Uses of biosensor technology in the development of probes for viral diagnosis

ELSEVIER

Clinical and Diagnostic Virology 5 (1996) 111-119

Clinical and Diagnostic Virology

Uses of biosensor technology in the development of probes for viral diagnosis Pascale M. Richalet-S6cordel a, Francis Poisson b, Marc H.V. Van Regenmortel a'* aLaboratoire d'Immunochimie des Peptides et des Virus, lnstitut de Biologic Molbculaire et Cellulaire, CNRS, 15 rue Ren~ Descartes, 67084 Strasbourg, France bDkpartement de Microbiologie Mkdieale et MolOculaire, URA CNRS 1334, Hdpital Bretonneau, 2 boulevard TonnellO, 37044 Tours, France

Received 10 October 1995; accepted 21 January 1996

Abstract Background: Since 1990, a new biosensor technology based on surface plasmon resonance makes it possible to visualize molecular recognition as a function of time, in terms of change in mass concentration occurring on a sensor chip surface. One of the reactants is immobilized on a dextran matrix while the other is introduced in a flow passing over the surface. The binding is followed in real time by the increase in refractive index caused by the mass of bound species. Objectives: In the present review, the applications of this new technology for developing probes intended for viral diagnosis will be described. Study design: In contrast with other immunoassay systems, the biosensor technique preserves the conformational integrity of the reactants since no labelling is required. It also makes it possible to follow every step of a multiple-layer assay and allows interaction measurements in real time. Suitable antigen and antibody probes can be selected on the basis of the conditions of the diagnostic assay that is being developed, especially in terms of affinity and specificity. Results: Our results suggest that when the cyclic peptide 209-222 of the E1 protein of hepatitis C virus (HCV) is immobilized on the sensor chip via a biotin moiety, it retains a constrained conformation which is better recognized by HCV antibodies than the linear form. Data are presented which indicate that the biosensor technique facilitates the screening and selection of anti HIV-1 antibodies that are likely to possess the most potent neutralizing potential. Conclusion: Since there is a good correlation between BIAcore and ELISA data, it seems likely that the biosensor technology will be increasingly used for developing reagents intended for viral diagnosis. Keywords: Biosensor technology; Probes; Viral diagnosis

1. Introduction T h e i n t r o d u c t i o n in 1990 o f a new b i o s e n s o r t e c h n o l o g y b a s e d on surface p l a s m o n r e s o n a n c e * Corresponding author. Tel.: + 33 88417022; fax: + 33 88610680,

has truly r e v o l u t i o n i z e d the m e a s u r e m e n t o f b i n d ing i n t e r a c t i o n s in biology. This new t e c h n o l o g y , c o m m o n l y k n o w n as b i o m o l e c u l a r i n t e r a c t i o n analysis (BIA), m a k e s it p o s s i b l e to visualize the p r o g r e s s o f b i n d i n g o f a b i o m o l e c u l e on a c o m p u t e r screen as a f u n c t i o n o f time, in terms o f c h a n g e in m a s s c o n c e n t r a t i o n o c c u r r i n g o n a sen-

0928-0197/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PI1 S0928-0197(96)00212-7

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sor chip surface. One of the interacting partners is immobilized to the surface of the chip and the binding of the other is followed by the increase in refractive index caused by the mass of bound species. None of the reactants needs to be labelled, which avoids the artefactual changes in binding properties that often result from the labelling process. Several reviews describing the new technology have been published (F~gerstam and Karlsson, 1994; J6nsson and Malmqvist, 1992) and special issues of journals have been devoted to the many applications of biosensors in molecular biology and immunology (Granzow, 1994; Van Regenmortel, 1995). In the present review, we will concentrate on the applications of BIA in virology and in particular on the uses of this technology for developing probes intended for viral diagnosis.

2. Description of the BIAcore instrument The most commonly used biosensor instrument today is the BIAcorC M developed by Pharmacia Biosensor (Uppsala) of which an upgraded version, the BIAcore 2000, was released in 1995. A simpler, manual version of the instrument, BIAlite, is also available. Another instrument based on a resonant mirror system and called IAsys is distributed by Fisons Applied Sensor Technology (Fisons, Cambridge, UK) (Cush et al., 1993; George et al., 1995). A comparison between the available systems has been published (Hodgson, 1994). The BIAcore instrument consists of an optical detector system, exchangeable sensor chips, a processing unit and a personal computer for control and evaluation. The processing unit contains the surface plasmon resonance (SPR) monitor, an integrated microfluidic cartridge and an autosampler for dispensing samples automatically (J6nsson and Malmqvist, 1992). The sensor chip consists of a glass slide, coated on one side with a 50 nm thick gold film, which is covered with a dextran layer that extends about 100 nm out from the surface. By using a carboxymethylated dextran, it is possible to immobilize substances containing primary amine functions after activating

