Int Wkshop HIV Drug Res 1994 Aug 2-5;3:6 (abstract no. 5) Ron Swanstrom, Terri Smith, Steve Pettit, David Irlbeck, Wei Shao, Robert Wehbie, Ravi Sawhney, Lorri Everitt, and John Erickson* Virus has been selected which can grow in the presence of approximately 100 times the IC₅₀ value of inhibitor compared to the unmutated parental virus. Sequence changes associated with these two selections will be presented. These results suggest that virus evolution to higher level resistance can occur.
6	MODELING STUDIES. ON RESISTANCE USING STRUCTURAL DATA ON HIV-l PROTEASE – INHIBITOR COMPLEXES Int Wkshop HIV Drug Res 1994 Aug 2-5;3:7 (abstract no. 6) B. Govinda Rao, Eunice E. Kim, Joshua Boger, Mark A. Murcko, and Manuel A. Navia These mutants were chosen because: (1) these residues are in the middle of the active site and are in direct contact with all the three inhibitors; (2) the changes are minimal and appropriate for testing these methods, which use static coordinates of the enzyme-inhibitor complexes; and (3) experimental Ki changes of some of these mutants are available for comparison. The results are displayed graphically to provide visual summary of possible susceptibility of the enzyme's active site residues to mutation in the presence of an inhibitor.
7	IN VITRO SELECTION AND CHARACTERIZATION OF HIV-l VIRAL ISOLATES WITH REDUCED SENSITIVITY TO INHIBITORS OF HIV PROTEASE Int Wkshop HIV Drug Res 1994 Aug 2-5;3:8 (abstract no. 7) J.A. Partaledis, K. Yamaguchi, R. A. Byrn* and D.J. Livingston* The proviral DNA region encoding the VB-11,328-resistant HIV-1 protease was amplified from infected cell lysates by PCR and was sequenced directly. The amino acid substitutions identified within protease were L10F, M46I, I47V, I50V and I84V. The biochemical properties of these mutants are under investigation.
8	MUTATIONS CONFERRING VIRAL RESISTANCE TO HIV-1 PROTEASE INHIBITORS CAN LIE OUTSIDE, AS WELL AS WITHIN, THE ENZYME ACTIVE SITE & BINDING POCKETS Int Wkshop HIV Drug Res 1994 Aug 2-5;3:9 (abstract no. 8) B. Maschera, C. Blance, D. Brown & E.D. Blair The crystal structure of wild-type HIV-1 protease with A75925 (Brookhaven Database) shows that the V32I change affects the interaction of enzyme with the valine side chains of the inhibitor in P2 and P2´, and allows us to predict that V32I would also be cross-resistant to similar C2 inhibitors, e.g. P9941 (DuPont Merck), A-77003. Proteases containing V32I and others mutations have been expressed in E. coli and appear to self-process as well as wild-type enzyme. We await biochemical and structural data to confirm our modelling of the atomic basis of HIV drug resistance.
9	MOLECULAR CHARACTERIZATION OF HIV-1 VARIANTS RESISTANT TO SPECIFIC VIRAL PROTEASE INHIBITORS Int Wkshop HIV Drug Res 1994 Aug 2-5;3:10 (abstract no. 9) D, Lamarre, G, Croteau, L. Pilote, P. Rousseau and L. Doyon The relevance of the different mutations to the resistance phenotype will be discussed. These data suggest that emergence of resistance may occur in vivo with HIV-1 protease inhibitors but the relevance of these observations to the clinical setting remains to be established.
10	A METHOD FOR CONSTRUCTION OF INFECTIOUS MOLECULAR CLONES OF HIV-1 CONTAINING DEFINED MUTATIONS IN THE HIV PROTEASE GENE Int Wkshop HIV Drug Res 1994 Aug 2-5;3:11 (abstract no. 10) E.D. Anton, L.T. Bacheler, R.A. Horlick, R.J. Zagursky, R.J. Tritch and D.L. Winslow Infectious recombinant virus is then recovered by cotransfecting 5´ and 3´ half-virus plasmids linearized at their common Ncol sites into mammalian cells. This method was successfully applied to constructing viruses containing various substitutions in HIV protease.
*SESSION 2: PROTEASE INHIBITORS: CROSS RESISTANCE & IN VIVO* OBSERVATIONS** Co-chairs: John Erickson and Doug Mayers Abstract 11 thru 18, Pages 12 to 19
11	AN HIV-1 (RF) VARIANT ENCODING A PROTEASE WITH V82F AND 184V MUTATIONS EXHIBITS RESISTANCE TO CYCLIC UREA AND C₂ LINEAR DIOl PROTEASE INHIBITORS Int Wkshop HIV Drug Res 1994 Aug 2-5;3:12 (abstract no. 11) Robert W. King, Sena Garber, Carol D. Reid and Michael J. Otto When this isolate was tested for its susceptibility to several protease inhibitors, it exhibited a >50-fold decrease in its susceptibility to XM323 as well as a 3-fold decrease in susceptibility to P9941, a C₂ symmetrical diol. These mutations did not alter the susceptibility of the variant to the Roche inhibitor, R031-8959, or to the Abbott inhibitor, A-8098.
12	PATTERNS OF SPECIFIC MUTATIONS IN HIV-1 PROTEASE THAT CONFER RESISTANCE TO PROTEASE INHIBITORS IN CLINICAL DEVELOPMENT Int Wkshop HIV Drug Res 1994 Aug 2-5;3:13 (abstract no. 12) H. Mo, M. Markowitz, and D.D. Ho Certain amino acids in protease seemed to be hot spots for conferring resistance, including but not limited to residues 8, 48, 82, and 84. Furthermore, our early findings suggest that the protease inhibitors currently under clinical development fall into several different groups based on their resistance patterns, raising the notion that specific combinations of these agents could be useful in combating the resistance problem.
13	COMPREHENSIVE ANALYSIS OF HIV-1 VARIANTS INDIVIDUALLY SELECTED FOR RESISTANCE TO SIX HIV PROTEASE INHIBITORS Int Wkshop HIV Drug Res 1994 Aug 2-5;3:14 (abstract no. 13) M. Tisdale, R. Myers, N.R. Parry, N. Oliver, B. Maschera, and E. Blair Virus is currently being passed in the combined presence of 2 protease inhibitors in cell culture in an attempt to determine possible constraints on resistance development. In vitro resistance studies should provide useful information for future combination trials with protease and RT inhibitors.
