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•MVC has a novel target
•it targets a host receptor, CCR5 coreceptor, rather than a viral target. •As such it has unique resistance issues. Resistance to maraviroc can occur in the classical sense: where the phenotype changes by virus mutation resulting in viral entry and replication in the presence of the drug •HOWEVER since this drug targets the CCR5 receptor and will mechanistically work against only CCR5-tropic virus, the virus can bypass MVC by a tropism switch of the virus to use the X4 coreceptor or outgrowth of an already existing X4-tropic virus •And There are concerns that X4-tropic virus is more pathogenic •Our analysis examined both changes in tropism and the geno/pheno changes to MVC as mechanisms of viral resistance to MVC
GSS and PSS scores were used to determine susceptibility.
A score of 1 was given for each susceptible drug in OBT. Therefore, the higher the GSS and PSS score – the more susceptible drugs in OBT
•GSS and PSS scores were balanced across the three treatment groups in both studies with Median GSS=1 and PSS=2
•67% of subjects had on overall susceptibility scores <2 meaning they had 2 or less active drugs their in OBT 
•30% had only one potentially active drug in OBT and
•14% of subjects had no potentially active drug in their OBT.
•Consistent with a heavily treatment-experienced population.
•2560 subjects were screened; 56% had CCR5-tropic virus
•About 10% of viruses changed from R5-tropic to Dual-mixed or were non-phenotypable in the time period from screening until baseline.
•So 90% of the subjects enrolled had CCR5-tropic virus at Baseline
•Throughout these trials, it appears that the percentage of isolates which are non-phenotypable using the Monogram tropism assay ranged from 5 to 15%.
Why Did Subjects Fail MVC Treatment in Studies 1027 and 1028?
Reasons for Failure on MVC could include:
•Co-receptor switch from CCR5-tropic to CXCR4-tropic virus by mutation in the virus
•Outgrowth of CCR5-tropic viruses that are resistant to MVC
•Outgrowth of CXCR4-tropic viruses present at baseline but not detected with the standard tropism assay (because one of the limitations of the assay is that it is not able to detect with 100% sensitivity X4 when present below 10% levels in a viral mixture)
•Resistance to OBT
•Host CCR5 genotype – although not known at this point, it might be possible that MVC may not bind efficiently to some host CCR5 receptor genotypes
For resistance analyses, we use ‘as treated’ analyses.  Therefore, subjects are censored from the analysis if they
•discontinued with ≤400 copies/mL or
•if they discontinued with >400 copies/mL between Baseline and Week 4 or •if they discontinued between Baseline and Week 8 with at least 0.5 log decrease and no rebound (previous ≥2 log decrease with 1 log increase).  Forty-nine and thirty-nine subjects were censored from the analysis of studies 1027 and 1028, respectively = total of 88 censored
We first determined the percentage of virologic failures that had CCR5-tropic and CXCR4-tropic virus at time of failure. 
The analysis was done using two definitions of treatment failure
1) the protocol-defined treatment failure (PDTF) definition and
2) subjects with PDTF plus subjects with >400 copies/mL at Week 24 Regardless of the definition of treatment failure, more subjects (~50-60%) failed with CXCR4- or dual/mixed-tropic virus in the MVC arms, whereas >80% of the subjects in the placebo arm failed with CCR5-tropic virus.  A high percentage of treatment failure on MVC appears to be driven by tropism change from CCR5-tropic to CXCR4- or dual/mixed-tropic virus.  This supports the mechanism of action of MVC and suggests emergence of CXCR4-tropic virus is a prominent reason for failure on MVC.
Another reason for treatment failure could be resistance to the other drugs in the OBT.  Most subjects typically had low phenotypic and genotypic susceptibility scores at screening, indicating reduced susceptibility to their OBT.
Overall in studies 1027 and 1028, As the number of susceptible drugs in the OBT increased, reflected in an increased overall susceptibility score, the percent of subjects that achieved <400 copies/mL increased in the MVC arms (orange and blue) to 70% if 3 or more susceptible drugs in OBT. In the placebo arm (red), response rates were <20% with less than 2 active drugs, but increased to 61% when subjects had 3 or more susceptible drugs in OBT.
When examining the OBT in the Subjects who were Treatment Failures: •28% of treatment failure subjects had no susceptible drugs at baseline.  There was no difference between the MVC and placebo treatment arms. •When looking at changes in susceptibility to OBT on therapy, 43% of treatment failure subjects lost susceptibility to drugs in their OBT on treatment and again there was no difference between treatment arms.
Looking specifically at ENF use, ENF use in the failures was comparable between arms at 45%. About 70% of subjects developed ENF resistance mutations on treatment in the MVC QD and placebo arms. And There were significantly fewer ENF resistance mutations that developed in the MVC BID arm than in the placebo arm (p=0.01) or the MVC QD arm (p =0.02).
