AIDS RESEARCH AND HUMAN RETROVIRUSESVolume 4, Number 1, 1988Mary Ann Liebert, Inc., Publishers

Detection of HIV-1 Neutralizing Antibodies by a

Simple, Rapid, Colorimetric Assay

ALPHONSE J. LANGLOIS, THOMAS J. MATTHEWS, KENT J. WEINHOLD,SARAH CHAFFEE, MICHAEL HERSHFIELD, and DANI P. BOLOGNESI

ABSTRACT

A rapid, simple, reproducible and semi-quantitative assay to measure neutralizing anti-bodies has been developed. It employs a unique cell line which is exquisitively sensitive toinfection with all HIV isolates tested. The assay is amenable to microtiter formulation aswell as analysis by automation.

INTRODUCTION

There are currently numerous approaches to measure neutralizing antibodies against HIV. Themajor variables lie in (a) the characteristics of the virus preparation, (b) the nature of the target cell,

and (c) the end-point of the assay. Thus the infecting agent might be HIV itself or pseudo-types of HIV andvesicular stomatitis virus (VSV).1 Target cells may differ on the basis of lineage, level of expression ofcD4, the HIV receptor2,3 and susceptibility to viral cytopathic effects. Accordingly, some of the end-pointsare directly related to the macroscopic effects of the virus on the target cells, such as syncytium formationand/or cell death. Other end-points directly measure the release of virus particles on the basis of markers inthe culture supernatant, such as reverse transcriptase (RT)4 or p24, the major internal core antigen of thevirus.5

The ultimate goal in this area of research is to develop a simple rapid, quantitative, and reproducibleassay which is adaptable to diverse laboratories working in the field. Several of these assays are available,which approach one or more of these criteria but, to date, none embody all of them. For accuracy andreproducibility, the VSV pseudotype assay stands alone. However, it is more complex than simple and thusnot easily translatable to common practice. Reasonably quantitative assays based on syncytium formation6,7have also emerged and show considerable promise. However, not all HIV isolates form syncytia at thesame rate, if at all.8 Release of virus from infected cells is generally reproducible but difficult to quantitatein relation to the input virus titer or the serum titer. The timing of virus release is also highly variable,depending on infectious dose and various properties of the target cell, particularly its CD4 content andgrowth rate.

Duke University Medical Center, Department of Surgery, Durham, NC 27710.

63

LANGLOIS ET AL.

We report here a new test which is rapid, reproducible, and exhibits an easily measurable end-point. Itmakes use of a unique cell line which is rapidly infectable by all HIV strains tested, including some fieldisolates. Although quantitation is not ideal, its positive features make it quite useful for most studies relatedto neutralizing antibodies.

MATERIALS AND METHODSCell cultures

AA-5 cells, a subclone of the AA cell line, which was derived from an EBV infected B-cell line, was

obtained from Dr. Michael Hershfield, Duke University Medical Center, Durham, NC 27710. Its deriva-tion and properties will be described elsewhere (S. Chaffee, J. Leeds, M.S. Hershfield, in preparation).

Seed cultures of H-9 cells and HIV-IIIB infected H-9 cells were obtained from Dr. R.C. Gallo, Labora-tory of Tumor Cell Biology, Bethesda, MD. Stock cultures were seeded at 3 x 105/ml of growth mediumand passed at 2-3-day intervals. Cells were grown in RPMI 1640 containing 100 U/ml penicillin, 100pg/ml streptomycin and 20% heat inactivated (56°C/30 min) fetal calf serum (FCS) (GIBCO, Grand Island,NY). Cell cultures (20 ml vol) contained in 75 cm2 plastic T-flasks (#3075 Costar, 205 broadway, Cam-bridge, MA 02139) were grown at 37°C in a 5% C02-humidified incubator.