14	PHENOTYPIC AND GENOTYPIC CHARACTERIZATION OF HIV-1 VARIANTS SELECTED DURING TREATMENT WITH THE PROTEASE INHIBITOR L-735,524 Int Wkshop HIV Drug Res 1994 Aug 2-5;3:15 (abstract no. 14) E.A. Emini, W.A. Schleif, D. Graham, P. Deutsch, F. Massari, H. Teppler, K. Squires# andJ.H. Condra* Alteration at this residue position was uniformly affiliated with the appearance of resistance. Substitutions at position 82 were introduced into the NL4-3 provirus. However, the derived mutant viruses were not resistant to L-735,524, suggesting that the context into which the substitutions are introduced influences the phenotypic expression of resistance. Studies to define the nature of this influence are in progress.
15	GENOTYPIC CHARACTERISATION OF HIV-1 FROM PATIENTS AFTER PROLONGED TREATMENT WITH PROTEINASE INHIBITOR SAQUINAVIRr Int Wkshop HIV Drug Res 1994 Aug 2-5;3:16 (abstract no. 15) H. Jacobsen¹, F. Brun-Vezinet², I. Duncan³, M. Hänggi¹, M. Ott¹. S. Vella⁴, J. Weber⁵ and J. Mous¹ Mutations which have been previously shown to lead to reduced sensitivity to proteinase inhibitor Saquinavir in vitro could also be detected in vivo after prolonged treatment with high doses of inhibitor.
16	COMBINATION THERAPY WITH TWO PROTEASE INHIBITORS AS AN APPROACH TO ANTIVIRAL THERAPY Int Wkshop HIV Drug Res 1994 Aug 2-5;3:17 (abstract no. 16) B. Rose, J. Greytok, C. Bechtold, M. Alam, B. Terry, Y.-F. Gong, K. DeVore, A. Patick, R. Colonno and P.-F. Lin Genotypic analysis of the RF resistant variants identified several new mutations within the protease gene. The significance of these mutations are currently under study using HIV-1 recombinant viruses. These initial experiments investigating the viability of HIV-1 variants containing more than 2 protease mutations are quite encouraging. They suggest that treatment with 2 protease inhibitors having distinct resistance profiles may be an effective approach to antiviral therapy.
17	LIMITED SEQUENCE DIVERSITY OF THE HIV-1 PROTEASE GENE FROM CLINICAL ISOLATES AND IN VITRO SUSCEPTIBILITY TO HIV PROTEASE INHIBITORS Int Wkshop HIV Drug Res 1994 Aug 2-5;3:18 (abstract no. 17) D.L. Winslow, R. King, S. Stack, H. Scarnati, and M.J. Otto All clinical isolates demonstrated in vitro susceptibility to the protease inhibitors. There was no significant difference between the susceptibility of the reference strains and the clinical isolates. These data suggest that the variable regions of protease, as defined by these isolates, do not contain sites which are critical for interactions with the inhibitors tested.
18	IDENTIFICATION OF A CLINICAL ISOLATE OF HIV-1 FROM NORTHERN THAILAND WITH A NATURALLY OCCURRING ISOLEUCINE AT POSITION 82 OF THE PROTEASE Int Wkshop HIV Drug Res 1994 Aug 2-5;3:19 (abstract no. 18) Robert W. King, Dean L. Winslow, Sena Garber, Helen Scarnati, Sylvia Stack, and Michael J. Otto When HIV-1 (Th 16) was tested for its susceptibility to examples of several classes of protease inhibitors (XM323, Ro31-8959, P9941, and A-80987), it exhibited the same sensitivities as the V82 wild type. Thus, unlike V82A or V82F, the V82I difference does not alter the susceptibility of the virus to these inhibitors of HIV-1 protease.
SESSION 3: DETECTION AND PREVALENCE OF DRUG RESISTANT MUTANTS Co-chairs: Roberta Black and Charles Boucher Abstract 19 thru 27, Pages 20 to 28
19	A NEW METHOD FOR RAPID SEQUENCE ANALYSIS: DETECTION OF HIV REVERSE TRANSCRIPTASE MUTATIONS Int Wkshop HIV Drug Res 1994 Aug 2-5;3:20 (abstract no. 19) M.S. Chee, R. Yang, N. Shen, M. Kozal and T. Gingeras An 831 bp fragment spanning the HIV reverse transcriptase coding sequence was amplifIed by PCR,using primers tagged with T3 and T7 promoter sequences. Labelled RNA was prepared by incorporation of fluorescent nucleotides, fragmented, and hybridized to chips. We analyzed viral clones and clinical samples and were able to identify drug resistance mutations and strain differences.
20	NATURAL OCCURRENCE OF RESISTANCE MUTATIONS IN THE pol GENE OF HIV-1 ISOLATES Int Wkshop HIV Drug Res 1994 Aug 2-5;3:21 (abstract no. 20) I. Najera1^, D.D Richman², I. Olivares¹, J.M. Rojas¹, R Najera1 and C. Lopez-Galindez¹* The appearance of these mutations occurred in the absence of drug-selective pressure, thus pointing out the contribution that the extensive genetic variability plays in the frequent emergence of HIV-1 RT inhibitors-resistance.
21	SEXUAL TRANSMISSION OF ZIDOVUDINE RESISTANT HIV-1 Int Wkshop HIV Drug Res 1994 Aug 2-5;3:22 (abstract no. 21) A. Imrie¹, A. Carr¹, C. Duncombe², J. Vizzard³, N. Zheng¹, R. Finlayson⁴, L. Hurren¹, M. Roggensack¹, J. Kaldor³, R. Penny¹, David A.Cooper^1,3, and the Sydney Primary HIV Infection Study Group Despite ZDV therapy for treatment of PHI, HIV-1 wild-type at codon 215 had replaced the resistant variants within 3 months of transmission.This rate of transmission (1/27, 3.7%) is not significantly different at the 0.05 level from the proportion of people living withmv-1 infection in Australia who were estimated to have developed zidovudine resistance. 12 months after seroconversion this patient is clinically well, with category A2 HIV disease.
22	IN VITRO GENERATION AND CHARACTERISATION OF FOSCARNET-RESISTANT HIV-1 Int Wkshop HIV Drug Res 1994 Aug 2-5;3:23 (abstract no. 22) G. Tachedjian, A. Gurusinghe, D. Hooker, N. Deacon, J. Mills and C. Birch* Compared to foscarnet-sensitive HIV-1, variants with the 89 or 92 changes showed hypersensitivity to azido-based nucleoside analogues and non-nucleoside reverse transcriptase inhibitors, while identical sensitivity profiles were observed in the presence of ddC and ddI. These results and the potential for Fos-R virus to arise in vivo, have important implications in treatment strategies involving foscarnet.
