# Presentation: 236094

## Neutralizing Antibody Assays for HIV-1, SIV and SHIV:  Recent Advances in Technology

**David C. Montefiori, Ph.D.**
**Laboratory for AIDS Vaccine Research & Development**
**Duke University Medical Center**
**Durham, NC**
**monte@duke.edu**

## Why Neutralizing Antibodies are Considered Important to HIV/AIDS Vaccines

** ****Pre-existing neutralizing antibodies (active and passive ****	****immunization) can prevent AIDS virus infection through ****	****intravenous, vaginal, rectal and oral routes of challenge in ****	****nonhuman primates.**
** ****A rapid secondary responses to infection that is primed by prior ****	****vaccination might control virus replication, prevent early ****	****immunologic damage, prolong survival and reduce the probability ****	****of transmitting virus.**

## Key Parameters of the Neutralizing Antibody Response to Monitor

**Magnitude**
**Breadth**
**Duration**
**Kinetics**
**Epitope specificity**
**Escape**
**Systemic & mucosal**
**Correlate of immunity**

## Stages of HIV-1 Entry as Targets for Neutralization

**Stages of HIV-1 Entry as Targets for Neutralization**

**NAbs are entry inhibitors**

## Assay Requirements

**  ****Sensitive, quantitative, reproducible, high ****	****throughput ****	****and have **_**correlative value**_
**  ****Optimized and validated to meet GCLP requirements ****	****for human clinical trials**
**  ****Reagents need to be standardized and traceable**
**  ****Assay needs to be transferable to multiple labs**

## Various Assays Formats 

**Virus + Ab**

**Add cells**

**Measure infection**

**1 hr**

**days**

** ****TCLA**
** ****Primary isolates**
** ****TCLA and primary isolates**

** ****CD4****+**** cell lines**
** ****PBMC**
** ****Genetically engineered cell lines expressing HIV entry receptors and containing reporter genes**

** ****Syncytia**
** ****Cell-killing**
** ****Plaques**

** ****Gag Ag ELISA or FACS**
** ****RT activity**
** ****luciferase**
** ****green fluorescence protein**
** ****secreted alk. phosphatase**
** ****B-gal**

**Various Assays Formats **

## PBMC Assay

** ****Advantages:**
**  ****Gold standard for many years**
**  ****Broadly susceptible to infection by primary isolates**
**  ****Correlative value in passive Ab studies**
** ****Disadvantages:**
**  ****Time consuming and labor intensive**
**  ****Expensive**
**  ****Lacks precision**
**  ****Difficult to validate (e.g., PBMC from different donors, mixed cell ****	****population, viral quasispecies)**

## Latest Technology

**Tat-Regulated Reporter Gene Assays in Genetically Engineereed Cell Lines Using Molecularly Cloned Env-Pseudotyped Viruses**

## Luciferase Reporter Gene Assay in TZM-bl Cells Based on Single-Round Infection with Molecularly Cloned Env-Pseudotyped Viruses

**TZM-bl (JC53-bl) is a genetically engineered HeLa cell line that expresses CD4, CXCR4 and CCR5 and contains Tat-inducible Luc and ********-Gal reporter genes: **
**High success rate in single-round infections**
**Increased assay capacity (2-day assay)**
**Increased precision (accurately measure 50% neutralization)**
**Improved level of standardization (stable cell line)**
**Optimized and validated**

## SEQUENTIAL EVENTS IN DETECTING NEUTRALIZATION OF ENV-PSEUDOTYPED VIRUSES IN TZM-BL CELLS 

**SEQUENTIAL EVENTS IN DETECTING NEUTRALIZATION OF ENV-PSEUDOTYPED VIRUSES IN TZM-BL CELLS **

## SEQUENTIAL EVENTS IN DETECTING NEUTRALIZATION OF ENV-PSEUDOTYPED VIRUSES IN TZM-BL CELLS 

**SEQUENTIAL EVENTS IN DETECTING NEUTRALIZATION OF ENV-PSEUDOTYPED VIRUSES IN TZM-BL CELLS **

## OPTIMIZATION OF THE TZM-BL ASSAY

**  ****Cell culture conditions**
**  ****Range of isolates that infect adequately**
**  ****Cell number**
**  ****Virus dose**
**  ****Incubation time**
**  ****Choice of 96-well plates for luminescence**
**  ****Luminescence readings**
**  ****DEAE-dextran**
**  ****Indinavir**
**  ****Uncloned vs cloned virus**