An examination of overall susceptibility scores of the treatment failures by tropism shows that •80% of subjects who failed with X4-tropic virus had susceptibility scores of 0-1 compared to only 3% that had 3 or more active drugs in OBT. •50-60% of subjects who failed with R5 or dual/mixed tropic virus had susceptibility scores of 0-1 compared to less than 20% with susceptibility scores of 3 or more.
•A comprehensive analysis was requested for subjects who had experienced treatment failure and/or had a changes in their coreceptor tropism •Given the complexity and exploratory nature of the tropism and resistance analyses, substudies from Clinical Studies A4001027 and A4001028 were proposed. 
A subset of subjects failing with CCR5-tropic virus were analyzed to identify possible phenotypic and genotypic markers associated with MVC resistance in vivo.
•Including determining MVC susceptibility in cell culture
•Nucleotide sequence of gp120 to identify aa substitutions that might contribute to MVC resistance •Nucleotide sequence of protease and RT to identify resistance to drugs in OBT
A subset of subjects failing with X4-tropic virus were analyzed to ascertain whether the CXCR4-tropic virus emerged from undetected X4-tropic virus at screening or as a result of mutations in a CCR5 tropic virus cauing a tropism switch  while on MVC:  The evaluation included •Clonal evaluation of virus at baseline and on treatment samples to determine the relative number of CXCR4-tropic and CCR5-tropic viral isolates •Nucleotide sequence analysis of the gp120 region to identify amino acid changes that may contribute to a coreceptor switch from R5 to X4 •Phylogenetic analysis to determine the relationship of emerging CXCR4-tropic virus to the CCR5-tropic virus at baseline (to determine if X4 virus was distinct from R5 virus at baseline) •In addition, PR and RT were sequenced to determine resistance to OBT
In the virology subgroup analysis the sponsor selected 267 subjects on blinded therapy from studies 1027 and 1028.
From the pool of 267 subject, there were 38 who failed with CCR5-tropic virus 13 subjects were randomized to receive MVC (6 BID: 7 QD) and 25 subjects were on placebo. The genotype and phenotypic susceptibility to MVC of Env recombinant pseudoviruses was analyzed from these 38 subjects
Virus from 2 failure subjects had 3-fold shifts in MCV susceptibility at the time of failure. 
All other subjects on MVC had FC <2-fold within the normal range of the Monogram assay.
Viruses from 5 subjects failing MVC treatment with CCR5 tropic virus showed evidence of a lower plateau in maximum percentage inhibition rather than fold changes in EC50 values The results support previous findings from selection of MVC-resistant virus in cell culture and isolates from Phase 2 MVC studies that lower plateaus in the maximum percentage inhibition (MPI) were associated with subjects failing a MVC-containing regimen rather than changes in EC50 values. All 5 subjects had amino acid changes in the V3 loop of gp120 which were present at failure timepoints but were not present at BL.
•These 5 subjects also had lower Cmin values <75 ng/mL
The V3 loop sequences reflected the heterogeneity associated with the V3 region of gp120. All the failure clones had multiple different V3 amino acid changes However, Changes at either amino position 13 or 26 were seen in the V3 loop of gp120 in all five of the subjects with MVC-associated lower plateaus in MPI. 
•The role of the V3 Loop Amino Acid Substitutions in MVC Resistance was confirmed by site-Directed Mutagenesis •Mutating amino acids in baseline clones to those seen in MVC resistant clones resulted in the MVC resistance phenotype of <95% MPI
•Showing that these mutations contributed to MVC resistance
•Back mutation of aa changes of failure clones resulted in MVC sensitive phenotype
Not all subjects failing MVC treatment with CCR5-tropic virus had phenotypic markers of MVC.  •Seven of the subjects receiving MVC did not show phenotypic markers of MVC resistance in the Monogram assay •The majority of these subjects (5/7) had evidence of reduced susceptibility to one or more drugs within their OBT at screening and/or at failure. •CCR5 receptor genotype should be examined from these subjects to see if MVC can bind to these receptors
A subset of 20 subjects failing with CXCR4-tropic virus were analyzed: 16 were in a MVC treatment group and 4 in the placebo group.
192 pre-treatment and 48 on-treatment clones from each of the 20 subjects with X4-Tropic Virus were analyzed for tropism.
