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Cerebrospinal Fluid _CSF_ Drug Resistant HIV

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Cerebrospinal Fluid _CSF_ Drug Resistant HIV Powered By Docstoc
					  Cerebrospinal Fluid (CSF) Drug
Resistant HIV Compartmentalization
   Detected during Primary HIV-1
Infection by Ultra Deep Sequencing

Arjet Gega1, Michael Kozal1, Jennifer Chiarella1, Evelyn Lee2, Julia Peterson2,
 Elizabeth St. John3, Birgitte Simen3, Richard W. Price2 and Serena Spudich1

1Yale   University School of Medicine, New Haven, CT, USA; 2University of California San
        Francisco, San Francisco, CA, USA; 3454 Life Sciences, Branford, CT, USA.
                 Pilot Study Goals


• Demonstrate feasibility of ultra deep sequencing (UDS)
  of HIV species derived from cerebrospinal fluid (CSF)
• Use UDS to investigate early CNS compartmentalization
  of HIV
• Examine for low abundance transmitted drug resistant
  variants in early CNS-derived HIV
                                         Background
• Prior methods (heteroduplex tracking assay, single
  genome amplification, Sanger sequencing) have shown
  compartmentalization of CSF-derived HIV RNA species
   – CNS compartmentalization of env strongly associated with HIV
     associated dementia in advanced disease

   – CSF compartmentalization detected in minority of subjects
     during primary infection

• Recent studies suggest relevance of drug resistant HIV
  species in CSF for CNS disease in setting of cART
• Presence of low abundance transmitted drug resistant
  variants in plasma predicts systemic virologic failure in
  setting of cART

   Ritola J Virology 2005; Schnell J Virology 2010, Canestri CID 2010, Bogoch J Infection 2011, Simen JID 2009
              HIV RNA in Plasma and CSF in Primary
                            Infection

                   8
                               HIV RNA Levels
                                                                      plasma
                   7
log 10 copies/ml




                   6
                   5
                   4
                   3
                   2
                   1
                       0       100      200      300
                           Days Post HIV Transmission
                                                        Spudich et al., JID, In press.
              HIV RNA in Plasma and CSF in Primary
                            Infection

                   8
                               HIV RNA Levels
                                                                      plasma
                   7
                                                                      CSF
log 10 copies/ml




                   6
                   5
                   4
                   3
                   2
                   1
                       0       100      200      300
                           Days Post HIV Transmission
                                                        Spudich et al., JID, In press.
            Characteristics of Study Subjects
 ID    Sex Age    Days post     CD4          CD8        Plasma       CSF HIV      CSF         CSF/
                     HIV      (cells/ul)   (cells/ul)   HIV RNA       RNA         WBC         Serum
                 Transmission                             (log10      (log 10     (cells/ul) Albumin
                                                        copies/ml)   copies/ml)


9055   M   32        104          619        924          5.38         3.51         12      6.42

9039   M   33         36          539       1591          5.57         4.30         53         -

9044   M   25        126          533        575          4.82         3.49         20      4.30

9024   M   33        217          974       1840          4.75         4.29         12      5.74

9058   M   29        109          237        872          5.18         3.67         4       2.44


  All subjects MSM from San Francisco, USA, enrolled
  between 2008 and 2010
    Drug Resistant Viral Variants Detected by
            Ultra Deep Sequencing
                                                          Population based          Ultra-deep
    Viral Quasispecies                                    Sanger Sequencing         Sequencing
                                                    100
                                                     90
                                % of HIV variants    80
                                                     70         WT
                                                     60         viral
                                                     50         variants
                                                     40
                                                     30
Within-host virus
                                                     20
population consists of
                                                     10
different viral variants
                                                      0                                               NNRTI
that are evolutionarily
related.
                                                                                                      resistant
                      New technologies provide the ability to sequence unique
                      individual viral genomes
  Kozal MJ. Next Gen Dx Summit. Washington DC 2010 & Paredes R. Asian Pasic Next Gen Conf. Hong Kong 2010
                                Ultra Deep Sequencing                                       Micro-reactors

   A                                         Add PCR Reagents
                                               & emulsion oil


    B
 Amplicon pool           Mix amplicons                                            PCR in “water-in-oil”             Isolate DNA containing
                        & capture beads                                               emulsion                               beads




 Load beads onto                                        Load Enzyme
 PicoTiter™Plate                                           Beads




Load PTP on Sequencer


                                           DNA                                                               Read Flowgram
                            Pyro-         Capture
                          sequence                                           T
                                                                     APS PPi
                                           Bead

                                                    Sulfurylase
                                                                     ATP
                                                    Luciferase                       luciferin
                                                                  Light + oxyluciferin



                                                                                                    Gega & Kozal. Future Virology 2011
 Subject 9055: 104 Days Post Transmission
32 yo MSM, CD4 619 CSF WBC 12, CSF/Serum Albumin 6.42
         VL                   PI                             RT
                Reads    %     PI ML   Codon   Reads    %     RT ML   Codon
                        Mut            Reads           Mut            Reads
                        Var                            Var
Plasma          8206          Wild Type        2876          Wild Type
         5.38
         log


CSF             6995          Wild Type        2369          Wild Type
         3.51
         log



 VL: HIV RNA level
 % Mut Var: % mutant variants detected by UDS
 ML: estimated mutational load = mutant variant frequency x HIV
 RNA level (VL)
   Subject 9039: 36 Days Post Transmission
33 yo MSM, CD4 539, CSF WBC 53
         VL                      PI                                 RT
                Reads   % Mut         PI ML    Codon     Reads   % Mut   RT   Codon
                         Var                   Reads              Var    ML   Reads
Plasma          6368    D30N           D30N     D30N     2524        Wild Type
         4.75           (0.69)         (388)   (6,378)
         log            V82A           V82A     V82A
                         (0.3)         (169)   (6,589)
                        N83D           N83D     N83D
                         (0.3)         (169)   (8,599)
                         I85V          I85V     I85V
                        (0.27)         (152)   (8,599)
CSF             7471             Wild Type               2575        Wild Type
         4.29
         log
   Subject 9044: 126 Days Post Transmission

