634
A Simple Antigen-Reduction Assay for the Measurement of Neutralizing Antibodies to Hepatitis A Virus David L. Krah, Raju D. Amin, David R. Nalin, and Philip J. Provost
Departments of Cellular and Molecular Biologyand of Clinical Research, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania
Humoral immune responses to hepatitis A virus (HAV), the causative agentof viral hepatitisA, have been measured by methods including radioimmunoassay [1-3], immune adherencehemagglutination assay [4], ELISA [5], and hemagglutination inhibition [6] and serum neutralization [5, 7-11] assays. Onlythe serumneutralization assayidentifies biologicallyrelevant antibodies [9, 12] and is therefore the preferred testformeasuring antibodies to HAV, particularly those generated after vaccination. However, existing neutralization assays are relatively cumbersome and unreliable. Wedescribea simple and reliableHAV antigen-reduction neutralization assay (HAVARNA) and its use to measureantibodies in sera from recipients of a live-attenuated HAV vaccine. Materials and Methods Cells. MRC-5 (fetalhumandiploidlung)cells, populationdoubling level 27-31, were seeded as 6 x 1()4 cells in l-ml volumes of Eagle's basal medium (with Hanks' salts) with galactose (formula 78-0264PJ; GIBCO, GrandIsland,NY), 10%fetalcalfserum(FCS, not heat inactivated), 50 JLg/ml neomycin (OIBCO), and 2 mM L-glutamine (GIBCO) in 24-well plates (3424, MarkIl; Costar, Cambridge, MA). Cultureswere incubated at 35°C in a humidified 5% C02 atmosphere. Medium was removed on day 4 (±1 day) and
Received 5 July 1990; revised 17 September 1990. Presentedin part: International Symposium on Viral Hepatitis and Liver Disease, Houston, April 1990 (abstract 30). All participants gave informed consent. Reprints or correspondence: Dr. David L. Krah, Departmentof Cellular andMolecular Biology, MerckSharp& DohmeResearch Laboratories, Bldg. 16, Rm. 203, West Point, PA 19486. The Journal of Infectious Diseases 1991;163:634-637 © 1991 by The University of Chicago. All rights reserved. 0022-1899/91/6303-0035$01.00
replaced with l-ml maintenance medium(MM; Williams'medium E [GIBCO], 0.5% heat-inactivated FCS, 50 JLg/ml neomycin, and 2 mM L-glutamine). Confluent monolayers were used for infection (7-11 days after planting). Virus. The prototype indicator virus for the neutralization assay was Merck HAV strain CR326F(variantF') of live-attenuated HAV vaccine [2]. This strainhad beenadaptedto growthin MRC-5 cellsat 35°C. Aliquots (l-ml) from a standard preparation were stored in the vapor phase of a liquid nitrogen refrigerator. Sera. Sera were from healthy adults who received the variant F'vaccine. A standardglobulinpreparation(World HealthOrganization [WHO] reference anti-HAY globulin 1), obtained from the Center for Biologics Evaluation and Research, Foodand Drug Administration (Bethesda, MD), was reconstituted and diluted 1:5 in PBS, and aliquots werestoredat -70°C. Titersreportedfor this globulin were corrected for this initial 1:5 dilution. Modified HAVAR assay. The modified HAVAB assay for antibodies to HAV was done as described elsewhere [1]. Neutralization assay. A standardHAV preparation, containing 107 TCIDso/ml in Williams' mediumE with 50 JLg/ml neomycin and 2 mM glutamine, was mixed with 0.1 volumes of 15% (wt/vol, in distilled water) octylglucoside ([OG] 0-8001; SigmaChemical,St. Louis)in 2-mlcryovials (Corning [NY] GlassWorks) and incubated for 30 min at 4°C to increasethe susceptibility of HAV to neutralization. Virus was then diluted 1:40 in MM, and 300-JLI volumes were mixed with 300 ~l of dilutions of heat-inactivated (56°C, 30 min) sera in MM. Appropriate amounts of the 1:40 HAV dilution were diluted 1:2 with MM for use as a "no serum"control and in the"virusdilution series" (VDS). Samples wereincubated for16-20 h at 4°C, thenfor 1hat 35°C, followed bya 1:2.5dilutionwith 900-~1 MM (1:200 final HAV dilution, 100% viral inoculum concentration). The use of a viral concentrate (1:40 dilution, vs. 1:200in the final solution) duringthe serum incubations provided increased stability of viral infectivity. Samples used for the VDS were diluted further to provide viral concentrations of 80%, 70%, 60 %, 50%, 40%, 30%, and 20% of the neutralization inoculum (that mimics different amounts of viral neutralization). Mediumwas removed from MRC-5 cells in 24-well plates, and
