HIV Neutralization Assays: p24 PBMC Assays as Compared with the Pseudovirus (TZM-bl) Assay Using Multiple HIV-1 Subtypes

Transcription

1 HIV Neutralization Assays: p24 PBMC Assays as Compared with the Pseudovirus (TZM-bl) Assay Using Multiple HIV-1 Subtypes Vicky Polonis The USMHRP: Walter Reed Institute of Research and The Henry M. Jackson Foundation WHO Global Neutralization Workshop, March 17, 2007, Varese, Italy

2 NeutNet Project: Reagents and Assays Employed A methods comparison in numerous international labs using 11 viruses (7 PV) and 4 reagents: 4E10, D, TriMab, scd4 PBMC: stimulated (3-4 days, PHA), Nabs +/- virus inc 30 min, 150,000/well added over night, washed and cultured (4-6 days), p24 meas. by ag. capture. % Neutralization is calculated as: [(p24 in control - p24 with Ab) X 100 p24 control] TZM-bl assay with pseudoviruses

3

4 NeutNet: IC 50 for 4 Reagents (7 viruses) 25 -PB -TZ TriMab * 25 4E10 * IC50 (ug/ml) PBMC Assay TZM-bl Assay IC50 (ug/ml) PBMC Assay TZM-bl Assay VI 191 SF162 MN(P) DU174 CM244 QH0692 AC10 Viral Isolate 0 VI 191 SF162 MN(P) DU174 CM244 QH0692 AC10 Viral Isolate D-52D 10 * scd IC50 (ug/ml) PBMC Assay TZM-bl Assay IC50 (ug/ml) 6 4 PBMC Assay TZM-bl Assay VI 191 SF162 MN(P) DU174 CM244 QH0692 AC10 Viral Isolate 0 VI 191 SF162 MN(P) DU174 CM244 QH0692 AC10 Viral Isolate

5 Summary: NeutNet Data Both EC p24 PBMC and TZM-bl pseudovirus assays run for 7 viruses. In general, IC 50 s/ic 80 s lower in TZM-bl pseudovirus assay- more sensitive. While the qualitative patterns of neutralization are similar for some reagents in both assays (ie, TriMab and 447 anti V3), distinct examples exist (ie. 4E10, Zeptometrix plasmas) where the 2 assays provide different results Recommend continuing parallel capacity for primary cell based assays and PV assays in cell lines.

6 SF162 Bal ID50: GMT of 6 HIV-1 + Serum Samples Bx08 W61D ADX Du151 TV1 CM Luc-UAB Luc-Duke Luc-ViroLogic PBMC-VRC PBMC-Duke ID50

7 ID50: Based on GMT of 6 HIV-1 + sera Frequency of Outliers Bal SF162 Bx08 W61D ADX Du151 TV1 CM244 PBMC-Duke +++ PBMC-VRC Luc-ViroLogic + Luc-Duke ++ Luc-UAB Pairwise comparisons of all groups tested by ANOVA (alpha =.01). Significant differences with one or more assays are represented by +.

8 Conclusions Uniform trends were seen in how each assay rank-ordered the neutralizationsensitivity of virus isolates and the neutralization-potency of serologic reagents, i.e., no indication of general qualitative differences in outcomes

9 The USMHRP Multi-clade Virus Panel and Cross-clade NAb 60 Pure clade Full-length sequenced isolates (10 each from clades A-D, CRF01, CRF02) The majority are from chronic infection 6 clade-specific plasma pools from subjects presumed to have pure clade/crf infections (Full-length sequenced from PBMC (Francine McCutchan s lab) Cloning and sequencing: functional gp160 env clones from 56/60 virus stocks

10 Env gp160 phylogenetic analysis: Panel of 60 Viruses CRF01_AE NP1695 NI1149 NI1052 NI1046 M02138 NP1525 NP1251 CM244 TH253 CM240 CM235 J H D G K F (Isolates from 15 countries, 10 from each of 6 clade/crfs 100 CF CRF02_AG 0002BBY 0014BBY MSC BBY 1970LE SE BBY KNH BBY 0005BBY KNH MV KER2018 UG029 DJ264 UG037 KER2008 DJ263 KNH1209 IbNG TZ02 KSM4030 KEQ23 KNH1144 KNH1135 RW TZ A03349 UG065 UG57128 A07412 E08364 A08483 UG114 D26830 NKU3006 E13613 J32228 UGE2343 KE2059 Red: Current or candidate vaccine strains, (Brown et al., J Virol 79: , 2005.) RL42 NP1538 US873 BAL LAI BX08 BZ167 US1 US33931 RF MN US4 BK132 B A BW0504 IN905 IN20635 SM145 TZ05 ZA151DU TZ_BD9 MW965 SE364 TZ04 TZA246 ET14 ET288 TZA125 MSC5016 C

11 The USMHRP Multi-clade Virus Panel and Cross-clade NAb Tested all 60 viruses using each of the 6 clade pools, a US pool, scd4 and Tri-mAb PBMC assay: reduction of p24 production at day 4; 50% and 80% endpoint neutralization titers were determined Prepared Pseudoviruses from the 56 functional env clones and tested them in the luciferase reporter, TZM-bl cell assay (at day 2); 50% and 80% endpoint titers were determined

12 International Panel: IC80s PBMC vs TZM-bl PBMC vs TZMbl titers IC R 2 = Titers of pseudoviruses on TZMbl cells Titers of primary isolates on PBMC (6 Plasma Pools) R 2 =0.004