23	NOVEL MUTATIONS IN HIV-1 REVERSE TRANSCRIPTASE CONFER RESISTANCE TO FOSCARNET IN LABORATORY AND CLINICAL ISOLATES Int Wkshop HIV Drug Res 1994 Aug 2-5;3:24 (abstract no. 23) J. Mellors¹, H. Bazmi¹, J. Weir², E. Arnold³, R. Schinazi⁴, D. Mayers² Foscarnet (FOS) is a DNA polymerase inhibitor with in vivo activity against CMV and HIV-l. Resistance of HIV-1 to FOS has not been reported despite long-term FOS therapy of HIV-infected patients with CMV infection.
24	RAPID AND SIMPLE METHOD TO DETERMINE DRUG SUSCEPTIBILITY OF CLINICAL HIV-1 ISOLATES Int Wkshop HIV Drug Res 1994 Aug 2-5;3:25 (abstract no. 24) Nicole K.T. Back, Toon van Bommel, Dorien de Jong, Pauline Schipper, Menno de Jong and Charles A.B. Boucher The combination of the recombinant virus assay using a syncytium inducing deletion clone (HXB2RT) with the MTT assay provides a rapid and simple format with automated read-out for drug susceptibility testing of large numbers of clinical samples.
25	RAPID SCREEN OF CLINICAL SPECIMENS FOR DRUG RESISTANT HIV PHENOTYPES DURING VIRUS ISOLATION Int Wkshop HIV Drug Res 1994 Aug 2-5;3:26 (abstract no. 25) Douglas L. Mayers, J. Lane^* and O.S. Weislow^* This initial study suggests the RDPS will permit differentiation of ZDV-sensitive and resistant HIV phenotypes with results in 2-3 weeks. HIV isolates which produce p24Ag levels >25% no drug control in the presence of 0.2 µM ZDV in this assay should have IC₅₀ values ≥1.0 µM using the ACTG/DoD assay.
26	A QUANTITATIVE POINT MUTATION ASSAY: PERFORMANCE PARAMETERS AND APPLICATION TO THE ASSAY OF GENOTYPIC DRUG RESISTANCE Int Wkshop HIV Drug Res 1994 Aug 2-5;3:27 (abstract no. 26) S. Kaye, C. Loveday, E. Comber and R.S. Tedder The samples assayed have been taken from subjects on open therapy and participants in controlled trials of both monotherapy and combination therapy. The assay fulfils the criteria needed for the monitoring of genotypic drug resistance in clinical material, is formatted to conveniently handle large numbers of samples and is easily adapted to the assay of many mutations.
27	A COMPARISON OF CULTURE AND PCR FOR THE QUANTIFICATION OF HIV-1 LOAD AND ZIDOVUDINE (ZDV) RESISTANCE IN PATIENTS FROM THE CONCORDE TRIAL Int Wkshop HIV Drug Res 1994 Aug 2-5;3:28 (abstract no. 27) Clive Loveday on behalf MRC UK Virology Working Party The prevalence of codon mutations in serum RNA, tissue culture RNA and proviral DNA from PBMC were ranked by ZDV IC₅₀ RNA genomic resistance showed a correlation with phenotypic resistance, the mutations in RNA preceded that in DNA. The genotypic resistance in RNA derived from tissue culture demonstrated closer ranking with IC₅₀. This study shows good correlation between viral culture and molecular technologies for the quantification of HIV-1 load and ZDV resistance.
SESSION 4: STRUCTURAL & FUNCTIONAL CONSEQUENCES OF RT INHIBITOR RESISTANCE Co-chairs: Rich D’Aquila and Brendan Larder Abstract 28 thru 37, Pages 29 to 38
28	HIGH RESOLUTION REFINED STRUCTURES OF HIV-1 REVERSE TRANSCRIPTASE-INHIBITOR COMPLEXES: IMPLICATIONS FOR DRUG RESISTANCE Int Wkshop HIV Drug Res 1994 Aug 2-5;3:29 (abstract no. 28) D.I. Stuart^†, J.S. Rent^†, R. Esnouf^†, E.Y. Jones^†, C. Ross^, D. Somers^ and D.K. Stammers^* This has lead to a refined structure at 2.2 Å with R=O.187. The four inhibitors bind in a common site and adopt very similar overlapping conformations inspite of being chemically different. The location of residues that undergo mutation to give rise to resistance are in close proximity to the inhibitors.
29	IMPLICATIONS OF THE THREE-DIMENSIONAL STRUCTURE OF HIV-1 REVERSE TRANSCRIPTASE FOR RESISTANCE TO ANTIVIRAL DRUGS Int Wkshop HIV Drug Res 1994 Aug 2-5;3:30 (abstract no. 29) Edward Arnold¹, Christopher Tantillo¹, Paul Boyer², Jianping Ding¹ , Birgit M. Roy¹, Arthur D. Clark Jr.¹, Rudy Pauwels³, Koen Andries³, Paul A.J. Janssen³, John Mellors⁴, Nicholas Deacon⁵ and Stephen H. Hughes² An analysis of the locations of mutations that confer resistance to phosphonoformate (Mellors et al., unpublished) suggests that resistance to PFA may occur through a similar mechanism as with nucleoside analogs. We are pursuing the crystal structure determination of HIV-1 RT mutants to illuminate the structural basis of drug resistance.
30	MECHANISMS OF RESISTANCE OF HIV-1 RT TO NUCLEOSIDE AND NON-NUCLEOSIDE INHIBITORS Int Wkshop HIV Drug Res 1994 Aug 2-5;3:31 (abstract no. 30) P.L. Boyer¹, C. Tantillo², J. Ding², A.L. Ferris¹, P. Clark³, E. Arnold², and S.H. Hughes¹ We have studied two mutations, Leu 74Val and Glu89Gly, which confer resistance only to dideoxy nucleoside triphosphates. Neither of these amino acid positions are near the polymerase active site; however, both are in positions where they appear to interact with nucleic acid. Biochemical analyses suggest that both mutations affect the precise positioning of the template/prime and, as a consequence, affect the ability of the enzyme to discriminate between a deoxy and a dideoxy nucleoside triphosphate.
31	REDUCED IN VITRO POLYMERASE ACTIVITY OF 3TC-RESISTANT REVERSE TRANSCRIPTASE ENZYMES Int Wkshop HIV Drug Res 1994 Aug 2-5;3:32 (abstract no. 31) Nicole K.T. Back, Monique Nijhuis, Charles A.B. Boucher, Dorien de Jong, Rob Schuurman, Belinda Oude Essink, and Ben Berkhout These results suggest that 3TC-resistant RT is less processive in vitro. The M184V RT protein was slightly more active in the polyA: dT assay compared to the M184I variant, consistent with the in vivo outgrowth of the M184V virus.
