469 _NA_TU_R_E_VO_L_.3_32_31_M_A_RC_H_19_88 _____PRODUCT REVI EW------------_ Quantitative infectivity assay for HIV-1 and -2 P. L. Nara and P.J. Fischinger The development of a cytomorphologic infectivity microbioassay for human immunodeficiency virus (HIV) allows rapid, sensitive, and specific characterization of neutralizing antibodies or antiviral agents. INFECTION with the human immuno­ Syncytlal-Formlng Mlcroassay a deficiency virus (HIV) leads to a plethora of humoural and cellular immune res­ ponses 1, which ultimately leads to AIDS in humans. The characterization and understanding of these immune responses in humans may allow for the identification of various states of protection from disease, opening up new avenues for Plated CEM cell future vaccine development and for inter­ ventive strategies such as antiviral agents and biological response modifiers. To this end, a dissection of host responses by sen­ sitive, reproducible infectivity bioassays CEM cell {24-48 hr) gp120/fuslon protein presenl on cell will be of obvious importance. membrane Just prior to vlrion production. In the study of bacterial viruses, the production of plaques by single virus l particles (bacteriophages) on a uniform l~l bacterial layer was the earliest and most Unlnf.cted CEM cells multiply and become Incorporated Into tuslgenlc lnfacled CEM cell$. sensitive method for. evaluating various Key to aHey Is the pl'9sence of uninfacted CEM cells at the time Infected cell is tuslgenlc. anti-bacteriophage antisera'. Dulbecco introduced to animal virology a modifica­ I tion of the bacteriophage plaque assay lc-:::?~C-:?J that is now used and applied widely both CEM syncytlum on CEM monolayer. for the quantitation of human and animal Fig. 1 a, Schematic of the CEM-SS virus-induced syncytium forming assay (reprinted with viruses and for neutralizing antibody'A_ permission'). b, Microtitre well containing 35 typical HIV virally-induced syncytia. Approxi­ The virus neutralization/infectivity test mately 30 per cent of the well is shown. c, Five HIV-induced syncytia embedded in a monomorphic is the most sensitive and specific sero­ CEM-SS mono layer. Magnification, 100 x. d, Infectious cell centre assay. The five syncytia shown logical procedure for demonstrating the result from contact with virus-producing cells for 18 to 24 hours. Magnification, 200 X. quantitative relationship between virus and indicator cells. In such a test, a single cell line (CEM-SS) monolayer leads to a 0.5 per cent Nystatin ( optional). The cells infectious unit of virus infects a single cell population of virus-infected (fusigenic) are counted, then gently pelleted at 800 and initiates a focal cytomorphologic HIV envelope-expressing cells (Fig. la). r.p.m. for five minutes. The original expression of that infection, such as syn­ These cells contact the remaining pre­ volume of tissue culture medium is cytium formation, plaque formation, titred population of uninfected CEM-SS decanted and replaced with one-tenth the cell transformation or lysis (Fig. 1). cells, allowing the early recruitment of volume of complete RPMI 1640 medium Because a single infectious unit leads to a uninfected cells into a detectable and containing 25 µg ml- 1 diethylaminoethyl­ single discrete response, a linear relation­ quantifiable syncytium. With this method, dextran (DEAE-D) for 30 minutes. ship exists between the number of virally­ agarose overlays are not needed because DEAE-D may be omitted depending induced cytopathic effects and the first satellite syncytia from first- and second­ upon experimental conditions, but its order of virus concentration'-". When this generation virus progeny are not produced presence assures maximum and more uni­ test is applied in assays for virus inactiva­ within the assay period. form viral kinetic infection of the cells. tion or neutralization, the complex In the assay, individual wells of a The cells are washed twice and resuspended interactions of virus and antibody can be 96-well, flat-bottomed microtitre plate in medium, and plated in the PLL­ 4 systematically and accurately evaluated • (Costar) are coated with 50 µl of a 50 µg pretreated microtitre plates at a final cell The present assay' has been developed to ml- 1 poly-L-lysine solution (PLL) (Sigma) concentration of 50,000 cells per well in a provide a relatively rapid, quantitative, and allowed to stand at room temperature volume of 50 µI. Ten ml of 1.0 x 10" cells and reproducible bioassay for the detection for 30 to 60 minutes, after which residual per ml is appropriate for one 96-well and characterization of neutralizing anti­ PLL is removed by two washes with microtitre plate. The cells are allowed to body and other candidate antiviral agents. physiologic buffered saline solution (PBS) attach for 30 minutes at 100 per cent or medium. During the PLL treatment of humidity, 37 °C and five per cent CO,. Syncitium-forming assay the microtitre plates, the