25 yo MSM, CD4 533, CSF WBC 20, CSF/Serum Albumin 4.3
         VL                      PI                                 RT
                Reads   % Mut     PI ML       Codon      Reads   % Mut   RT   Codon
                         Var                  Reads               Var    ML   Reads
Plasma          7237     M46I         M46I     M46I      2876        Wild Type
         4.82           (0.46)        (304)   (6,130)
         log             I47V         I47V     I47V
                        (1.16)        (766)   (6,130)

CSF             8645    V82A          V82A      V82A     4394        Wild Type
         3.49           (0.39)         (12)   (10,644)
         log
  Subject 9024: 217 Days Post Transmission
33 yo MSM, CD4 974, CSF WBC 12, CSF/Serum Albumin 5.74
         VL                      PI                                   RT
                Reads   % Mut     PI ML   Codon     Reads   % Mut      RT ML      Codon
                         Var              Reads              Var                  Reads
Plasma          7352             Wild Type          1381    M184V*     M184V      M184V
         5.57                                               (0.33)     (1,226)    (1,196)
         log                                                T215D*     T215D      T215D
                                                            (99.41)    (369K)     (1,196)
                                                            T227L      T227L      T227L
                                                            (0.42)     (1,560)    (1,196)
CSF             6608    V82A      V82A    V82A      2396    F77L       F77L       F77L
         4.3            (0.52)    (104)   (7,917)           (0.29)     (58)       (3,095)
         log                                                T215D*     T215D      T215D
                        N83D      N83D    N83D              (99.43)    (19,838)   (2,277)
                        (0.27)    (54)    (7,917)           T227L*     T227L      T227L
                                                            (0.26)     (52)       (2,277)

                                                                *linkage present
      Subject 9058: 109 Days Post Transmission
 29 yo MSM, CD4 237, CSF WBC 4, CSF/Serum Albumin 2.44
         VL                       PI                                         RT
                Reads   % Mut PI ML             Codon      Reads   % Mut      RT ML      Codon
                         Var                    Reads               Var                  Reads
Plasma          6567              Wild Type                2642    D67G         D67G      D67G
         5.18                                                      (0.44)       (666)    (2,972)
         log                                                       T69N         T69N      T69N
                                                                   (1.31)      (1,983)   (2,972)
                                                                   G190E       G190E     G190E
                                                                   (0.34)       (515)    (2,619)
                                                                   T227L       T227L     T227L
                                                                   (0.27)       (409)    (2,619)
CSF             5582    D30N*          D30N      D30N      2628    L210W       L210W     L210W*
         3.37           (9.77)         (451)    (4,604)            (0.59)       (27)     (2,891)
         log            M46I*           M46I      M46I             G190E       G190E     G190E*
                         (8.1)         (374)    (4,604)            (1.56)       (72)     (2,891)
                        G73S           G73S      G73S
                        ( 5.16)        ( 238)   (6,589)
                        V82A*          V82A       V82A
                        (0.27)          (12)    ( 6,589)
                                                                            *linkage present
Longitudinal Plasma and CSF HIV RNA Levels
                           6
                                                Plasma HIV RNA
                                                                           9055
                           5                                               9039

       log 10 copies/ml
                           4                                               9044
                                                                           9024
                           3                                               9058
                           2

                           1

                           0
                               0          250        500       750         1000

                           6                     CSF HIV RNA
                           5
        log 10 copies/ml




                           4                                 DRV/RTV/RAL

                           3           TDF/FTC/RAL

                           2

                           1       TDF/FTC/DRV/RTV
                           0
                               0          250        500       750         1000
                                           Days Post HIV Transmission
                            Limitations
• Pilot, ‘proof of concept’ study with 5 subjects only
• Subjects with high CSF HIV RNA picked from cohort
• 0.2% chosen as lowest detection level as >3x greater than
  PCR error rate:
   – Error occurring in early rounds of amplification may still affect results
• Mutational load values only an estimate:
   – Calculated by multiplying the results from separate molecular
     assays using different primer sets
• As sample HIV RNA level decreases, the ability to obtain a
  fully representative sample of HIV RNA templates declines:
   – Levels of mutations may represent the proportion of sequenced
     PCR amplicons containing the mutation vs. the actual proportion in
     the sample
                      Conclusions

• UDS of HIV from CSF samples was successful in 5
  subjects with CSF HIV RNA > 3000 copies/ml.

• Marked differences can exist between quasispecies in
  blood and CSF at a median < 4 months after HIV
  transmission.

• Four patients had substantial differences in resistance
  mutations in plasma and CSF HIV variants which could
  potentially impact clinical response to cART.

• Purely descriptive study; larger and longitudinal studies
  are needed to determine clinical significance.
                Acknowledgements
UCSF/San Francisco:
• Study Volunteers
• Richard W. Price
• Evelyn Lee
• Julia Peterson
• Frederick Hecht
• Christopher Pilcher
• UCSF Options Study Staff
• Magnet Staff/Volunteers
• Teri Liegler
• SFGH/GIVI Virology Lab

Yale:
•   Arjet Gega               NIH/NIMH/NIAID
•   Michael Kozal
•   Jennifer Chiarella       SF AIDS Research Institute
                             UCSF REAC/Academic Senate
454 Life Sciences:
•   Birgitte Simen
•   Elizabeth St. John
•   Elizabeth Moreno

				
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posted:11/30/2012
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