Techniques in HIV Research PDF
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TECHNIQUES In H I V RESEARCH TECHNIQUES . In H I V RESEARCH Edited by Anna Aldovini and Bruce D. Walker M stockton press New York London Tokyo Melbourne Hong Kong Cover Photo Reprinted with permission of © 1989 George V. KelvinlScience Graphics © Stockton Press, 1990 All rights reserved. No part of this publication may be reproduced, or transmitted, in any form or by any means, without permission. Published in the United States and Canada by Stockton Press 15 East 26th Street, New York, N.V. 10010 Library of Congress Cataloglng-in-Publication Data Techniques in HIV research 1 edited by Anna Aldovini and Bruce D. Walker. p. cm. ISBN 978-1-349-11890-8 ISBN 978-1-349-11888-5 (eBook) DOI 10.1007/978-1-349-11888-5 1. HIV (Viruses)-Laboratory manuals. I. Aldovini, Anna, 1957-. II. Walker, Bruce D., 1952- QR414.6.H58T431990 616.97'92027 -dc20 90-9481 CIP Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990 Brunei Road, Houndmills, Basingstroke, Hants RG21 2XS, England British Library Cataloguing in Publication Data Techniques of HIV research. 1. Man. AIDS virus. Diagnosis. Laboratory techniques I. Aldovini, Anna II. Walker, Bruce 616.9792075 UK ISBN 978-0-333-51806-9 U.S. ISBN 978-0-333-51806-9 987654321 Contributors Jeffrey Ahlers Marlo Clerici Metabolism Branch, National Cancer Experimental Immunology Branch, National Institute, National Institutes of Health Cancer Institute, National Institutes of Health Bethesda, MD Bethesda, MD Anna Aldovini Brian Conway Whitehead Institute Infectious Disease Unit, Massachusetts Cambridge, MA General Hospital and Harvard Medical School Boston, MA Salvatore J. Arrigo and Ottawa General Hospital, University Division of Hematology-Oncology, of Ottawa Departments of Medicine and Microbiology Ottawa, Canada and Immunology UCLA School of Medicine and Jonsson Ronald C. Desrosiers Comprehensive Cancer Center Department of Microbiology, New England Los Angeles, CA Regional Primate Research Center, Harvard Medical School, Southborough, MA Roy E. Byington Infectious Disease Unit, Massachusetts Steven Dewhurst General Hospital and Harvard Department of Cancer Biology Medical School Harvard School of Public Health Boston, MA Boston, MA Jay A. Berzofsky Mark Feinberg Metabolism Branch, National Cancer Insti Whitehead Institute tute, National Institutes of Health Cambridge, MA Bethesda, MD Suzanne Gartner Kevin Byron Virologyllmmunology Department, Primate Department of Pediatrics, Research Institute, New Mexico State Uni University of Massachusetts Medical School versity Worcester, MA Holloman, NM Irvin S. V. Chen Alan S. Go Division of Hematology-Oncology, Division of Hematology-Oncology, Departments of Medicine and Microbiology Departments of Medicine and Microbiology and Immunology and Immunology UCLA School of Medicine and Jonsson UCLA School of Medicine and Jonsson Comprehensive Cancer Center Comprehensive Cancer Center Los Angeles, CA Los Angeles, CA v vi CONTRIBUTORS David D. Ho Mlkulas Popovic Department of Medicine, UCLA Virology/Immunology Department, Primate School of Medicine, Infectious Diseases Research Institute, New Mexico Division, Cedars-Sinai Medical Center State University Los Angeles, CA Holloman, NM Anne Hosmalin Barbara J. Potts Metabolism Branch, National Cancer Repligen Corporation Institute, National Institutes of Health Cambridge, MA Bethesda, MD Marjorie Robert-Guroff Victoria A. Johnson Laboratory of Tumor Cell Biology, Infectious Disease Unit, Massachusetts National Cancer Institute, National Institutes General Hospital and Harvard of Health Medical School Bethesda, MD Boston, MA Gene M. Shearer Phyllis Kankl Experimental Immunology Branch, National Department of Cancer Biology, Harvard Cancer Institute, National Institutes of Health School of Public Health Bethesda, MD Boston, MA Robert H. Singer Joan C. Kaplan Department of Cell Biology, University of Infectious Disease Unit, Massachusetts Massachusetts Medical School