32	IN VITRO STUDIES WITH 3TC-RESISTANT HIV-1: GROWTH·PROPERTIES AND CROSS-RESISTANCE PROFILE Int Wkshop HIV Drug Res 1994 Aug 2-5;3:33 (abstract no. 32) S.D. Kemp and B.A. Larder As 3TC-resistant virus remains susceptible to AZT we have attempted to select dual resistant virus. Passage of the AZT-resistant cloned strain HIVRTMN (41L+215Y) in 3TC alone, resulted in selection of highly 3TC resistant virus, with restored AZT sensitivity, presumably due to suppression by mutation at codon 184. Similar passage experiments in the presence of both 3TC and AZT have so far failed to select dual resistance. These data suggest that both 3TC and AZT may be good candidates for combination therapy.
33	IN VITRO SELECTION OF HIV-1 VARIANTS RESISTANT TO 3TC AND A SECOND REVERSE TRANSCRIPTASE INHIBITOR Int Wkshop HIV Drug Res 1994 Aug 2-5;3:34 (abstract no. 33) N. Cammack, M.G. Goulden, K.C. Viner, P. Hopewell, C.R. Penn and J.M. Cameron This HIV-1 variant demonstrated 3TC™-resistance with restored susceptibility to AZT as has previously been described. An HIV-1 variant passaged in nevirapine with maintained 3TC™ drug pressure revealed a virus with demonstrable phenotypic resistance to both drugs. Genotypic characterisation of these HIV-1 variants is under way and will be reported. The results have implications for the treatment of patients with a combination of 3TC™ and other HIV inhibitors.
34	BIOCHEMICAL ANALYSIS OF HIV-1 REVERSE TRANSCRIPTASE WITH MUTATIONS AT MET 184 IN THE HIGHLY CONSERVED YMDD REGION Int Wkshop HIV Drug Res 1994 Aug 2-5;3:35 (abstract no. 34) JE Wilson^, TB Caligan^, JL Martin^, A. Aulabaugh^, LL Wright^, S McPherson^, JK Wakefield^, S Jablonski^, CD Morrow^, and PA Funnan^ The K_i values exhibited by the corresponding 1-ß-L configuration were elevated a 100- to 1000-fold under equivalent conditions. The origin of the extreme stereochemical dependence of substrate recognition and utilization by the variant enzyme is presently under study.
35	MECHANISMS OF ddC RESISTANCE OF THE K65R SUBSTITUTION IN HIV-1 REVERSE TRANSCRIPTASE Int Wkshop HIV Drug Res 1994 Aug 2-5;3:36 (abstract no. 35) Z. Gu, E. Arts, X. Li, M. Parniak and M.A. Wainberg Recombinant HIV RTs containing only K65R or both the K65R and M184V mutations yielded significantly more (-) strong-stop product in the presence of ddCTP, 3TC-TP and ddATP than did wt HIV-1 RT. A slight decrease in degree of chain termination was observed with each of AZT-TP and ddITP. Altered nucleoside-analog recognition and chain termination are likely involved in drug resistance mechanisms for K65R.
36	THE BIOLOGICAL ROLE AND THERAPEUTIC SIGNIFICANCE OF DNA IN HIV-1 VIRIONS Int Wkshop HIV Drug Res 1994 Aug 2-5;3:37 (abstract no. 36) P. Krogstad¹, J.A. Zack², and I.S.Y. Chen³ Limiting dilution analysis revealed no difference in the infectivity of virus stocks produced in the presence or absence of azidothymidine. We conclude that reverse transcriptase inhibitors interfere with virus replication by inhibiting de novo synthesis of viral DNA occuring after virions enter PBL, and that virion-associated DNA appears to play, at most, a minor role in the HIV-1 lifecycle.
37	RETROVIRAL RECOMBINATION CAN LEAD TO LINKAGE OF REVERSE TRANSCRIPTASE MUTATIONS THAT CONFER INCREASED ZIDOVUDINE RESISTANCE Int Wkshop HIV Drug Res 1994 Aug 2-5;3:38 (abstract no. 37) B.A. Larder and P. Kellam No evidence of de novo generation of mutations at codons 41 or 215 (or the other AZT resistance mutations) was seen. Sensitivity analysis of the passaged virus populations did not show large changes in AZT IC₅₀ values. However, the observed changes were consistent with susceptibility analysis of defined virus mixtures. This study provides an example of the role of retroviral recombination in the development of HIV-1 variants with increased drug resistance.
SESSION 5: NNRTIs Co-chairs: Françoise Brun-Vézinet and Doug Richman Abstract 38 thru 43, Pages 39 to 44
38	SELECTION OF NOVEL RT MUTATIONS UPON CONTINUED EXPOSURE OF NNRTI-RESISTANT HIV-1 MUTANT STRAINS TO NON-NUCLEOSIDE REVERSE TRANSCRIPTASE INHIBITORS (NNRTIs) Int Wkshop HIV Drug Res 1994 Aug 2-5;3:39 (abstract no. 38) J. Balzarini¹ , A. Karlsson², V.V. Sardana³, E.A. Emini³ and E. De Clercq¹ Our data indicate that continued exposure of HIV-1 mutants containing the 181 Tyr → Cys mutation in their RT to high concentrations of NNRTIs result in the emergence of highly resistant HIV-1 mutants that contain novel mutations in the same codon (181 Cys → Ile).
39	SUBGROUPS OF HIV-1-SPECIFIC INHIBITOR-RESISTANT ISOLATES ARE DEFINED BY THEIR RESPECTIVE RESISTANCE-ENGENDERING MUTATIONS Int Wkshop HIV Drug Res 1994 Aug 2-5;3:40 (abstract no. 39) R W. Buckheit Jr. and V. Fliakas-Boltz We have also determined that a given class of compounds (i.e., HEPT) generate mutations which yield isolates belonging to different subgroups. Finally, phenotypic cross-resistance data served to identify specific combinations of compounds which might be therapeutically useful. The results of ongoing studies with selected drug combinations will be presented.
40	PROBING THE MECHANISM FOR THE IN VITRO DRUG RESISTANCE-SENSITIZATION MEDIATED BY THE PROLINE TO LEUCINE CHANGE AT 236 OF HIV-1 REVERSE TRANSCRIPTASE Int Wkshop HIV Drug Res 1994 Aug 2-5;3:41 (abstract no. 40) S.K. Sharma, N. Fan, D.B. Evans, K.B. Rank and W.G. Tarpley^* The studies with the P236 HIV-1 RT mutants demonstrate a direct correlation between sensitization to L-697,661 and resistance to the BHAPs. It is concluded that susceptibility and resistance to these RT inhibitors are the consequence of the precise geometry of the inhibitor-binding pocket.