Downloaded from http://jid.oxfordjournals.org/ at University of Sussex on July 19, 2015
A simplified hepatitis A virus (HAY) antigen-reduction neutralization assay (HAVARNA) was developed to permit the measurement of biologically active antibodies in recipients of candidate HAV vaccines. Degrees of neutralization were measured from the reduction in the amount of HAV antigen synthesized by 7-10days after infection ofMRC-5 (fetal human diploid lung) cell cultures. Sera producing a ~50% reduction in viral infectivity were scored as neutralizing. The assay was applied to demonstrateserum HAV neutralizingactivity in 10of 10and 9 of 10recipients of 107 and 1()6 TCIDso doses, respectively, of the Merck CR326F (F' variant) liveattenuated vaccine. The dilution end points of selected sera ranged from 1:10 to 1:640. The dilution end point of the World Health Organization reference globulin no. 1 was 1:530,000 (0.2 mlU/ml of HAV antibody). The HAVARNA provided a rapid, sensitive,and reproducible means to measure neutralizing antibodies to HAV.
1ID 1991;163 (March)
Concise Communications
Results The variant F' live-attenuated HAV vaccine was chosen as the prototype indicator virus for the development of the HAVARNA becauseit lackedvirulencein humans,chimpanzees, and marmosets [2], produced more reproducible antigen under the describedassayconditions thanhigherpassage preparations(data not shown), and wasavailable in sufficient amountsto providea standard virus for use in neutralization assays.
HAV was pretreated with the nonionic detergent, 00, to enhancevirus susceptibility to neutralization. 00 treatment of HAV increasedneutralization activity by the WHO referenceglobulin10-fold and reduced the neutralization-resistant fraction from 40% to
A Simple Antigen-Reduction Assay for the Measurement of Neutralizing Antibodies to Hepatitis A Virus David L. Krah, Raju D. Amin, David R. Nalin, and Philip J. Provost
Departments of Cellular and Molecular Biologyand of Clinical Research, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania
Humoral immune responses to hepatitis A virus (HAV), the causative agentof viral hepatitisA, have been measured by methods including radioimmunoassay [1-3], immune adherencehemagglutination assay [4], ELISA [5], and hemagglutination inhibition [6] and serum neutralization [5, 7-11] assays. Onlythe serumneutralization assayidentifies biologicallyrelevant antibodies [9, 12] and is therefore the preferred testformeasuring antibodies to HAV, particularly those generated after vaccination. However, existing neutralization assays are relatively cumbersome and unreliable. Wedescribea simple and reliableHAV antigen-reduction neutralization assay (HAVARNA) and its use to measureantibodies in sera from recipients of a live-attenuated HAV vaccine. Materials and Methods Cells. MRC-5 (fetalhumandiploidlung)cells, populationdoubling level 27-31, were seeded as 6 x 1()4 cells in l-ml volumes of Eagle's basal medium (with Hanks' salts) with galactose (formula 78-0264PJ; GIBCO, GrandIsland,NY), 10%fetalcalfserum(FCS, not heat inactivated), 50 JLg/ml neomycin (OIBCO), and 2 mM L-glutamine (GIBCO) in 24-well plates (3424, MarkIl; Costar, Cambridge, MA). Cultureswere incubated at 35°C in a humidified 5% C02 atmosphere. Medium was removed on day 4 (±1 day) and
Received 5 July 1990; revised 17 September 1990. Presentedin part: International Symposium on Viral Hepatitis and Liver Disease, Houston, April 1990 (abstract 30). All participants gave informed consent. Reprints or correspondence: Dr. David L. Krah, Departmentof Cellular andMolecular Biology, MerckSharp& DohmeResearch Laboratories, Bldg. 16, Rm. 203, West Point, PA 19486. The Journal of Infectious Diseases 1991;163:634-637 © 1991 by The University of Chicago. All rights reserved. 0022-1899/91/6303-0035$01.00