Virus poolH-9 cells chronically infected with HIV-IIIB were seeded at 7 x 105 cells/ml in growth medium. The

cell culture (200 ml vol) contained in a 1-1 Erlenmeyer flask closed with a rubber stopper were incubated at37°C on a gyrotary shaker oscillating at 60-70 rpm. The following morning, approximately 18 h afterseeding, the cultures were examined microscopically and a cell count was made. The cultures contained 12X 105 cells/ml with less than 5% nonviable cells as determined by trypan blue dye exclusion. The cellswere removed by centrifugation at 150 x g in a PR6 centrifuge. The supernatant was clarified by filtrationthrough a 0.45 p Millipore filter (Millipore Corp., Bedford, MA) aliquoted in 2 ml volumes, shell frozenin an ethanol dry ice bath and stored at -70°C.

Titration of virus poolThe infectious titer of the virus pool was determined by making fourfold dilutions of virus stock in

growth medium. 100 (jlI of individual virus dilutions were mixed with 200 pi of medium containing 1 x105 AA-5 cells in 6 well plates (#3046 6 well multi-well tissue culture plate with lid; Falcon Labware).The cultures were incubated at 37°C at a 30° angle in a humidified 5% C02 incubator. At 1 day post-infec-tion 500 (xl of fresh medium were added to the wells. On the third day of cultivation 2 ml of growthmedium were again added. On the sixth day the cultures were transferred to 15 ml conical centrifuge tubes,sedimented at 150 x g, and the supernatant was assayed for RT activity.

Neutralization assay

The stock virus pool was diluted with growth medium to give 800 infectious units (IU) per 100 pi andadded to 48-well plates (#3548, Costar, 205 Broadway, Cambridge, MA 02139). Serum was diluted withgrowth medium (fourfold dilutions) and 100 pi were mixed with the virus in the wells. The virus serum

mixtures were incubated for 30 min,, in a 37°C, 5% C02 humidified incubator. AA-5 cells, 2 x 104/100pi medium were then added. Fresh medium 500 p.1 free of NaHC03 containing a fivefold x concentrationof phenol red (25 mg/ L) amd 0.0034M NA2HP04: was added to the wells 2 days after seeding. Six dayspost-seeding 100 pi of the culture supernatant were transferred to 96-well plates, 10 pi of 5% Triton-XlOOin PBS were added, and the optical density (O.D.) read in an ELISA plate reader (540 nm). The wells wererinsed with 2 ml of medium, cells were removed by centrifugation, and RT assays were performed on thesupernatant.

64

DETECTION OF HIV-1 ANTIBODIES

NHS

AIDS 1

AIDS 2

NGS

G anti gplóO

G anti PB1

SERUM DILUTIONS1:8 1:32 1:128 1:512 1:2048

NO VIRUSVIRUS

•••

FIG. 1. Visual appearance of the assay in a 48-well plate. Sera tested represent normal human serum (NHS), a weakneutralizing screen from a patient with AIDS (AIDS-1), a more potent human neutralizing serum (AIDS-2), a normalgoat serum (NGS), a goat serum prepared against the entire HIV envelope gene expressed in a baculovirus system10 (Ganti-gpl60), and a goat serum to a fragment of the HIV envelope expressed in E. coli which contains the majorneutralizing epitope10,11 (G anti-PBl). "NO VIRUS" represents uninfected AA-5 cells, "VIRUS" their infectedcounterparts in the absence of serum.

Table 1. Correlation Between Visual Color and Reverse Transcriptase Activity

Sera used Serum dilutions No serum

NHS

AIDS-1

AIDS-2

NGS

G anti-gpl60

G anti-PBl

Color1RT2ColorRTColorRTColorRTColorRTColorRT

1:8Red

43,200Yellow

4700Yellow

4900Red

28,400Yellow

4800Yellow

5200

1:32Red

32,200Orange[60,200|Yellow

4900Red

34,500Yellow

5000Yellow

5100

1:128Red

31,500Red

33,400Orange|51,200|Red

31,500Orange|58,9001Yellow

4300

1:512Red

38,000Red

37,800Red

38,200Red

31,700Red

32,300Orange|62,900|

1:2048Red

27,100Red

29,600Red

37,600Red

30,200Red

33,200Red

31,600

No virusYellow

6,100Yellow

6000Yellow

5900Yellow

5500

Yellow6500

Yellow6200

VirusRed

27,200Red

30,000Red

28,900Red

26,800Red

32,100Red

34,300

From Fig. 1.2Reverse transcriptase activity in counts/min x 10~3 (avg. of two determinations).

