15

Journal of Virological Methods, 39 (1992) 15-26 0 1992 Elsevier Science Publishers B.V. / All rights reserved / 0166-0934/92/%05.00 VIRMET 01359

A sensitive assay for detection and measurement of neutralising antibody to human immunodeficiency virus Robert

Osbornea,

Helen Masona, Michael BrowningaT’, and William Jarretta

Ruthven

Mitchellb

aMRC Retrovirus Research Laboratory, Department of Veterinary Pathology, University of Glasgow, Glasgow (UK) and bGIasgow and West of Scotland Blood Transfusion Service, Law Hospital, Carluke. Lanarkshire (UK) (Accepted

12 February

1992)

Summary An assay based on the inhibition of syncytium formation in C8166 cells was developed to measure low levels of neutralising antibody (NT-AB) to human immunodeficiency virus (HIV) and to detect cross-reactivity between virus strains.The relationship between virus challenge and antibody titre was represented by a tripartite curve which was essentially linear over moderate levels of virus input. Based on these findings, antibody titres were standardised against 100 TCIDso of challenge virus. However, lower virus inocula were found to detect minimum levels of antibody. Reproducibility of antibody titres between tests was high, with variation generally lying within one dilution step.The improved sensitivity of the technique allowed detection of NT-ABs in animals immunised with immune-stimulating complexes (ISCOMS) incorporating HIV antigens. Consistent levels of cross-reactivity between HIV strains was demonstrated, indicating the presence of distinct viral groups, from which dominant isolates may be chosen for use in vaccination studies. HIV-neutralizing

antibody, low levels of; Cross-reactivity

between strains

Correspondence to: R. Osborne, MRC Retrovirus Research Laboratory, Department of Veterinary Pathology, University of Glasgow, Bearsden, Glasgow, G61 IQH, UK. ‘Present address: ICRF Cancer Immunology Laboratory, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK.

16

Introduction Several quanta1 and quantitative procedures have been described for detection and measurement of neutralising antibodies (NT-AB) against lentiviruses, particularly HIV, simian immunodeficiency virus (SIV) (Weber and Schild, 1990) and feline immunodeticiency virus (FIV) (Osborne et al., in preparation). With HIV these techniques have exploited many parameters to quantify the reduction of viral infectivity. These have included cytopathic effects (CPE) in a variety of cell types observed as syncytiation (Clapham et al., 1989; Weiss et al., 1985; Katzenstein et al., 1990) or cell death (Emini et al., 1990; Skinner et al., 1988; Nakashima et al., 1989), plaque formation either directly with HIV (Nara et al., 1987; McKeating et al., 1989) or with vesicular stomatitis pseudotypes of HIV (Thomas et al., 1988; Weiss et al., 1985), the uptake of tritiated thymidine (Ranki et al., 1987 ; Harada et al., 1986), immunofluorescence (Matsushita et al., 1988; Lasky et al., 1986) and or detection of gag (Matsushita et al., 1988; Looney et al., 1988; Robert-Guroff et al., 1985) or pol (Matthews et al., 1986; Cheng-Mayer et al., 1988) gene products. The objective of the work presented here was the development of a technique for screening HIV antibody in sera obtained during vaccine development studies. Investigations focussed on the inhibition of syncytial formation by infected cells (Clapham et al., 1989) and this method was refined to detect low levels of NT-AB and to investigate cross-reactivity between strains of HIV.

Materials and Methods Cell Culture C8166 (Popovic medium penicillin

(Maddon et al., 1986; Sodroski et al., 1984) and H9 T cell lines et al., 1984; Mann et al., 1989) were maintained in RPMI-1640 containing 10% foetal calf serum, 0.2 M glutamine, plus 100 units of and 100 micrograms of streptomycin per ml.

Viruses

Stocks of HIV strain RF (Popovic et al., 1984) IIIB (HTLV-IIIB) (Popovic et al., 1984) and SF2 (ARV-2) (Levy et al., 1984) were obtained through the Medical Research Council Aids Directed Programme (MRC-ADP). Human sera

Sera were supplied by the Scottish Blood Transfusion anonymous local collections or through the MRC-ADP.