**OPTIMIZATION OF THE TZM-BL ASSAY**

## VALIDATION OF THE TZM-BL ASSAY

_**Specificity**_**:**
**  ****Background activity of normal human serum and plasma**
_**Accuracy**_**:**
**  ****Comparisons have been made to other in-house assays and assays performed in other labs **
_**Precision**_**:**
**  ****Well-to-well variability in cell control, virus control and test wells**
**  ****Intra- and inter-assay variability**
**  ****Intra- and inter-operator variability**
_**Limits of Quantitation**_**:**
**  ****Upper and lower limits established **
_**Linearity & Range**_**:**
**  ****Neutralization curves generated with positive serum samples and mAbs show a consistent pattern of linearity over a range of 20-85% reductions in RLU.  Values in this range are directly proportional to the concentration of neutralizing antibodies in the sample.**
_**Ruggedness & Robustness**_**:**
**  ****Stability of CD4, CCR5 and CXCR4 expression**
**  ****Stability of TZM-bl infectivity after multiple passages**
**  ****Effect of DEAE-dextran on neutralizing antibody activity**
**  ****Effect of heat-inactivation on neutralizing antibody activity**
**  ****Serum vs plasma**
**  ****Uniformity of multiple luminometers**

**VALIDATION OF THE TZM-BL ASSAY**

## Linear Range of Infection in TZM-bl Cells

**Relative luminescence units (RLU)**

**TCID****50**** added per well**

**Env-pseudotyped virus**

**Linear Range of Infection in TZM-bl Cells**

**Cell killing at high virus input**

## Neutralization Curves Under Optimal TZM-bl Assay Conditions

**% Reduction in RLU**

**Concentration (********g/ml)**

**Neutralization Curves Under Optimal TZM-bl Assay Conditions**

**Env-pseudotyped virus QH0692.42**

** ****200 TCID50**
** ****10,000 cells/well**
** ****30 ********g/ml DEAE dextran**
** ****RLU measured after 48 hrs**

**IgG1b12 - circle**
**2G12 - triangle**
**2F5 - square**

***Control RLU = 197,433***
***Background RLU = 1,029***
***Range = 196,404 RLU***

## Examples of Inter-Assay and Inter-Operator Variability in the TZM-bl Assay: Neutralizing Activity of TriMab

**Three operators:  HG, NH and BW**

## Examples of Intra-Assay Variation:

**Examples of Intra-Assay Variation:**
** ****Comparison of Two Luciferase Kits (PerkinElmer vs Promega)**

**SF162.LS**

## Internal Proficiency Test with an External Panel of Reagents

**Six operators assayed 7 positive serologic reagents against 6 reference strains of Env-pseudotyped HIV-1 in TZM-bl cells (SOP HVTN02-A0009):**
**  ****Mean variance =  32 ******** 16% of mean titers**
**  ****Range =  10 - 79% of mean titers**