41	PHENOTYPIC AND GENOTYPIC CHARACTERIZATION OF HIV-1 VIRAL ISOLATES FROM PATIENTS TREATED WITH COMBINED AlT AND DELAVIRDINE MESYLATE (DLV) THERAPY Int Wkshop HIV Drug Res 1994 Aug 2-5;3:42 (abstract no. 41) L.K. Wathen, W.W. Freimuth, D.H. Batts, S.R. Cox With increasing DLV IC₅₀ there was a decrease in AZT IC₅₀, despite the pre-existence of a Thr215Tyr/Asn in 4/14 and the treatment emergence of Lys219Gln in 2/14. Further studies will be needed to determine the time course of change in DLV IC₅₀ and full characterization and clinical significance of the resulting mutations.
42	IN VITRO AND IN VIVO CORRELATES OF ZDV/ddI/NEVIRAPINE THERAPY OF HIV-1 INFECTION Int Wkshop HIV Drug Res 1994 Aug 2-5;3:43 (abstract no. 42) John L. Sullivan¹, Frank Brewster¹, Mohan Somasundaran¹, Thomas Greenough¹, Dan Pauletti², Cheng-Kon Shih², Johanna Griffm², Maureen Myers², Katherine Luzuriaga¹ Reduction in viral load without complete suppression of viral replication was observed both in vitro and in vivo after the introduction of ZDV/NVP/ddI therapy; the rapidity and extent of viral load reduction appeared to be dependent on prior antiretroviral experience. Funded in part by Boehringer-Ingelheirn Pharmaceuticals.
43	IN VITRO EVALUATION OF N-α-ACETYL-NONA-D-ARGININE (ALX40-4C): ANTIVIRAL EFFICACY AND EMERGENCE OF RESISTANCE Int Wkshop HIV Drug Res 1994 Aug 2-5;3:44 (abstract no. 43) B. Conway¹, O. Mpanju¹ , Y. Zheni², S.C. Climie² and M. Sumner-Smith² In conclusion, reduction in viral load without complete suppression of viral replication was observed both in vitro and in vivo after the introduction of ZDV/NVP/ddI therapy; the rapidity and extent of viral load reduction appeared to be dependent on prior antiretroviral experience. Funded in part by Boehringer-Ingelheirn Pharmaceuticals.
SESSION 6: Biological and Clinical Aspects of Resistance During Monotherapy Co-chairs: David Cooper and Mike Ussery Abstract 44 thru 58, Pages 45 to 59
44	ISOLATION, BIOLOGICAL PHENOTYPE AND AZT SUSCEPTIBILITY OF HIV ISOLATED FROM POSTMORTEM TISSUE Int Wkshop HIV Drug Res 1994 Aug 2-5;3:45 (abstract no. 44) S.A. Land, K.M. McGavin, K.L. Sebire and C.J. Birch The isolation rate of HIV from post-mortem tissues was influenced by the site and time to specimen processing. Although the syncytial inducing phenotype is supposedly associated with disease progression, less than one third of the isolates studied induced syncytial formation in MT-2 cells. AZT resistant virus could be isolated from tissues of infected patients treated with the drug. Emergence of phenotypically sensitive virus was seen in the tissues of patients who had ceased drug for several months prior to death.
45	DISCORDANCE OF RT SEQUENCES CONFERRING ZDV RESISTANCE IN PROVIRAL DNA FROM BRAIN AND SPLEEN Int Wkshop HIV Drug Res 1994 Aug 2-5;3:46 (abstract no. 45) J.K. Wong, N.J.S. Fitch, F. Torriani, D. Havlir and D.O. Richman We conclude that the viral populations in brain and spleen can differ in their RT sequences with regards to mutations conferring ZDV resistance. This observation confirms previous data based on env sequence analysis that distinct genotypic and phenotypic populations of virus are maintained in different organs and further, suggests differences in the evolution of ZDV resistance mutations within different tissue compartments.
46	CHARACTERISATION OF HIV ISOLATED FROM PATIENTS ENROLLED IN THE ALPHA ddI TRIAL Int Wkshop HIV Drug Res 1994 Aug 2-5;3:47 (abstract no. 46) N.N. Zheng¹, S. Simasathiansophan¹, P.W. McQueen², L. Hurren², L. Evans³, S.F. Delaney¹, R. Penny² and D.A. Cooper³ A high percentage of NSI isolates at week 12 suggests that ddI may be useful for delaying the emergence of SI virus during the early stages of therapy. Although trends for individuals patients varied, in general there was stability of ZDV sensitivity and an increase in ddI resistance with time. This resistance may be conferred by novel mutations as well as known mutations at codons 74 and 184.
47	RELATIONSHIP OF SI/NSI STATUS TO DEVELOPMENT OF ZDV RESISTANCE AND CLINICAL OUTCOMES IN PATIENTS ON ZDV THERAPY Int Wkshop HIV Drug Res 1994 Aug 2-5;3:48 (abstract no. 47) Douglas L. Mayers, K.F. Wagner, R.C.Y. Chung, M.T. Vahey, O.S. Weislow, J.L. Lennox, S.Y.J. Zhou, D.S. Burke and the RV 43 STUDY GROUP SI phenotype has been associated with more rapid CD4 decline and rapid emergence of ZDV resistance in HIV-infected patients. There was no significant relationship of SI phenotype with either development of new 4 OIs or death in this cohort of patients. These anomalous results need to be reconciled to understand the mechanisms of clinical failure on ZDV monotherapy.
48	AZT RESISTANCE: A CASE-CONTROL STUDY IN THE CONCORDE TRIAL Int Wkshop HIV Drug Res 1994 Aug 2-5;3:49 (abstract no. 48) F. Brun-Vézinet, S. Kaye^, F. Ferchal, C. Loveday^, C. Buffet-Janvresse, R. Tedder^, J.H. Darbyshire†, J.P. Aboulker‡* The analysis of this case-control study, based on the PCR results, showed a significant association (p=0.001) between emergence of mutation at codon 215 and clinical progression. Resistant viral populations were detected at least 6-9 months before the clinical event. Statistical analyses of the correlation between AZT resistance and clinical progression, adjusted on CD4 cell counts and viral load will be presented.
49	NOVEL MUTATIONS ASSOCIATED WITH A RAPID DECLINE IN CD4 CELLS COUNT Int Wkshop HIV Drug Res 1994 Aug 2-5;3:50 (abstract no. 49) Ruiz, L.¹, Erice, A.², Gómez, M.¹, Sannerud, K.² and B. CLotet¹ The appearance of mutations, not associated to ZDV resistance, can influence the CD4 cells count and a more rapid progression of the disease. This could be related to different parameters, amongst which could be cellular factors particular to each host, which regulate the replication of HIV, together with cellular and/or humoral immunity, etc. Its expression as poorer prognosis of the disease shows us once again that it does not only depend on the type of strain that is transmitted, but also depends on the immunity and cellular mechanisms of the host. It is necessary to sequence the gene which codifies for RT in a large number of infected patients to estimate the real significance of all the mutations.