General Hospital and Harvard Worcester, MA Medical School Boston, MA John L. Sullivan Department of Pediatrics, University of Richard A. Koup Massachusetts Medical School Departments of Medicine and Pediatrics, Worcester, MA Program in Molecular Medicine, University of Massachusetts Medical School Karin li'avers Worcester, MA Department of Cancer Biology, Harvard School of Public Health Jeanne B. Lawrence Boston, MA Department of Cell Biology, University of Massachusetts Medical School Bruce D. Walker Worcester, MA Infectious Disease Unit, Massachusetts General Hospital and Harvard Lynne Lederman Medical School Applied bioTechnology, Inc. Boston, MA Cambridge, MA Kent J. Weinhold James I. Mullins Department of Surgery, Duke University Department of Microbiology and Medical Center Immunology, Stanford University School Boston, MA of Medicine Stanford, CA Sam Yin Applied bioTechnology Peter L. Nara Cambridge, MA National Cancer Institute Frederick Cancer Research Facility Jerome A. Zack Frederick, MD Division of Hematology-Oncology, Departments of Medicine and Microbiology Julie Overbaugh and Immunology, Department of Microbiology, University UCLA School of Medicine and Jonsson of Washington Comprehensive Cancer Center Seattle, WA Los Angeles, CA Table of Contents Preface x SECTION I VIRAL DIAGNOSTICS 1 Chapter 1 HIV Antibody Detection in Serum 3 Karin Travers and Phyllis J. Kanki Enzyme-Linked Immunosorbent Assay (ELISA) 4 Western Blot (Immunoblot) 6 Radioimmunoprecipitation (RIP) 10 Chapter 2 Detection of HIV in Clinical Specimens 15 HIV-1 p24 Core Profile ELISA 15 Du Pont NEN Research Products, In situ Hybridization to Detect HIV: Elements of a Nonradioactive Test 30 Robert H. Singer, Kevin Byron, Jeanne B. Lawrence, John L Sullivan, Sam Yin, Lynne Ledermann Detection of HIV-1 by PCR in Clinical Specimens 40 Brian Conway Southern Blot Analysis 47 Anna A/dovini SECTION" BASIC VIROLOGIC TECHNIQUES 51 Chapter 3 Virus Isolation and Production 53 Suzanne Gartner and Mikulas Popovic HIV-1 Isolation from Peripheral Blood 54 HIV-1 Isolation from Tissues 63 Virus Production in Stable Cell Lines 66 Chapter 4 Quantitative Assays for Virus Infectivity 71 Infectivity Assay (Virus Yield Assay) 71 Victoria A. Johnson and Roy E. Byington n Quantitative Infectivity Synctium-Forming Microassay Peter L Nara Chapter 5 Quantitative Assays for Virus Detection 87 HIV Indirect Immunoflourescence Assays 87 Victoria A. Johnson and Roy E. Byington HIV-Infected Cell Fusion Assay 92 Victoria A. Johnson and Bruce D. Walker vii vIIi TABLE OF CONTENTS Modified HIV-1 p24 Antigen ELISA 95 Victoria A. Johnson and Roy E. Byington Reverse Transcriptase (RT) Activity Assay 98 Victoria A. Johnson, Roy E. Byington, and Joan C. Kaplan "Mini" Reverse Transcriptase Assay 103 Barbara J. Potts Quantitative Culture Assay for HIV-1 in Peripheral Blood 107 Richard A. Koup and David D. Ho Quantitative Polymerase Chain Reaction 113 Alan S. Go, Jerome A. Zack, Salvatore J. Arrigo, and Irvin S. Y. Chen Chapter 6 Virus Purification, Preparation of Infectious Virus Stock, and Virus Storage 121 Ronald C. Desrosiers Virus Purification 121 Use of Purified Virons for ELISA 123 Western Blot 126 Preparation and Storage of Infectious Virus Stock 126 SECTION III MOLECULAR BIOLOGY 129 Chapter 7 Proviral DNA Cloning 131 Julie Overbaugh, Steven Dewhurst and James I. Mullins Genomic Cloning in Bacteriophage A 132 Cloning via Polymerase Chain Reaction (PCR) 139 Chapter 8 Transfection of Molecularly Cloned HIV Genomes 147 Anna Aldovini and Mark B. Feinberg DNA Transfection 147 Selection of Stable Cell Lines 159 SECTION IV VIRUS-SPECIFIC HOST IMMUNE RESPONSE 177 Chapter 9 Neutralizing Antibodies 179 Marjorie Robert-Guroff Chapter 10 Nonrestricted Forms of Anti-HIV-1 Cytoxicity 187 Kent J. Weinhold Anti-HIV-1 ADCC (Indirect ADCC) 188 Anti-HIV-1 CMC (Direct ADCC) 194 Chapter 11 HIV-1-Specific Cytoxic T Lymphocytes 201 Bruce D. Walker Chapter 12 T Helper Cell Responses 211 Jeffrey Ahlers, Mario Clerici, Anne Hosmalin, Gene