Service

from

17

Rabbit sera

Rabbit sera to gp120 were raised by immunising New Zealand white rabbits with mammalian cell-derived recombinant (r-) gp120 (strain IIIB; MRC AIDS Reagent Project). Two rabbits (658 and 659) received 10 ,ug r-gp120 in ISCOMS by intramuscular inoculation at 0, 4 and 8 weeks. One rabbit (660) received 100 pg r-gp120 in Freund’s complete adjuvant (FCA) at time 0, followed by boosting with the same dose incorporated into incomplete Freund’s adjuvant at week 4, and subsequently with soluble gp120 (100 pg i.v.) at week 8. Neutralisation

assay

Virus stocks were prepared from cell-free culture supernatants of acutely infected H9 cells. Culture fluids were harvested at peak periods of syncytial formation (usually two to three days after infection), clarified by centrifugation (800 x g for 10 min) and stored at -70°C. For strain SF*, supernatant was derived from freshly growing cell cultures since viral infectivity was labile to freezing and thawing (Clapham, P. personal communication); in this case infectivity titres of the virus were established simultaneously to neutralisation. Acute infection was induced by inoculating one volume of infected cells on to four volumes of uninfected cells and if necessary passaging at 3-day intervals in the same ratio. All antisera were inactivated before use by heating for two periods of 30 min at 56°C. The neutralisation assay utilised a quanta1 microtitre format with a unit volume of 50 ,ul. Serial 0.5 loglo dilutions of pretitrated (except SF2) virus and antiserum (0.3 loglo series) were allowed to react for 1.5 h at 37°C prior to the addition of C8166 indicator cells (5 x lo5 ml-‘). The reactants were incubated for a further 6 days at 37°C with the addition of a fresh volume of growth media after 3-4 days. The end-point was recorded as the serum dilution which completely inhibited syncytium formation. Titres were determined from the Karber formula and regression analysis. HIV antigen assay

Commercial

kits for p24 were employed

(Coulter).

Infectivity of HIV follows single-hit kinetics

Two strains, RF and IIIB, svere used in parallel. Infectivity end-points were determined microscopically, with syncytia indicating the presence of infectious virus. Following virus titrations, attempts were made to recover virus by

18

culture and subsequent antigen assays in apparently negative cultures both within the end-point dilution and beyond. Virus was detected only occasionally in replicates of the same, or rarely, within the 0.5 log,, step immediately beyond the end point dilution. From these observations it appeared unlikely that infectious non-syncytium forming virus was produced to any significant extent beyond the end point. Syncytium formation was shown to follow single hit kinetics, since a linear relationship was demonstrated between final virus infectivity and dilution of stock inoculum (Table 1). These results indicate that across the working dilutions of antigen only infectious virus could induce syncytia while components such as free gp120 did not influence viral end-point readings. Antibody titre of human sera is dependant on virus dose in neutralisation Fig, 1 represents the results of chequer-board titrations of virus antigens and a human HIV-l seropositive serum; these results were typical for several sera examined. Regression analysis showed a tripartite relationship between antigen challenge and serum titre; with moderate levels of virus input (1.5 to 2.5 log,* TCID& the plot was essentially linear but curved at extremes of antigen challenge. When detecting minimal antibody levels low virus challenges considerably increased the sensitivity of the technique. At a challenge dose of 1.0 log10 TCIDSO sera from seronegative individuals have titres of ~0.6 loglo. For strain differentiation studies NT-AB titres were optimised against 100 TCID50 of virus. This viral dose lies approximately midway along the linear part of the curve and is also used in many virus neutralisation systems. In contrast, at high virus inputs with the convergance of regression curves and suppression of serum titres, strain differences are reduced.

TABLE 1 Relationship

between infectivity titre and dilution of inoculum

Strain

Dilution

Actual titre

Equivalent

RF

Undiluted 10-l 10-z 1O-3

4.25 3.5 2.25 1.38

4.25 4.5 4.25 4.38

IIIB

Undilyted

4.2

4.2

2.16 3.16 1.25

4.16 4.25

&* 1o-3

titre

Titres expressed as log,0 TCIDm. Actual titre = observed infectivity titre. Equivalent titre = actual titre corrected for dilution factor.

19

3.5

REQRESSION CURVE OF HIV-l w HUMAN SERUM

1

TYPICAL RESULTS

CURVING

3.0

Tut

1

Twt 1 To.12 T-1

2

8 1 0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

ANTIQEN CHALLENQE - Log10 TCIDSO

Fig. 1. Relationship between virus challenge and serum titre.