65

LANGLOIS ET AL.

In later studies the assay was adapted to 96-well plates [flat-bottom wells, Costar, #3956, 205Broadway, Cambridge MA 02139] eliminating the necessity of transferring the tissue culture fluid for O.D.determination. Each well consisted of 30 p,l of twofold serum dilutions, 30 p.1 of virus (480 IU) and 30 p.1of AA-5 target cells representing 6 x 103 total cells. Two days post-seeding, 150 p.1 of growth mediumcontaining a fivefold concentration of phenol red, and 0.0034M NA2NP04 was added. On the fifth day, theplates were transferred to a humidified incubator with C02 and allowed to remain at 37CC overnight. Thefollowing morning the wells were treated with Triton-XlOO and the optical density determined directly inan ELISA plate reader.

RESULTS

The assay is based on color changes that occur in the culture medium resulting from accumulation ofnonvolatile acid generated by actively metabolizing growing cells. These changes can be intensified byinclusion of a higher than normal concentration of phenol red in the medium. The color develops overnightfollowing removal from an atmosphere of C02 and the escape of the gas from the culture medium. Ayellow color is indicative of cell growth while a red color represents either no cell growth or cell death. Aneasily distinguishable orange color represented intermediate situations between these extremes, that is, lessthan normal cell growth. Fig. 1 shows the color changes observed in a typical neutralization assay usingvarious sera under investigation in our laboratory. In brief, cells infected wtih HIV that remain untreatedsuccumb to the cytopathic effects of the virus, and do not release sufficient metabolic products to changethe color of the medium. The same is true for infected cells treated with either an HIV seronegative humanserum, or preimmune or nonimmune goat sera. In each instance the color of these wells is red. In markedcontrast, uninfected cells generate a distinct yellow color under conditions of the experiment. Protection

1:8 1=32 1:128 1=512 1:2048SERUM DILUTION

FIG. 2. Optical density measurements of the well supernatant shown in Fig. 1. 100 pi aliquots of individual wellswere transferred to a 96-well plate and treated with 10 pi of 5% Triton-X100. The latter was then placed in an ELISAplate reader and the optical density (O.D.) at 540 mM was obtained.

The 50% end-point (E.P.) titers of the serums were calculated as follows:

O.D. virus infected O.D. noninfected50% E.P. = (control serum)

—

(control serum) + O.D. noninfected (control serum)12

6c

O"<*W>>-

enZ

<I—o_O

zjur

200

150

100

50

66

DETECTION OF HIV-1 ANTIBODIES

from infection by incubation with sera possessing the capacity to neutralize HIV is also exhibited by a

yellow color. Thus sera from infected patients or goat sera prepared against components of the envelope ofHIV demonstrate this property and exhibit varying titers which are consistent with other measurements (seebelow). Examination of the degree of cell growth in each of the wells revealed none, or sparse cells in wellsexhibiting a red color, optimal cell growth in those represented by yellow, and intermediate cell growth inorange-colored wells. Thus when the antibodies are tested for virus-neutralizing capacity a yellow colorwould represent a significant degree of neutralizing activity, an orange color some neutralizing activity, butinsufficient to prevent virus breakthrough, whereas a red color would be indicative of an absence of neu-

tralizing potential.To substantiate these observations the supernatant medium of each of the wells was analyzed for the

presence of sedimentable reverse transcriptase (RT) activity indicative of release of mature virions. Theresults (Table 1) indicate a 1:1 correlation with the visual color examination. Thus all yellow wells were

devoid of RT activity while all red wells exhibited RT activity. Orange wells were also positive for RT butquantitatively higher than red wells, rather than an expected lower value. The possible significance of thisobservation will be discussed below.

