1. Mixed HCV Genotype Infection and Response to anti-HCV treatment in HIV/HCV Co-Infected Patients Lucy Porrino, Sabrina Bagaglio, Giulia Morsica, Giulia Gallotta, Hamid Hasson, Laura Galli,Vega Rusconi, Adriano Lazzarin, Caterina Uberti-Foppa Infectious Diseases Dept, San Raffaele Scientific Institute, Milan, Italy WEAB0102

2. Background The frequency of mixed infection in HCV monoinfection is about 2- 13% (Schroter et al. 2003; Qian et al. 2000) There is a single study on viral infections with different genotypes in HIV/HCV coinfected patients and has not been evaluated the performance of the genotype during the anti-viral therapy (Soriano et al. 2005) Previous reports from Japan identified a mutational pattern within the core protein (70Q-91M) of HCV-1b related to the responsiveness to standard treatment for HCV This finding possibly mediated by an interaction with interferon signalling cellular protein STAT-1

3. To determine the HCV genotype pattern and core domain in HIV/HCV coinfected patients during anti-HCV treatment AIM

4. Study Patients A pilot clinical trial (Kamon2) was conducted to compare LPV/r monotherapy and LPV/r-based HAART during anti-HCV therapy A virological sub-study was performed on patients HIV/HCV coinfected enrolled in Kamon2 Patients were analyzed according to response to anti-HCV treatment HCV-RNA negativity at 24-weeks of treatment and maintainment up to 72 weeks Sustained Virological Response = (SVR) ≤2 Log decrease of HCV-RNA at 12-weeks No Response (NR) = HCV-RNA positivity during follow up Relapse (RE) post-treatment =

5. Methods Sequence analysis of HCV-5’ UTR was performed in plasma at different time points: baseline (BL), during 48 weeks treatment and after treatment The entire sequence of core was inferred in plasma at BL, during treatment and/or after treatment. All these sequences were compared with the respective prototype (based on HCV genotyping)

6. Baseline Characteristics of HIV/HCV Coinfected Individuals According to Anti-HCV Treatment Response All patients (N=22) SVR (N=11) NR(NR+RE) (N=11) P (SVR vs NR) Age (years) 42 (40-44)44 (41-45)42 (39-43)0.95 Males/Females13/96/57/41 ALT (U/I) 79.5 (59-164)95 (74-150)59 (45-137)0.77 AST (U/I) 48 (36-88)51 (41-84)39 (29-83)0.80 CD4 + cells count (cell/mmc) 592 (414-794) 596 (421-739) 514 (407-921) 0.70 CD8 + cells count (cell/mmc) 925 (693-1167) 926 (837-1116) 935 (595-1258) 0.70 HCV-RNA (Log IU/ml) 6.05 (4.83-6.34) 5.37 (4055-6.03) 6.34 (6.11-6.42) 0.05 HCV Genotype (1-4 vs 2-3) 13/92/90/110.0002

7. HCV Genotype Pattern in NR during Follow-Up BLW4W12W24W36W48W60W72 NR 11aNA3a NR 24a3a4a 4c/d NR 31bNA1b3aNA 11b NR 41b 3a2a1b RE 11b3aNAneg 1b  6 (55%) maintained the same HCV genotype during follow up  5 (45%) showed an alternance of HCV genotype in plasma lost in follow up

8. Core sequence analysis at BL We obtained the core sequences of 10 SVR and 8 NR Different distribution of amino acids (aa) mutations in different genotype (G) HCV Genotype (n)Median number of aa mutations (IQR) G1 (7)1 (0-4.5) G2 (2)4 (3-5) G3 (6)6 (6-12) G4 (3)4 (4-4.50) G1-G4 vs G2-G3p= 0.0276 G1 vs G3p= 0.018

9. Core sequence analysis during follow up in 8 NR patients 1pt showed the same sequence at each time point 7 pts showed at least one aa substitution during follow up This aa mutation were recurrent in specific position aa position2036707175110 N°pts6/83/82/83/86/85/8

10. ...in particular 6/8 showed at least aa substitution in 1-20* and 69-75 **region 5/8 showed at least aa substitution in 110-115* region 110 120 130 140....|....|....|....|....|....|....|....|....| RGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLG 10 20 30 40 50....|....|....|....|....|....|....|....|....|....| MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATR 60 70 80 90 100....|....|....|....|....|....|....|....|....|....| KTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSP *epitope regions **peptide-specific CD4+ T-cell

11. 5/8 pts showed 70Q and/or 91M »70Q was undetectable at BL in 3pts and appeared during treatment »in 1pt this mutation was detectable at BL and disapperead during treatment »1pt had 91M at BL and maintained during treatment

12. Characteristic mutational pattern in 2 RE at BL... NR vs REp= 0.0056...and disappeared during follow up

13. Conclusions - 5’ UTR analysis The dynamic pattern of HCV genotypes in half of NR suggests a mixed HCV-infection The anti-HCV treatment may induce a change balance of dominant strain Work in progress: ultradeep sequencing (pyrosequencing) will be performed to verify viral population in plasma

14. Conclusions - Core domain analysis At BL difficult to treat genotypes are more similar to the respective prototype than “favourable”genotypes On treatment the most prominent variability of specific residues in specific regions of the core domain could be consequent to the pressure exerted by interferon and/or ribavirin

15. Thanks to Infectious Diseases Dept, San Raffaele Scientific Institute, Milan Giulia Morsica Sabrina Bagaglio Marco Merli Alice Dadda Hamid Hasson Giulia Gallotta Laura Galli Vega Rusconi Caterina Uberti-Foppa Adriano Lazzarin National Institute of Infectious Diseases, Spallanzani, Roma Infectious Diseases Section, Hospital SS Annunziata, Firenze Institute of Infectious Diseases, University of Bari, Bari