50	EVOLUTIONARY ANALYSIS OF THE DEVELOPMENT OF AZT RESISTANCE WITHIN PATIENTS Int Wkshop HIV Drug Res 1994 Aug 2-5;3:51 (abstract no. 50) A. Cleland, H. Watson^, L.Q. Zhang, E. Harvey and A.I. Leigh Brown* Resistance-associated mutations which arise uniquely in the viral population may be particularly useful for targeting in convergent therapy. Such mutations can be identified by a phylogenetic analysis as applied here.
51	SIMILAR RATE OF DEVELOPMENT OF ZIDOVUDINE RESISTANCE CONFERRING RT MUTATIONS IN SI- AND NSI CLONES OBTAINED FROM INDIVIDUALS HARBOURING BOTH HIV-1 VARIANTS Int Wkshop HIV Drug Res 1994 Aug 2-5;3:52 (abstract no. 51) M.D. de Jong, A.B. van’t Wout^, J. Veenstra^, N.A. Kootstra^, R. Schuurman, P. Reiss, CAB. Boucher, H. Schuitemaker^* With respect to the rate of development of the 215- and 41 RT mutation, NSI clones from individuals harbouring both HIV-1 variants resemble their co-existing SI counterparts, rather than NSI clones from individuals solely harbouring NSI variants. This suggests that the differences in the rate of ZDV resistance development between individuals solely harbouring NSI variants and individuals also harbouring SI variants can not be attributed directly to the syncytium-inducing capacity of HIV-1.
52	A NOVEL IN VITRO HIV PROPERTY IN PEDIATRIC ISOLATES Int Wkshop HIV Drug Res 1994 Aug 2-5;3:53 (abstract no. 52) C.L. Vavro¹, Marty St. Clair¹, K.N. Pennington¹, M. Valentine² and R.E. McKinney² Further investigation of NSI+, including antibody neutralization studies and genetic analysis of the envelope region of HIV, may elucidate its significance in HIV disease.
53	VIROLOGIC EVALUATION OF HIV-INFECTED CANADIAN CHILDREN Int Wkshop HIV Drug Res 1994 Aug 2-5;3:54 (abstract no. 53) B. Conway, U. Allen, Marshall, J. Tobin, J. Forbes, D. Moore, J. Gilmour, S. King, N. Lapointe, S. Read There was a trend toward SI phenotype being more associated with higher titres than lower titres though this was not statistically significant (P=0.10, 1-tailed). A similar trend did not apply for the relationship between SI/NSI and ZDV resistance (P=0.44, 1-tailed). Of strains with low titres (≤25 TCID₅₀/10⁶ MCs) were susceptible to ZDV. In comparison, 11/17 high titre (≥125 TCID₅₀/10⁶ MCs) isolates were susceptible, showing no clear association between titre and ZDV resistance (P=0.50). It is likely that all three virologic parameters will have to be interpreted in concert to provide optimal information for clinical applications.
54	INFECTION OF NEWBORN RHESUS MACAQUES WITH AZT-RESISTANT SIMIAN IMMUNODEFICIENCY VIRUS (SIV) Int Wkshop HIV Drug Res 1994 Aug 2-5;3:55 (abstract no. 54) Koen Van Rompay, M. Otsyula, M. Marthas, C. Miller, M. McChesney, N. Pedersen The results show that this AZT-resistant SIV mutant replicates well in newborn macaques, even in the presence of AZT, and that its early course of infection resembles that of AZT-sensitive SIVmac. Further monitoring of the animals' clinical status and their viral/immunological parameters will reveal whether this AZT-resistant mutant is pathogenic and whether its AZT-resistant phenotype limits the efficacy of AZT treatment.
55	EFFECT OF A GLU TO LYS MUTATION AT POSITION 202 OF FELINE IMMUNODEFICIENCY VIRUS REVERSE TRANSCRIPTASE UPON RESISTANCE TO AZT Int Wkshop HIV Drug Res 1994 Aug 2-5;3:56 (abstract no. 55) T.W. North, K.M. Remington, Y.-Q Zhu and T.R. Phillps^* We have hypothesized that either the 2939 mutation is not responsible for AZT-resistance and there is another mutation outside of the RT-encoding region that is responsible; or that the phenotypic revertants contain a suppressor mutation that maps outside of the RT-encoding region. In order to determine the role of the 2939 lesion in AZT-resistance we have introduced it into a molecular clone of FIV, 34TF10. Properties of virus obtained from this construct will be presented.
56	CORRELATION OF IN VITRO AZT RESISTANT TESTED HIV-1 STRAINS WITH CLINICAL AND SURROGATE MARKERS IN AIDS PATIENTS Int Wkshop HIV Drug Res 1994 Aug 2-5;3:57 (abstract no. 56) H.G. Maxeiner⁺⁺, Th. Grünewald⁺, W. Schüller-Mauè⁺, H.D. Pohle⁺, and K.O. Habermehl⁺⁺ It is not possible to predict the degree of resistance of the HIV-1 strains in a patient under therapy by any of the widely used surrogate parameters.
57	INCIDENCE OF AZT RESISTANCE IN THE CONCORDE TRIAL Int Wkshop HIV Drug Res 1994 Aug 2-5;3:58 (abstract no. 57) F. Brun-Vézinet, B. Masquelier, ^†V. Joumot, L. Déforges, D. Ingrand, M.P. Schmitt, C. Buffet-Janvresse, F. Ferchal et al In these asymptomatic patients with high CD4 cell number (mean = 604) at the start of therapy, the incidence of resistance was 30% after 2 years7#8217; treatment. No association between resistance and clinical progression was observed in this cross-sectional study.
58	THE ACQUISITION OF RESISTANCE MUTATIONS IN SERUM HIV-1 RNA PREDICTS THEIR ACQUISITION IN PBMC PROVIRAL DNA Int Wkshop HIV Drug Res 1994 Aug 2-5;3:59 (abstract no. 58) S. Kaye, Clive Loveday, E. Comber and R.S. Tedder Our results show changes in serum RNA precede PBMC DNA and implies: transcription is a greater source of drug-induced mutational changes than reverse transcription or, PBMC may not be the major source of serum HIV-1 RNA and they may not be genetically representative of total HIV-1 infected cell population. This latter hypothesis is compatible with the observation of a higher level of viral activity in lymphoid tissue at certain stages of disease compared to peripheral blood reported by other groups.
