1. Novel assay using PCR and mass spectrometry for quantification of minor populations of HIV-1 carrying drug-resistant mutations Kato Shingo, Koji Sudo, Rie Tanaka Department of Microbiology and Immunology, Keio University School of Medicine,Tokyo, Japan

2. Background Detection and quantification of a minor population of drug- resistant HIV-1 allow us to understand the spread of drug- resistant virus in primary infection and may select a better combination of antiretroviral drugs in salvage therapy. Direct sequencing cannot detect minority of <20%. A number of methods have been developed –Restriction digestion, heteroduplex tracking assay, oligonucleotide tracking assay, pyrosequencing, allele-specific real-time PCR assay Among them, allele-specific real-time PCR assay is the most sensitive (down to 0.1%) and the least laborious, and has been commonly used to study the dynamics of HIV-1 populations before and after antiretroviral treatment, mother- to-child transmission, and STI (structured treatment interruption).

3. Problems of allele-specific real-time PCR Affected by frequent cross-reactions in transition-type point-mutations (A ↔ G; C ↔ T) Cannot test three types of point-mutations (ex., A → G, C, or T) simultaneously Cannot test consecutive two-base mutations such as T215Y (ACC → TAC)

4. Allele-specific real-time-PCR L90 selectiveL90M selective T A A A X A T T T X selective primer unselective primer WT L90 sequence mutant L90M sequence WT L90 sequence mutant L90M sequence In the case of L90M

5. Objective We aimed to develop a new method to quantify a minor population of HIV-1 with combinational use of PCR and LC-MS (liquid chromatography-mass spectrometry). –K103N (RT) –M184V (RT) –T215Y (RT) –L90M (PR)

6. Standard HIV-1 isolates Culture media of following virus isolates were used. –Wild-typeIIIB –VariantK2901 M41L, K103N, M184V, T215Y in RT I54V, G73S, V82A, L90M in PR It was confirmed that these two isolates had not contain a detectable minor population by using sequencing and the present method.

7. Procedure (1) 1.Mix the wild-type strain and the drug-resisitant variant at various ratios. 2.Extract RNA by silica matrix kits. 3.Reverse transcription and PCR to amplify only the target nucleotide. 4.PCR to add an recognition signal (RS) of type I restriction endonuclease Acu I. 5.Digest PCR product with Acu I. 6.Qunatify the digested-fragment by LC-MS Mutation locus Viral RNA cDNA RT PCR RS

8. Procedure (2) 5.Digest PCR product with Acu I. In the case of M184V, Wild-type Mutant 6.Separate and quantify the digested DNA fragments by LC-MS CAATAC GTTA TGTAC CAATAC TGAT ATGGA CAATAC GTTA TGCAC CAATAC TGAT GTGGA 14 bp 16 bp CTGAAG GACTTC CTTCAG GAAGTC CTGAAG GACTTC CTTCAG GAAGTC

9. Mass spectrograms of wild-type and mutant sequence fragments CACGT 772.0 amu 184V (mutant) CATGT 779.5 amu 184M (WT)

10. Linearity of quantification of drug-resistant mutant virus

11. Precision and accuracy in intra- and inter-assays Precision and accuracy for quantifying samples which contained 50%, 5%, and 0.5% mutants of K103N, M184V, T215Y, and L90M were evaluated. Coefficients of variation were in the range from 3% to 64%, a half of which were within 15%. Accuracy was in the range from -18% to 42%, a half of which were within ±15%.

12. Distinction of dinucleotides with a different order (TA and AT) T215Y is caused by a mutation at two consecutive nucleotides from ACC to TAC. The Acu I-digested antisense fragment from a mutant is TAAA, which is indistinguishable from ATAA in mass spectrometry. But these diffent dinucleoitides can be distinguished in liquid chromatography. TAAAATAA

13. Transition of wild-type and mutant virus populations during treantment interruption d4T+3TC+NFV No drug d4T+3TC+NFV d4T+ddI+EFV 184M 184V 215Y 215T <0.2%

14. Conclusions The present method combines PCR and LC-MS and enables us to quantify minor HIV-1 variants with point mutations of K103N, M184V, T215Y (RT) and L90M (PR) down to 0.2% or 0.5%. This method can distinguish three types of point mutations and detect a consecutive two-base mutation (T215Y). Because the fragments analyzed in LC-MS were designed to have different sizes, mutations at the four sites can be simultaneously quantified. The present assay may be useful for accurate quantification of minor populations of HIV-1 variants in clinical samples and contribute to drug resistance reserch.

15. Acknowledgements Collaborated with clinical staffs at Department of Infectious Disease of Keio University Hospital in Tokyo, Japan. –Dr. Masayoshi Negishi –Dr. Naoki Hasegawa –Ms Yoshiko Tomaki Supported by grants from the Ministry of Health, Labour and Welfare of Japan.