non-adherent Frozen HTLV-III 8 and HTLV-IIIRF The assay is based on the previously CEM-SS cells' are prepared for plating. (isolates of HIV-I) stocks are made from reported interaction between fusigenic To standardize plating conditions and recently infected H9 cells by centrifuga­ virus-infected cells expressing the HIV assure a population of logarithmically tion. The virus-containing media is ali­ envelope gene products and uninfected growing cells, the CEM-SS cell cultures quoted into 1 ml volumes and frozen adjacent cells bearing CD4 molecules". are split in a 1:2 ratio 24 hours prior to at - 70 °C. Viral stocks are then thawed The conditions are optimized in vitro so plating in RPMI 1640 medium containing and evaluated for syncytium forming units that a virus inoculum introduced onto a 10 per cent fetal calf serum, one per cent (SFU) by twofold endpoint dilution anal­ syncitium-sensitive Leu 3a-positive CEM antibiotics (PSN lOOX Gibco Labs) and ysis in the CEM-SS assay. For confirma- _47_0 __________________ PRODUCT REVIEW----------N_A_T_U_R_E_V_0_L_.3_32_3_J_M_A_R_C_H_l9_ 88 tion, tests for reverse transcriptase and after virus neutralization (V.) to the 1.0 p24 content by radioimmunoassay can be number of syncytia induced by the virus ) performed simultaneously. Other HIV inoculum (V0 is obtained. The data are variants - such as HTLV-MN, HTLV­ represented as classical multiplicity CC and HIV-2NrHz - are labile and often curves". The virus-surviving fractions (V,I lose titre on freezing, and must be main­ Vo) from each twofold serum dilution are ~ tained as freshly infected cultures. Opti­ then plotted on the logarithmic Y axis, f mal tissue culture conditions have been and the reciprocal of the serum dilutions -~ standardized, and reproducible virus are plotted on the arithmetic X axis. Each ~ yields can be achieved on a weekly basis curve represents the virus-surviving frac- ~ and be titred directly in the assays. tion at progressive twofold serum dilu- -~ Frozen, pre-titred or fresh virus stocks tions (Fig. 2). Neutralization titres can be -~ are diluted in medium to yield approxi­ standardized at any level of per cent ~ mately 100 to 200 SFU per well. Fifty µI neutralization depending upon the indivi- 2 of approximately 100 SFU of virus (the dual experimental parameters. For HIV, > constant component) is added to 50 µI a 90 per cent (V jV., = 0.1) point of virus each of a serially prediluted sample of inhibition is recommended as a standard antiserum, purified immunoglobulin or neutralization index because of the other potentially virus-inactivating variable initial slope of the curve at high compounds under study. These virus­ serum dilutions. 8192 4096 2048 1024 512 256 128 64 32 16 8 antibody mixtures are incubated for Two minor modifications to the HIV-1 Fig. 2 Sequential analysis of neutralizing anti­ upon experi­ assay are necessary for HIV-2 because of body response in an experimentally inoculated, various times depending persistently infected chimpanzee. The dashed mental conditions. Following incubation, the more rapid viral-infectivity replicative line represents the 90 per cent neutralization each virus-antibody mixture is divided kinetics exhibited by HIV-2NrH z in this cell (Vj V0 = 0.1) serum titre (512-1024) from a into duplicate wells (50µ1 per well) con­ line. For HIV-2, 75 ,000 CEM-SS cells serum sample taken 2 yr(<)) after inoculation taining PLL-adherent CEM-SS cells (in instead of 50,000 cells should be plated per (Al). Prebleed (o), 1 month Al(•), 2 months which the medium has been removed well, and EIV- 2-induced syncytia should Al (6 ), 3 months Al (.6), 1 yr Al(•). gently) for 60 minutes. Following this be quantified on the third or fourth day adsorption phase, which lasts 60 minutes instead of on the fifth or sixth day as infected humans, chimpanzees, or other for most strains, the virus-media only and previously described. primates, medium containing either inter­ virus-antibody mixtures are removed and Several variants of simian immuno­ leukin-2 or phytohaemagglutinin - used replaced with 100 to 200 µl of media. deficiency virus (SIV) have been tested to support growth and viral expression - Some wells of plated CEM-SS cells are left with this assay to date. Several pathogenic do not adversely affect the CEM-SS cells. uninfected to serve as cytomorphologic strains of SIV/Delta have been found to Isolates of HIV-1 and -2 other than controls. The microtitre plates are stored replicate rapidly to maximal titres and to those reported here need to be evaluated in an incubator at 100 per cent humidity, produce quantifiable syncytia in the by twofold, endpoint-limiting dilution­ 37 °C and five per cent CO2 for five days. CEM-SS cell line (M. Murphy-Corb, per­ amplification analysis' for their quantita­ On the third day, 100-200 µI of complete sonal communication). The exact con­ tive relationship to syncytia formation and medium may be added per well.