Reproducibility Table 2 summarises results of repeated tests using standardised reactants. In the upper panel, titres are presented for two sera challenged by three individual stocks of the same virus strain, produced from H9 cells over a period of eighteen months. Overall the maximum range was f 0.2 around a mean of 2.16. Results from tests performed closely together, (e.g., using RF pass 17) showed more consistent correlation, suggesting some variation in sensitivity of indicator cells. Virus passage history did not significantly alter sensitivity for antiserum. The lower panel compares two sera challenged by the reference HIV strains RF and IIIB. Despite individual test variations, the strains were repeatedly distinguishable with titre differences of the same order of magnitude (5 to lofold). Distinction

of HIV strains by neutralisation

Local and WHO reference sera were examined by the refined neutralisation test protocol described above. Of these sera, four taken as representative, and collected from two local individuals, were investigated, with a view to demonstration of potentially cross-reactive and dominant HIV strains. In Table 3 the upper panel represents mean titres of neutralising antibody and the lower panel indicates the ratio of titres of the same sera to different strains of HIV. Sera 1 and 6 show a repeatable and similar pattern of neutralisation,

20

TABLE 2 Reproducibility

of neutralisation

assay

Within strains Virus strain/ passage

Antibody

titres (logic,)

Serum 1A No. of

RF p13 RFpll RF pl7

Serum 6B Mean titre

Range

tests 3 4 5

2.21 2.18 2.08

2.062.39 2.03-2.34 2.06-2.11

No. of tests

Mean titre

Range

2 5

2.19 2.04

2.18-2.20 1.97-2.11

Between strains - ratio of antiserum titres Virus/passage

Serum 1A

BIB plO/ RF p17

6B

No. of tests

Mean ratio

Range

No. of tests

Mean ratio

Range

4

0.11

0.084.19

4

0.12

O.lcHl.14

Titres expressed against 100 TCIDSO of virus. (Similar reproducibility IIIB.)

has been obtained with strain

irrespective of date of acquisition. Activity against RF was dominant by a factor of 10; this ratio of titre between RF and IIIB has been found in all but one of the ten local sera tested (results not shown). Titres against stain SF2 were not highly elevated but were less than two-fold below those to RF. Detection of HIV neutralising antibodies induced in immunised rabbits Figure 2 summarises the NT-AB response of rabbits to r-gp120 (strain IIIB) incorporated into ISCOMS or with Freund’s complete adjuvant. Low levels of activity were induced against the homologous ISCOM immunogen. In contrast, r-gp120 in FCA induced higher levels of antibody, which, on challenge by 100 TCIDSO, produced titres approaching those observed with reference serum from an infected patient. No activity was detected in preimmune sera or in the immune sera when tested against RF or SF2 virus strains. Parallel results from the MRC ADP Neutralisation Reference Laboratory showed neutralising activity to strain IIIB and only in the serum of the animal immunised with r-gp120 incorporated into FCA. However, these results are consistent with the data presented here since the Reference Laboratory used an assay with a challenge of lo3 TCID 5a, which would clearly miss low antibody levels (Fig. 2).

21

TABLE 3 Neutralking

antibody

titres of reference antisera challenged by standard

strains of HIV

Mean titre against virus strain Serum 1A

RF 2.06 & 0.01

RIB 1.10 f 0.12

:: 6B

2.07 1.91 f. + 0.01 1.99 & 0.01

0.93 1.11 f+ 0.01 0.06 1.06 + 0.08

1.89 + 1.74 f 0.13 0.06 1.80 f 0.03

IIIB/RF 0.10 0.11 0.10 0.11

IIIB/SF2 0.17 0.17 0.15 0.17

SF2/RF 0.61 0.65 0.68 0.64

SF2

1.85 f 0.08

Ratio of antiserum titres Serum 1A 1B 6A 6B

Titres expressed as log,, against 100 TCIDso of virus. (Mean titre = mean of titres from at least two tests). Sera 1A and lB were collected from the same individual, 12 months apart; sera 6A and 6B were collected from a second individual 4 months apart.

Discussion A quanta1 microtitre assay system is described for the detection of neutralising antibodies to HIV. The method relies on low viral challenge with residual infectivity determined by a distinctive cytopathology in C8166 indicator cells. The method has a number of advantages compared with techniques previously described. It is simple to perform, shows excellent reproducibility and is highly sensitive. Thus, in independent experiments using the same reactants, variations in antibody titre usually lie well within one (two-fold) dilution step. The sensitivity of this assay is considerably improved by reducing the virus input to moderate levels (1.5 to 2.5 log,,, TCIDSO, Fig. 1). Under such circumstances an essentially linear relationship is seen between virus challenge and serum titre. Where minimal neutralising activity is anticipated, further reduced virus challenges may be used, as employed here when examining immune sera raised against recombinant gp120. We used this assay to examine the cross-neutralising activity between different strains of HIV, antibody titres being quantified against a virus challenge of 100 TCID 50. The close reproducibility of serum titres allows a consistent, inter-strain relationship to be established in independent assays. Our results suggest that strains related to RF, rather than IIIB, are present in the blood donations collected and tested to date from the west of Scotland. The very high titres reported by Weiss et al. (1986) against strain SF2, in VSV pseudotypes, were not observed in the sera studied. Currently, there is a dearth of information comparing different strains of