SESSION 7: Drug Resistance and Viral Load During Mono- or Alternating Therapy Co-chairs: Tom Merrigan and John Sninsky Abstract 59 thru xx, Pages 60 to xx
59	RELATIONSHIP BETWEEN HIV-1 VIRAL LOAD AND APPEARANCE OF DRUG RESISTANCE MUTATIONS IN PATIENTS INITIALLY TREATED WITH 3TC AND SUBSEQUENTLY WITH A COMBINATION OF AZT AND 3TC Int Wkshop HIV Drug Res 1994 Aug 2-5;3:60 (abstract no. 59) Rob SChuunnan, Monique Nijhuis, Remko van Leeuwen, Pauline Schipper, Phil Collis, Sven Danner, John Mulder, Clive Loveday, Cindy Christopherson, Shirley Kwok, John Sninsky, and Charles A.B. Boucher 3TC monotherapy effectively inhibits the replication of HIV-1 during the first weeks of treatment. Subsequent increase in virus load in patients treated with 3TC is due to outgrowth of 3TC resistant virus variants. These variants were effectively but temporarily inhibited by AZT.
60	CHANGES IN SERUM HIV-1 RNA LOAD AND GENOTYPIC RESISTANCE IN EARLY ZIDOVUDINE (ZDV) THERAPY Int Wkshop HIV Drug Res 1994 Aug 2-5;3:61 (abstract no. 60) Clive Loveday, S. Kaye, M. Tennant Flowers^, M. Semple, U. Ayliffe, IVD Weller^, R.S. Tedder During early ZDV therapy there appeared to be a triphasic response in serum viral load. A prompt fall in days, followed by a return towards baseline in weeks, and a longer phase of viral suppression for months. These early changes were independent of ZDV resistance.
61	MUTATION AT CODON 215 0F HIV-1 REVERSE TRANSCRIPTASE IN PLASMA RNA AND DISEASE PROGRESSION IN PATIENTS RANDOMIZED TO AZT VS ddI Int Wkshop HIV Drug Res 1994 Aug 2-5;3:62 (abstract no. 61) R.W. Shafer¹, D.A. Amato², M.J. Kozal¹, M.A.Winters¹, T.C. Merigan¹ The AZT-resistance HIV RT codon 215 mutation predicted clinical progression in pts randomized to AZT but not in pts randomized to ddI: However, the presence of a correlation between previous AZT and the relative benefit of ddI even in pts without a codon 215 mutation suggests that other factors in addition to this mutation may explain the benefit observed in pts switching from AZT to ddI. These factors might include other AZT-resistance mutations or the differential activity ofAZT and ddI in various cell populations.
62	HIV-1 RESISTANCE GENOTYPES IN THE PLASMA RNA AND PBMC DNA DURING ZDV/ddI COMBINATION THERAPY. Int Wkshop HIV Drug Res 1994 Aug 2-5;3:63 (abstract no. 62) Eastman, P. Scott^, Boyer E^, Urdea M^, Kolberg J^, Mole L^ and Holodniy M^ The addition of ddI results in a decline of HIV RNA levels and codon 215 WT RNA in the plasma of some patients. Proviral load and codon 215 MUT and WT proviral populations appear not to be affected. Furthermore, mutation at sites other than codons 74, 135, and 184 must be responsible for the observed resistance to ZDV/ddI combination therapy.
63	ddI AND ZDV RESISTANCE MUATIONS IN 64 PATIENTS SWITCHED FROM ZDV TO ddI: RELATIONSHIP TO CD4 CELL DECLINE, VIRUS BURDEN AND DISEASE PROGRESSION Int Wkshop HIV Drug Res 1994 Aug 2-5;3:64 (abstract no. 63) M.J. Kozal, K. Kroodsma, M. Winters, R. Shafer, B. Efron, D. Katzenstein and T. Merigan Among HIV-1 infected patients with advanced disease who were changed from ZDV to ddI, more than one-half developed a mutation at codon 74 by 24 weeks of ddI therapy. Patients who developed the codon 74 MUT experienced a significantly greater decline in their CD4+ T cells after the development of the MUT and had a significantly greater serum virus burden than did patients without the codon 74 MUT.
64	OLIGONUCLEOTIDE MUTATION AT SITES 215,74 AND 184 OF THE HIV REVERSE TRANSCRIPTASE AND PROLONGED THERAPY WITH DIDANOSINE Int Wkshop HIV Drug Res 1994 Aug 2-5;3:65 (abstract no. 64) C. Jacomet, B. Burghoffer^, L. Morand-Joubert^, J.C. Petit^, W. Rozenbaum* While the 184 mutation is correlated to a weak level of phenotypic resistance in patients on ddI, this mutation is correlated to a high level resistance in patients on 3TC. A coherent therapeutic strategy would have to take into account these results.
65	QUANTIFICATION OF SERUM VIRAL LOAD AND RESISTANT MUTATIONS IN PATIENTS (MRC ALPHA TRIAL) STOPPING ZIDOVUDINE (ZDV) AND COMMENCING DIDANOSINE (ddI) Int Wkshop HIV Drug Res 1994 Aug 2-5;3:66 (abstract no. 65) Clive Loveday, E. Comber, U. Ayliffe, J.H. Darbyshire^, A. Babiker^, R.S. Tedder, A. Valentine, A. Pinching and The Alpha Coordinating Committee Serum viral load showed a mean fall of 0.77 log₁₀ over the study period as resistance at codon 74 increased from <2% to 45%. The acquisition of resistance at codon 74 was dose dependent. A reciprocal relationship between codon 215 and 74 was only seen if pretreatment 215 prevalence was submaximal.
SESSION 8: Drug Resistance and Viral Load During Combination Therapy Co-chairs: Joep Lange and Johanna Griffin Abstract 66 thru 73, Pages 67 to 74
66	HIV-1 RNA LEVEL IN PLASMA AND ASSOCIATION WITH DISEASE PROGRESSION, ZIDOVUDINE SENSITIVITY PHENOTYPE AND GENOTYPE, SYNCYTIUM-INDUCING PHENOTYPE, CD4+ CELL COUNT AND CLINICAL DIAGNOSIS OF AIDS Int Wkshop HIV Drug Res 1994 Aug 2-5;3:67 (abstract no. 66) C. Hooper, S. Welles, R. D’Aquila, A. Japour, V. Johnson, D. Kuritzkes, B. Jackson, V. DeGruttola, C. Crumpacker, D. Richman^, J. Kahn^, S. Kwok^$, J. Todd^¶, P. Reichelderfer and R. Coombs for the ACTG Virology PCR Validation and Resistance Working Groups The SI-inducing phenotype was not associated with the HIV-1 RNA copy number (P=0.65). In this study, an increase in HIV-1 RNA copy number was associated with clinical progression, genotypic resistance to ZDV, lower CD4+ cell count and diagnosis of AIDS at study entry but not with phenotypic resistance or SI phenotype. The higher virus load for subjects with genotypic ZDV resistant isolates may explain their increased risk of disease progression.