M. Shearer, and Jay A. Berzofsky Lymph Node T Cell Proliferation in Mice: Identification of Immunodominant Helper T Cell Sites Within the Human Immunodeficiency Virus (HIV) Envelope 212 Proliferation of Peripheral Blood T Cells from the Rhesus Monkey (Macaca Muletta) 214 Antigen-induced Proliferation and Interleukin 2 Production by Human Peripheral Blood Leukocytes 215 TABLE OF CONTENTS Ix SECTION V EVALUATION OF CANDIDATE ANTI-HIV AGENTS IN VITRO 223 Chapter 13 Evaluation of Candidate Anti-HIV Agents in vitro 225 Victoria A. Johnson APPENDICES Appendix 1 Source of Reagents 239 Appendix 2 Biosafety Issues - HIV 257 Agent Summary Statement for Human Immunodeficiency Viruses (HIVs) Including HTLV-III, LAV, HIV-1, and HIV-2 261 Occupationally Acquired Human Immunodeficiency Virus Infections in Laboratories Producing Virus Concentrates in Large Quantities: Conclusions and Recommendations of an Expert Team Convened by the Director of the National Institutes of Health (NIH) 279 Safety Guidelines - Vaccinia 285 INDEX 287 Preface Infection with HIV-1 and related retroviruses has become a global medical crisis, requiring an international scientific effort in an attempt to stem the spread of infection, as well as to modify the course of disease in those persons already infected. For these purposes, the number of laboratories investigating basic scientific aspects of viral pathogenesis has swelled, and numerous techniques adapted specifically to the study of molecular biologic, virologic, and immunologic aspects of HIV have been developed. Techniques in HIV Re search has been written with the help of active investigators in the field of HIV research in order to make these techniques widely available, not only to established investigators, but also to those now entering the field. The manual is divided into five broad sections. Section I deals with viral diagnostics, both using serum and tissue specimens. Section II includes a large variety of basic virologic techniques as applied to HIV, such as virus isolation and production, quantitative infectivity assays, quantitative assays for virus detection, and virus purification and storage. Section III deals with basic molecular biologic techniques used in HIV research, including proviral DNA cloning and transfection techniques. Numerous assays for virus-specific host immune responses are outlined in Section IV, and testing of candidate antiretroviral compounds is discussed in Section V. An appendix has been included with sections covering important biosafety issues as well as sources of specific reagents. Although the main focus of this book has been HIV research techniques, many of these are directly applicable to SIV research as well. The protocols in this manual have been prepared by the investigators who helped to develop these techniques, and in each case the authors have also discussed potential problems and pitfalls. We have attempted to include a broad spectrum of established techniques; space has not permitted the manual to be absolutely comprehensive, however, nor have we been able to include all existing alternative protocols to the ones presented. The user of this manual must also be aware that many of these techniques may undergo evolution and refinement in the future, but are printed now as the present "state of the art." We welcome and encourage suggestions and contributions of protocols not presently included for future expanded editions of this manual. We are indebted to the many distinguished contributors who have taken the time to provide these problem-oriented protocols, and thank them for their considerable efforts. We would also like to acknowledge the contribution of Ms. Carolyn C. Carpenter, who not only played a major administrative role in this endeavor, but also converted the entire content of the manual to camera-ready copy. Her expertise and dedication have been significant factors in the completion of this manual. Anna Aldovini Bruce D. Walker xl
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