ll1B-r~p12O/ISCOM

or FCA rabbit anlimws

L 0

0 FCA

0

= F,.““d’S

so 1

0 75

1

1 00

1 25

IlIE ANTIGEN

I

1 50 CHALLENGE

I

I

1 75

2 00

CornpI.,.

Ad,W.nt

I 225

_ Log10 TCIO50

Fig. 2. Detection of neutralising antibody m experimental antisera.

HIV using homologous and heterologous sera. When attempted, in general, homologous exceed heterologous reactions (Nara et al., 1987; Trembley and Wainberg, 1990; Bottiger et al., 1989) though exceptions have been described which may reflect common and strain-specific epitopes (Weiss et al., 1986). The presence of a restricted number of HIV serotypes has been mooted by ChengMayer et al. (1988); of significance the isolate MN appears to be particularly cross-reactive, perhaps explained by its great similarity to a consensus sequence at the principal neutralising determinant (Arthur et al., 1989; LaRosa et al., 1990). This information, together with our own results, suggests that it may be possible to characterise and classify strains serologically using neutralisation. Unfortunately there are a number of disadvantages to such a method: firstly, the requirement for homologous human antisera (Berman et al., 1981), and secondly, a necessity to adapt strains to the indicator cell, whether it be C8166, as in our case, or alternatives, such as CEM (Nara et al., 1987). Highly sensitive neutralisation techniques will be required to assess the efficacy of vaccination strategies by monitoring the development of antibody levels and also for detection of cross-reactivity. The former element is wellillustrated by our studies with r-gp120. When presented with FCA this immunogen induced readily detectable levels of neutralising antibody with titres approaching those seen in seropositive individuals. The detection of NTAB against the same immunogen incorporated into ISCOMS, although to very low levels, is encouraging, particularly since these animals received ten-fold less r-gp120 than did the control rabbit immunised with FCA. The use of varying antigen challenges, especially those of low magnitude, enables detection of

23

activity. Subsequent regression analysis allows an estimate of antibody levels against a fixed standard, such as 100 TCIDsc, where a titre may be below any starting serum dilution, particularly likely in early immune sera or if the reactants were from widely diverse strains. The protective potential of NT-AB during HIV infections is recognised (Weber and Schild, 1990; Tyrell, 1990) though its effectiveness remains unclear. Thus, higher levels of such antibodies are generally present in individuals with a of passive protracted clinical course (Ranki et al., 1987); administration antibody with neutralising activity is thought to have some therapeutic value (Jackson et al., 1988; Karpas et al., 1988; Dalgleish, 1988); during SIV vaccine studies resistance to challenge was observed in the presence, rather than absence, of NT-AB (Marx et al., 1986; Murphy-Corb et al., 1989; Desrosiers et al., 1989); and infection of chimpanzees could be prevented if cell-free virus were pre-mixed with specific NT-ABs (Emini et al., 1990). The mechanism of protection offered by NT-AB is likely to be through a cocktail of common and variable epitopes, recently summarised by Bolognesi (1990). Candidate epitopes include sites on the viral envelope, notably within the GPGR/V3 loop, the CD4 binding site (Lasky et al., 1987) and fusion sites on gp41 (Thomas et al., 1988; Chanh et al., 1986); epitopes on p17 have also been implicated (Weber and Schild, 1990; Schupbach, 1988). The importance of NT-AB to each site awaits evaluation, although the use of multiple inoculations using peptides derived from different strain V3 loops, broadening the neutralising antibody response, is significant (Palker et al., 1988). Of interest, the antibody response to r-gp120 described above appears to be recognising a linear epitope and so is unlikely to be against the GPGR loop in its normal configuration (Browning et al., in preparation).

Acknowledgements Many colleagues have kindly donated reagents or given valuable advice; the authors would particularly like to thank Professor 0. Jarrett and Drs. P. Clapham, B. Dow, R. Gallo and A.J. Garrett. This work was supported by the AIDS Directed Programme of the Medical Research Council.

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