13 Neutralization of HIV-1 Primary Isolates (PBMC assay) and Individual Pseudoviruses (TZM-bl assay) Using Clade-specific Plasma Pools Virus/ Clade: BZ167ec9/ B BZ167/ B ec2/ C 56313/ C GS16.ec1/ C GS16/ C Assay/Cells: TZM-bl PBMC TZM-bl PBMC TZM-bl PBMC Plasma Pool: A B C D CRF01_AE CRF02_AG US HIV # of AA Changes: Plasma dilution= 1:40

14 M47 M9 2F5 4E10 HIV+ P+ TZ - P+/- TZ++ P+ TZ++

15 HIV-1 Neutralization Using an anti-pip mab in the PBMC vs TZM-bl Assays A, B PBMC: A B C D C, D TZM-bl: C. Alving mab; Brown et al., J. Virol. 81: , 2007

16 Common features of Current Neutralization Assays Assay: Cells: Virus: Assay Length: Common endpoint: Rounds of Infection: Measures inhibition of Attachment/Entry- Cell-cell transmission- Coreceptors used: CCR5 density on cells: PBMC PBMC Uncloned primary 4-7 days EC or IC p24 Multiple Yes Yes R5, X4 (other?) Psuedovirus (PV) TZM-bl, JC53-BL13 Cloned env PV (or primary) 2-3 days Luciferase activity Single Yes No R5, X

17 CCR5 and CD4 Receptor Quantitation on TZM-bl Cells vs PBMC TZM-bl PBMC PHA-PBMC CD4 * * CCR5 * > 2 Log difference 6 Passages of TZM-bl, 6 different PBMC donors

18

19 The mechanism(s) of HIV entry into target cells: T lymphocytes: entry predominantly by classic retrovirus pathway of receptor dependent (ph independent) fusion at the plasma membrane Epithelial cells (HeLa and derivatives): 85-90% of virions enter by endocytosis, ph dependent and requires endosomal acidification (Schaeffer et al., W. Greene J. Virol. 78: , 2004) Others: J.V. Garcia et al., Ira Mellman

20 Why do some Antibodies function better in one assay than another? May be related to: Valency, avidity, affinity and effectiveness during the preattachment phase. Abs that function post-cd4 attachment and/or whose function is linked to coreceptor (CCR5) engagement may show greater assay-specific differences? Abs that can attach to the cell surface may show differences if endocytosis is a key entry pathway in epithelial cell models No single assay to date---what will best predict or reflect what occurs in natural targets in vivo? Dengue virus-->enhancement predicted by cell line model Thus, parallel assay evaluation is recommended

21 ACKNOWLEDGEMENTS The Trial Volunteers Siriraj Hospital Dr. Kovit Pattanapanyasat AFRIMS Dr. Sorachai Nittayaphan Dr. Mark desouza The JCRC staff K. Somsak Chantakulkij AIP Penprapa Chanbancherd International Sites Aventis Pasteur VaxGen DAIDS Janice M. Darden WRAIR/ HMJF Bruce Brown Lindsay Wieczorek Kara Lombardi Eric Odom Charline Bermudez Andrew RosaBorges Anita Gillis Dr. Merlin Robb Dr. Jerome Kim Dr. Deborah Birx VTC Dr. Punnee Pitisuttithum Dr. Prasert Thoncharoen TAVEG

22 Assay A, B, C Assay D, E, F

23 Carbohydrate Compound-Mediated Inhibition or Enhancement of HIV Globotriose PBMC 3'-sialyllactose PBMC Globotriose TZM-bl 3'-sialyllactose TZM-bl A B C D CRF Viral Clade

24 Why FL-Sequenced Pure HIV Panel? Loss of B vs E Serotype Observations Using a CRF01_ AE/B Recombinant Virus and AE or B Plasma % Neutralization E 2007se-E AE/B 2003se-B 1538-B Virus name - Subtype E pool B pool 1:20) (1623 AE/B : E gp120 / B gp41 )

25 NeutNet: IC 80 for 4 Reagents (7 viruses) -PB -TZ TriMab 4E IC80 (ug/ml) PBMC Assay TZM-bl Assay IC80 (ug/ml) PBMC Assay TZM-bl Assay VI 191 SF162 MN(P) DU174 CM244 QH0692 AC10 Viral Isolate 0 VI 191 SF162 MN(P) DU174 CM244 QH0692 AC10 Viral Isolate 447D-52D scd IC80 (ug/ml) PBMC Assay TZM-bl Assay IC80 (ug/ml) 6 4 PBMC Assay TZM-bl Assay VI 191 SF162 MN(P) DU174 CM244 QH0692 AC10 Viral Isolate 0 VI 191 SF162 MN(P) DU174 CM244 QH0692 AC10 Viral Isolate

26 Gold Standard PBMC Assay Primary human lymphocytes are stimulated in culture (3-4 days, PHA) and infected in the presence or absence of NAbs. The cells (150,000/well) are washed and cultured (4-6 days), the virus in culture fluids is collected, lysed and the core (gag) p24 is measured by antigen capture (kit). % Neutralization is calculated as: [(p24 in control - p24 with Ab) X 100 p24 control]

27 Neutralization of US-1/B Using Serum from Volunteer (BA) Prime-Boost B/B, Boost = o-gp160mn/b Pre 13% 11% Pre (duplicates) CD4-PE Post 0.5% 1% Post (duplicates) X % NT = 93% p24-fitc (day 182 post)

28 Neutralization of SI 2079 B/E Recomb. Virus (env BE) Value of double staining: Blocking CD4 down-regulation - X4 Uninf 55% 92% U+L=0.4% 92% 5% 4% Media 45% 91% CD4-PE E Pool 1:40 8% 6% 86% 47% 6% 47% B Pool 1:40 EC: B& E =0% CD4 down reg. blocked > 50% p24-fitc