By removing aliquots of the medium in each well it was also possible to more precisely determine colorintensity using photometric means. These results are shown in Fig. 2 where the O.D. at 540 nm is plottedagainst the respective serum dilutions. The results are entirely consistent with both the visual and RTdetermination (Table 1) and because of the linearity of the curves, it became possible to determine andend-point titer. For biohazard considerations virus infectivity can be eliminated by treatment with 10 pi of5% Triton-XlOO without affecting the existing color of the medium (unpublished data).

For convenience, an additional adaptation is to perform the assay in 96-well plates. While it is imprac-tical to measure RT activity because of the reduced volume, both visual inspection and direct photometric

800

700

E 600c

O¡o 500

I/OZ 400

< 300

Q-O 200

100

NHS

50%END POINT

G ANTI gp 160

ow/- 100 1:20 1:40 1:80 1:160 1:320 1:640 1:1280SERUM DILUTION

FIG. 3. Direct optical density measurements in 96-well plates. On the sixth day of cultivation, the wells were treatedwith Triton-X100 and the plate subjected to optical density scanning as described in Fig. 2.

67

LANGLOIS ET AL.

6 DAYS

800 200 50 12 3 0.75 0.18INPUT VIRUS (INFECTIOUS UNITS PER WELL)

0.04

FIG. 4. Relationship of input virus to RT activity as a function of time in culture. Various dilutions of the stock inputvirus were added to 4 sets of 8 individual wells each containing a constant number of AA-5 cells. At various intervalsafter infection the level of RT activity in the supernatant was determined for the entire series of input virus dilutions.

assessment is possible. A parallel analysis of the neutralization assay by direct scanning with an automatedmicroplate reader is shown in Fig. 3. This compares favorably with the results of Fig. 2, which representthe experiment performed in 48-well plates and the O.D. read after manual transfer to 96-well plates.

As mentioned earlier we observed an unusual phenomenon when the RT activity was measured in wellswhere the virus was partially neutralized, based on medium color change and degree of cell growth. Insteadof exhibiting intermediate levels of RT activity the amounts measured were actually higher (as much as

twofold) than the untreated virus-infected cultures. A possible explanation for this observation is thatincomplete neutralization resulted in a breakthrough virus which infected the cell culture at a slower ratethan the untreated input dose. This delay in infection would also allow for increased cell growth prior to theonset of virus cytopathic effects. In combination, these two events could conceivably lead to higher levelsof virus production prior to complete degeneration of the culture.

If this is correct, one would predict that exposure of the cells to lower doses of virus would generate a

higher level of RT activity within a given interval following infection. A titration of this sort is presented inFig. 4, which tends to substantiate this notion. Thus when examined on the sixth day post-infection, thehighest level of RT activity results from a dose of virus which is 16-fold lower than the maximum used inthis experiment. An alternative and equally plausible explanation for this effect is that one is dealing withdefective interfering (DI) particles in the high virus challenges that significantly reduce the level of infec-tivity. If these are neutralized or diluted out, the more infectious material becomes apparent.

DISCUSSION

These results demonstrate the rapidity, reproducibility, practicality, and flexibility of a newly developedculture system which has been adapted to assess infectivity of HIV by monitoring subsequent effects on cellgrowth and metabolism. In this report we have illustrated the applicability of the system to assay for HIVneutralizing antibodies, but, presumably, this could be extended to a variety of anti-viral agents.