## Intra-Laboratory Variability in the TZM-bl Assay:  Results of 3 independent operators 

**QH0692.42 -**

**AC10.0.29 -**

**PVO.4 -**

**WITO.33 -**

**THRO.18 -**

**CAAN.A2 -**

**Pool C**

**QH0692.42 -**

**AC10.0.29 -**

**PVO.4 -**

**WITO.33 -**

**THRO.18 -**

**CAAN.A2 -**

**2F5**

**QH0692.42 -**

**AC10.0.29 -**

**PVO.4 -**

**WITO.33 -**

**THRO.18 -**

**CAAN.A2 -**

**4E10**

**QH0692.42 -**

**AC10.0.29 -**

**PVO.4 -**

**WITO.33 -**

**THRO.18 -**

**CAAN.A2 -**

**TriMab**

**QH0692.42 -**

**AC10.0.29 -**

**PVO.4 -**

**WITO.33 -**

**THRO.18 -**

**CAAN.A2 -**

**Pool B**

**QH0692.42 -**

**AC10.0.29 -**

**PVO.4 -**

**WITO.33 -**

**THRO.18 -**

**CAAN.A2 -**

**2G12**

**Intra-Laboratory Variability in the TZM-bl Assay:  Results of 3 independent operators **

**QH0692.42 -**

**AC10.0.29 -**

**PVO.4 -**

**WITO.33 -**

**THRO.18 -**

**CAAN.A2 -**

**Neg. Serum**

**Inside bar = 2-fold variation from mean;  Outside bar = 3-fold variation from mean**

## Program of External Proficiency Testing for the TZM-bl Neutralizing Antibody Assay

**  ****Initial round of testing**
** ****Assess inter-laboratory variation under conditions of ****	****relaxed standardization**
**  ****Subsequent rounds of testing**
**  ****Confirm the key parameters that affect assay performance**
**  ****Revise and validate the assay SOP**
**  ****Develop an SOP for proficiency testing**
**  ****Validate the proficiency testing SOP**

## REFERENCES

**Wei, X., J. M. Decker, S. Wang, H. Hui, J. C. Kappes, X. Wu, J. F. Salazar-Gonzalez, M. G. Salazar, J. M. Kilby, M. S. Saag, N. L. Komarova, M. A. Nowak, B. H. Hahn, P. D. Kwong, and G. M. Shaw. 2003.  Antibody neutralization and escape.  **_**Nature**_** 422:307-312.**
**Montefiori, D.C.  (2004)  Evaluating neutralizing antibodies against HIV, SIV and SHIV in luciferase reporter gene assays.  **_**Current Protocols in Immunology**_**, (Coligan, J.E., A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W. Strober, and R. Coico, eds.), John Wiley & Sons, 12.11.1-12.11.15.**
**Mascola, J. R.,  P. D'Souza, P. Gilbert, B. Hahn, N. L. Haigwood, L. Morris, C. J. Petropoulos, V. R. Polonis, M. Sarzotti-Kelsoe, and D. C. Montefiori. (2005) Recommendations for the design and use of standard virus panels to assess the neutralizing antibody response elicited by candidate human immunodeficiency virus type 1 vaccines.  **_**J. Virol**_**. 79:10103-10107.**
**Li, M., F. Gao, J.R. Mascola, L. Stamatatos, V.R. Polonis, M. Koutsoukos, G. Voss, P. Goepfert, P. Gilbert, K.M. Greene, M. Bilska, D.L. Kothe, J.F. Salazar-Gonzalez, X. Wei, J.M. Decker, B.H. Hahn, and D.C. Montefiori.  (2005) Human immunodeficiency virus type 1 *****env***** clones from acute and early subtype B infections for standardized assessments of vaccine-elicited neutralizing antibodies.  **_**J. Virol**_**., 79:10108-10125.**
**Li, M,.  J.F. Salazar-Gonzalez, C.A. Derdeyn, L. Morris, C. Williamson, J.E. Robinson, J.M. Decker, Y. Li, M.G. Salazar,****  ****V.R. Polonis, K. Mlisana, S.A. Karim, K. Hong, K.M. Greene, M. Bilska, J.T. Zhou, S. Allen, E. Chomba, J. Mulenga, C. Vwalika, F. Gao, M. Zhang, B.T.M. Korber, E. Hunter, B.H. Hahn, and D.C. Montefiori.  (2006)  Genetic and neutralization properties of acute and early subtype C human immunodeficiency virus type 1 molecular *****env***** clones from heterosexually acquired infections in southern Africa.  **_**J. Virol**_**., in press.**

**REFERENCES**

## Dr. Montefiori’s laboratory is funded by:

_**Division of AIDS/NIAID/NIH**_**: **
** ****Primate Core Immunology Laboratory for AIDS Vaccine Research and Development (PCIL)**
** ****HIV Vaccine Trials Network (HVTN)**
** ****Center for HIV/AIDS Vaccine Immunology (CHAVI)**
_**Bill & Melinda Gates Foundation**_**:**
** ****Collaboration for AIDS Vaccine Discovery (CAVD)**