replaced with l-ml maintenance medium(MM; Williams'medium E [GIBCO], 0.5% heat-inactivated FCS, 50 JLg/ml neomycin, and 2 mM L-glutamine). Confluent monolayers were used for infection (7-11 days after planting). Virus. The prototype indicator virus for the neutralization assay was Merck HAV strain CR326F(variantF') of live-attenuated HAV vaccine [2]. This strainhad beenadaptedto growthin MRC-5 cellsat 35°C. Aliquots (l-ml) from a standard preparation were stored in the vapor phase of a liquid nitrogen refrigerator. Sera. Sera were from healthy adults who received the variant F'vaccine. A standardglobulinpreparation(World HealthOrganization [WHO] reference anti-HAY globulin 1), obtained from the Center for Biologics Evaluation and Research, Foodand Drug Administration (Bethesda, MD), was reconstituted and diluted 1:5 in PBS, and aliquots werestoredat -70°C. Titersreportedfor this globulin were corrected for this initial 1:5 dilution. Modified HAVAR assay. The modified HAVAB assay for antibodies to HAV was done as described elsewhere [1]. Neutralization assay. A standardHAV preparation, containing 107 TCIDso/ml in Williams' mediumE with 50 JLg/ml neomycin and 2 mM glutamine, was mixed with 0.1 volumes of 15% (wt/vol, in distilled water) octylglucoside ([OG] 0-8001; SigmaChemical,St. Louis)in 2-mlcryovials (Corning [NY] GlassWorks) and incubated for 30 min at 4°C to increasethe susceptibility of HAV to neutralization. Virus was then diluted 1:40 in MM, and 300-JLI volumes were mixed with 300 ~l of dilutions of heat-inactivated (56°C, 30 min) sera in MM. Appropriate amounts of the 1:40 HAV dilution were diluted 1:2 with MM for use as a "no serum"control and in the"virusdilution series" (VDS). Samples wereincubated for16-20 h at 4°C, thenfor 1hat 35°C, followed bya 1:2.5dilutionwith 900-~1 MM (1:200 final HAV dilution, 100% viral inoculum concentration). The use of a viral concentrate (1:40 dilution, vs. 1:200in the final solution) duringthe serum incubations provided increased stability of viral infectivity. Samples used for the VDS were diluted further to provide viral concentrations of 80%, 70%, 60 %, 50%, 40%, 30%, and 20% of the neutralization inoculum (that mimics different amounts of viral neutralization). Mediumwas removed from MRC-5 cells in 24-well plates, and
Downloaded from http://jid.oxfordjournals.org/ at University of Sussex on July 19, 2015
A simplified hepatitis A virus (HAY) antigen-reduction neutralization assay (HAVARNA) was developed to permit the measurement of biologically active antibodies in recipients of candidate HAV vaccines. Degrees of neutralization were measured from the reduction in the amount of HAV antigen synthesized by 7-10days after infection ofMRC-5 (fetal human diploid lung) cell cultures. Sera producing a ~50% reduction in viral infectivity were scored as neutralizing. The assay was applied to demonstrateserum HAV neutralizingactivity in 10of 10and 9 of 10recipients of 107 and 1()6 TCIDso doses, respectively, of the Merck CR326F (F' variant) liveattenuated vaccine. The dilution end points of selected sera ranged from 1:10 to 1:640. The dilution end point of the World Health Organization reference globulin no. 1 was 1:530,000 (0.2 mlU/ml of HAV antibody). The HAVARNA provided a rapid, sensitive,and reproducible means to measure neutralizing antibodies to HAV.
1ID 1991;163 (March)
Concise Communications
Results The variant F' live-attenuated HAV vaccine was chosen as the prototype indicator virus for the development of the HAVARNA becauseit lackedvirulencein humans,chimpanzees, and marmosets [2], produced more reproducible antigen under the describedassayconditions thanhigherpassage preparations(data not shown), and wasavailable in sufficient amountsto providea standard virus for use in neutralization assays.
HAV was pretreated with the nonionic detergent, 00, to enhancevirus susceptibility to neutralization. 00 treatment of HAV increasedneutralization activity by the WHO referenceglobulin10-fold and reduced the neutralization-resistant fraction from 40% to