The AA-5 cell line is a surprisingly sensitive target for HIV infection. The AA-5 clone were derivedfrom an EBV positive B-cell line. These cells exhibit CD4 antigen at the cell surface but in amounts that

68

DETECTION OF HIV-1 ANTIBODIES

are substantially reduced in comparison to standard T-cell lines (S. Chaffee, J. Leeds, M.S. Hershfield, inpreparation). Despite this, both infection and cytopathic effects occur rapidly and pervasively throughoutthe culture. Our results are consistent with reported studies likewise indicating susceptibility of EBV im-mortalized B cells to HIV-1.9

To date we have tested several prototype virus strains for rapid infectivity on the AA cells. We havefound that the AA-5 clone is susceptible to several prototype HIV-1 isolates such as IIIB, RFn, MN (S.Chaffee, J. Leeds, M.S. Hershfield, in preparation) as well as a number of other isolates originating fromthe laboratory of Dr. R.C. Gallo. Thus far all viruses infecting the AA-5 line, including field isolates are

susceptible to blockade by antibodies from HIV seropositive individuals. Studies are now in progress todetermine their susceptibility to other virus isolates including those that are macrophage tropic. Finally, we

have recently shown that the line is also susceptible to infection by HIV-2.

REFERENCES

1. Weiss RA, Clapham PR, Cheingsong-Popou R, Dalgleish AG, Carne CA, Weiler IVD, and Tedder RS: Neutral-ization of human T-lymphotropic virus type III by sera of AIDS and AIDS-risk patients. Nature 1985;316:69-72.

2. Dalgleish AG, Beverley PCL, Clapham PR, Crawford DH, Greaves MF, and Weiss RA: The CD4 (T4) antigen isan essential component of the receptor for the AIDS retrovirus. Nature 1984;312:763-766.

3. Klatzmann DE, Champagne E. Chamaret, S, Gruest J, Guetard D, Hercend T, Gluckman JC, and Montagnier L: Tlymphocyte T4 molecule behaves as the receptor for human retrovirus LAV. Nature 1984;312:767-768.

4. Poiesz BJ, Ruscetti FW, Gazdar AF, Bunn PA, Minna JD, and Gallo RC: Detection and isolation of type Cretrovirus particles from fresh and cultured lymphocytes in a patient with cutaneous T-cell lymphoma. Proc NatiAcad Sei USA 1980;77:7415-7419.

5. Sarngadharan MG, Popovic M, Bruch L, Schupbach J, and Gallo RC: Antibodies reactive with human T-lympho-tropic retroviruses (HTLV-III) in the serum of patients with AIDS. Science 1984;224:506-508.

6. Harada S, Koyanagi Y, and Yamamoto N: Infection of HTLV-III/LAV in HTLV-I-carrying cells MT-2 and MT-4and application in a plaque assay. Science 1985;229:563-590.

7. Nara PL, Hatch WC, Dunlop NM, Robey WG, Arthur LO, Gonda MA, and Fischinger PJ: Simple, rapid, quantal,syncytium-forming microassay for the detection of neutralizing antibody against infectious HTLV-III/LAV. AIDSRes and Hum Retroviruses 1987;3:283-302.

8. Evans LA, McHugh TM, Stites DP, and Levy JA: Differential ability of human immunodeficiency virus isolates to

productively infect human cells. J Immunol 1984;138:3415-3418.9. Dahl K, Martin K, and Miller G: Differences among human immunodeficiency virus strains in their capacities to

induce cytolysis or persistent infection of a lymphoblastoid cell line immortalized by Epstein-Barr virus. J Virol1987;61:1602-1608.

10. Rusche JR, Lynn DL, Robert-Guroff M, Langlois AJ, Lyerly HK, Carson H, Krohn K et al.: Humoral immuneresponse to the entire human immunodeficiency virus envelope glycoprotein made in insect cells. Proc Nati AcadSei USA 1987;84:6924-6928.

11. Putney SD, Matthews TJ, Robey WG, Lynn DL, Robert-Guroff M, Mueller WT, Langlois AJ et al.: HTLV-III/LAV-neutralizing antibodies to an£. coli produced fragment of the virus envelope. Science 1986;234:1392-1395.

Address reprint requests to:Alphonse J. Langlois

Department of SurgeryDuke University Medical Center

P.O. Box 2926Durham, NC 27710

69