67	COMPLEXITY OF HIV REVERSE TRANSCRIPTASE CHANGES IN PATIENTS RECEIVING THE COMBINATION OF AZT+ddI FOR 2 YEARS: ASSOCIATIONS BETWEEN DRUG RESISTANCE, VIRUS LOAD, AND CD4 COUNTS Int Wkshop HIV Drug Res 1994 Aug 2-5;3:68 (abstract no. 67) R.W. Shafer, A.K.N. Iversen, M.A. Winters, E. Aguiniga, D.A. Katzenstein, T.C. Merigan, and the ACTG 143 Virology Group 2 years of AZT+ddI combination therapy selected for mutations conferring high-level AZT-resistance (codons 41+215). Multidrug-resistance mutations at codons 75, 77, 116, 151 occurred in 3/39 pts. Other possibly drug-induced mutations occurred 3.5 times more often among the culture-positive HIV strains. HIV burden correlated with drug resistance, SI phenotype, and CD4 decline.
68	A PLACEBO CONTROLLED TRIAL OF AZT ALONE OR IN COMBINATION WITH ddI OR ddC: VIRAL LOAD AND AZT RESISTANCE Int Wkshop HIV Drug Res 1994 Aug 2-5;3:69 (abstract no. 68) P.R Harrigan, I. Kinghorn, R. Myers, M.H.·St. Clair^, B.A. Larder, and the 34,225-02 Collaborative Group* AZT treatment alone reduces median serum viral load, and combinations of AZT with ddI or, in particular, AZT with ddC were more effective at reducing HIV RNA levels than AZT monotherapy. Given the observed changes in viral load, ongoing larger trials designed to monitor clinical effects of these treatments will be of great interest.
69	VIRAL RESISTANCE IN ADULTS TREATED WITH NEVIRAPINE (NVP) ADDED TO ESTABLISHED NUCLEOSIDE RT INHIBITOR REGIMENS OF ZDV +ddC, ZDV + ddI, OR ddI ALONE Int Wkshop HIV Drug Res 1994 Aug 2-5;3:70 (abstract no. 69) V. Johnson¹ M. ·Saag¹, J. Decker¹, J.-P. Sommadossi¹, M. Myers², S. Cort², D. Ha1l², J. Griffin², J. Lifson³, G. Shaw¹ Addition of NVP therapy to ongoing RT inhibitor regimens resulted in significant reduction in viral burden and increases in CD4+ cell counts. Dual ZDV- and NVP-resistant HIV-1 was demonstrated in ZDV-experienced subjects treated with NVP who had loss of antiviral suppression. Triply resistant viral isolates were not identified. Evidence for ddI resistance was not seen phenotypically or genotypically despite a detailed genetic sequence analysis of viral nucleic acid from uncultured PBMC and plasma. Evaluation for drug resistance in subjects with sustained virologic and/or clinical responses is underway.
70	NUCLEOTIDE SEQUENCE ANALYSIS OF HIV-1 RNA AND DNA FROM PLASMA AND PBMCs OF PATIENTS TREATED WITH ZDV, ddI, AND NEVIRAPINE: RAPID HIV-1 TURNOVER AND RESISTANCE DEVELOPMENT IN VIVO Int Wkshop HIV Drug Res 1994 Aug 2-5;3:71 (abstract no. 70) G. Shaw¹, X. Wei¹, V. Johnson², M. Taylor¹, J. Decker¹, M. Kilby², J. Lifson³, B. Hahn¹, and M. Saag² In nucleoside analogue experienced patients, the addition of NVP to ZDV/ddI selects for NVP resistant virus that replicates readily in vivo. NVP associated resistance changes were found preferentially at RT codons 181, 188, and 190. The magnitude and rapidity of virus turnover in plasma and PBMCs suggest that ongoing de novo infection of, and virus production by, a relatively short-lived (or transiently expressing) lymphoreticular cell population is primarily responsible for the rapid appearance of resistant virus and contributes substantially to the persistence of viremia observed in chronic HIV-1 infection.
71	A PLACEBO CONTROLLED TRIAL OF AZT ALONE OR IN COMBINATION WITH ddI OR ddC: IN VITRO AZT RESISTANCE ANALYSIS AS DETERMINED BY PBMC ASSAY Int Wkshop HIV Drug Res 1994 Aug 2-5;3:72 (abstract no. 71) St. Clair^, M.H., Pennington^, K.N., Larder^#, B.A., and the Protocol 34,225-02 Collaborative Group Decreased serisitivity to AZT developed with AZT+ddI and AZT+ddC combinations as well as with AZT monotherapy arm. While combination chemotherapy may not prevent the development of AZT resistance, it is possible that it may delay it. Combination therapy, particularly AZT+ddI, delayed the appearance of high level AZT resistance.
72	A PLACEBO CONTROLLED TRIAL OF AZT ALONE OR IN COMBINATION WITH ddI OR ddC: GENOTYPIC AND PHENOTYPIC RESISTANCE ANALYSIS Int Wkshop HIV Drug Res 1994 Aug 2-5;3:73 (abstract no. 72) B.A. Larder, S.D. Kemp, I. Kinghorn, A. Kohli, S. Bloor, M.H. St. Clair^ and the Protocol 34,225-02 Collaborative Group* Very few ddI or ddC resistance mutations were observed at week 48 (1/39 74V and 1/39 184V in the +ddI group; 1/29 74V and 2/29 184V in the +ddC group). We conclude that combination therapy with AZT/ddI or AZT/ddC does not significantly retard the development of AZT resistance. However, the significant effects observed on CD4+ cell counts and viral load (see Harrigan et al.), may be due to the lack of ddI or ddC resistance mutations occurring during this treatment period.
73	ANTIVIRAL DRUG RESISTANCE IN VIVO: THE INTERACTION BETWEEN DRUG AND RESISTANCE IN CLINICAL TRIALS Int Wkshop HIV Drug Res 1994 Aug 2-5;3:74 (abstract no. 73) Edlin. B.R. When examined in this light, available data comparing zidovudine with didanosine suggest that in vitro resistance to zidovudine predicts a poorer prognosis overall, but-unless there is cross-resistance between the two drugs-not a diminished response to zidovudine.

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