•Shown here is the clonal evaluation of one subject
•Each square is a clone, pink=R5, blue=dual/mixed, green=X4, blank were non-functional clones •shows the proportion of R5 and X4 clones at pretreatment and on treatment •Most of the pretreatment clones were R5 consistent with this subject being classified as having R5-tropic virus •Then more of the on-treatment clones are X4 or dual/mixed consistent with this subject failing with X4-tropic virus •Each of these clones was sequenced – region sequenced was 290 bases encompassing the V3 loop of the env gene •Phylogenetic trees were generated using these sequences in order to investigate possible ancestry of the CXCR4-using clones - To determine if green X4 clones were related to any of these pink pretreatment R5 clones (which would suggest tropism switch) or if they were related to green X4 pretreatment clones  (which would suggest outgrowth of this X4-tropic virus) •Tropism of clones was confirmed in the validated format of the Trofile™ assay
•In 14 subjects, CXCR4-tropic clones in the ‘on-treatment’ samples shared a common ancestor with a pre-treatment X4-tropic virus indicating outgrowth of X4-tropic virus
•In the remaining 6 subjects, CXCR4-using env clones identified in the ‘on-treatment’ samples did not have a pretreatment X4 ancestor but they were also genetically distinct from both the ‘pre-treatment’ and ‘on-treatment’ R5 population.
•The V3 loop sequences of the these on-treatment X4-tropic clones differed by 7-17 amino acid residues from the V3 loop of the nearest R5 sequence on the phylogenetic tree.
ΨThe Monogram tropism assay has a sensitivity of 100% for detecting CXCR4-tropic or dual/mixed HIV if present at 10% or more.  If present at <10%, the sensitivity of the assay goes down (83% at a 5% mixture).
Although a tropism switch whereby CXCR4-using virus emerged on MVC resulting from these 7-17 amino acid substitutions in the CCR5 precursor cannot be ruled out from the review of the data submitted, From the amino acid sequence differences and phylogenetic tree data, the most likely explanation is the CXCR4-using clones in these 6 subjects emerged from a pre-existing CXCR4-using virus not detected by the assay at BL.
So why did subjects fail on MVC?
•Most (~50-60%) subjects failed with CXCR4- or dual/mixed-tropic virus in the MVC arms.
•The data from the virology substudies suggest that the most prominent reason for failure in these studies was outgrowth of CXCR4-using viruses not detected at screening. 
•Treatment failure on MVC with CCR5-tropic virus also occurred and resulted from phenotypic and genotypic resistance to MVC and resistance to OBT. 
•It remains to be determined if host CCR5 genotype also plays a role in MVC failure
The evolution to a CXCR4-utilizing HIV has been proposed to result in a more virulent virus. Therefore, there is a concern that using MVC will cause outgrowth of CXCR4-tropic virus and result in worse outcomes for patients.
An examination of CD4+ cell counts by Tropism at Failure showed that the mean and median change in CD4 cell counts from baseline using last observation carried forward was lower in subjects with X4 and dual/mixed tropic virus than those with R5-tropic virus
So we asked for follow-up on the subjects who failed with X4-tropic virus in studies 1027 and 1028
We requested:
Long term follow-up on the subjects viral loads, CD4+ cell counts, HIV coreceptor usage and AIDS defining events
•28 subjects failed with CXCR4 virus and were followed by the sponsor, 20 had at least one follow-up visit.
2/3 had changed tropism back to CCR5 or dual-mixed and 35% still had X4-tropic virus
For the subjects with R5- or dual/mixed-tropic virus at end of follow-up, the median time to last follow-up was approximately 5 months (range 18 days to 8 months). 
In contrast, the follow-up time for the subjects who remained X4-tropic at the last follow-up visit was one month or less (median time was approximately 11 days).
•Suggesting that over a longer period of follow-up, CCR5 viruses will outgrow X4 viruses in these subjects
In half of the subjects, CD4 cell counts also declined (mean change -21, median change -3) consistent with the ongoing viremia.
•Viral loads remained similar to the value at treatment failure
•No new category C AIDS-defining events were reported for any of the 20 subjects •If subjects went on a new ARV treatment, viral loads decreased.   •And CD4 increases were seen concomitant with the reduction in viral load. 
Most (50-60%) subjects failed with CXCR4- or dual/mixed-tropic virus in the MVC arms
Most prominent reason for failure in these studies was outgrowth of a minor CXCR4-tropic virus population not detected at screening
Treatment failure on MVC with CCR5-tropic virus also occurred and resulted from phenotypic and genotypic resistance to MVC and resistance to OBT The role of the CCR5 host receptor genotype is yet to be determined
Lower plateaus in maximum plateau inhibition were detected in viruses from 5 subjects failing maraviroc and fold changes in EC50 values to maraviroc were only seen in two subjects Changes in the V3 sequence of gp160 correlated with the presence of lower plateaus and Maraviroc resistance. There is heterogeneity of the envelope protein and likely multiple pathways to MVC resistance.