the matrix with carbodiimide-N-hydroxysuccinimide. Various other immobilization strategies can also be used (Johnsson et al., 1995). The optical detection system uses the SPR phenomenon which detects changes in refractive index at the surface of the chip. Since the refractive index is directly correlated with the concentration of material in the medium, the system can detect the binding between a ligand immobilized on the sensor chip and an analyte introduced in a flow passing over the surface. A microflow cell with a volume of 0.06/~1 is used and the bulk flow rate is in the range of 1-10 /~l/min, allowing a typical interaction analysis to be performed in 10 min. Four independent flow cells are present on each sensor chip. Changes in the SPR signal and thus in the concentration of bound molecules are monitored continuously and are presented as a plot of signal (resonance units or RU) versus time, called a sensorgram. A signal of 1000 RU corresponds to a surface concentration change of 1 ng/mm 2. After each analytical cycle, the sensor surface can be regenerated by introducing a small volume of a dissociating agent that removes the analyte from the immobilized ligand. As many as 50-100 analytical cycles can be performed on the same surface (O'Shannessy et al., 1992).

3. Advantages of the biosensor technique compared to other immunoassay systems For over 15 years, the most popular immunoassays in virology were solid-phase assays which have the advantage of allowing unreacted reagents to be removed from the immobilized probe by simple washing. The biosensor technique shares this advantage by having one of the reactants immobilized on the sensor chip. However, the immobilized probe is covalently bound to the hydrophilic dextran matrix and thus usually preserves the conformation of the molecule. In contrast, in classical solid-phase assays, the probe is immobilized by adsorption to plastic which tends to induce a~ least partial denaturation of proteins with a concomitant alteration of epitopes (Butler, 1992). Since the conformational integrity of a viral protein is usually preserved on the sensor

P.M. Richalet-Skcordel et al./ Clinical and Diagnostic Virology 5 (1996) 111-119

chip, it is possible to map conformational epitopes (Dubs et al., 1991; Tarrab et al., 1995; VanCott et al., 1995) and to detect the induction of conformational changes in the viral antigen caused by the binding of a first antibody ligand (Dubs et al., 1992; Saunal and Van Regenmortel, 1995a). A major advantage of the biosensor technique is that none of the reactants needs to be labelled and that every step in a multiple-layer assay can be directly visualized. In ELISA, only the last step is detectable and if the end result is unsatisfactory, each component of the system must be analyzed separately to discover which of the many steps is at fault. Using the BIAcore, it will be immediately apparent if monoclonal antibodies (Mabs) are suitable for a diagnostic assay. The new BIAcore 2000 instrument makes it possible to detect the binding of molecules of MW 1-2 x 103 at a concentration of 1-2 pg/mm 2 (Karlsson and Stahlberg, 1995) which is a significant improvement in sensitivity compared to that of the earlier BIAcore instrument. Multi-channel analysis facilities reduce sample consumption and in the case of the BIAcore 2000, sample recovery facilities make it possible to recover the material after the analysis is completed. Another major advantage of biosensors is that they measure interactions in real time. This allows kinetic rate constants and equilibrium affinity constants to be calculated (Altschuh et al., 1992; Karlsson et al., 1991; Karlsson et al., 1992; Pellequer and Van Regenmortel, 1993; Zeder-Lutz et al., 1993a). Knowledge of the affinity of viral antibodies is important for the proper design of immunoassays and for understanding the molecular basis of viral epitope recognition and infectivity neutralization by antibodies (Gruen et al., 1993; VanCott et al., 1994; Van Regenmortel et al., 1994).

4. Selection of antibody probes The biosensor instruments that are commercially available at present have been designed as powerful, multipurpose, analytical instruments and they are not suitable for the large scale,

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routine detection of infectious agents or their antibody markers. Within the next few years, however, it is likely that less expensive biosensor instruments will become available, dedicated to single diagnostic tasks. At the moment instruments such as BIAcore are useful in the field of diagnosis because they make it possible to select, on a rational basis, the diagnostic reagents that are most suitable for current immunoassays. Mabs intended for use in ELISA should have a reasonably fast association rate constant (in the range of 104-105 M - l s - l ) and a sufficiently low dissociation rate c o n s t a n t (10 -4 S 1) to prevent them from dissociating from the antigen during the washing step. The kinetic rate constants of Mabs can be determined from a few microlitres of culture supernatant during hybridoma cell culture and clones producing unsuitable Mabs can thus be discarded at an early stage during the selection of M a b reagents. If Mabs need to be labelled for the assay, it is also possible to assess if the labelling had any deleterious effect on the rate constants. Recombinant DNA technology allows antibody fragments to be obtained in various expression systems (Pliickthun, 1994) and by site-directed mutagenesis. It may then be possible to modify the binding characteristics to make the antibody more suitable for particular therapeutic or diagnostic applications (Malmqvist, 1994). BIAcore is also useful for assessing the binding characteristics of bispecific Mabs (Horenstein et al., 1994) and of recombinant antibodies obtained by chain shuffling (Marks et al., 1992). For double antibody sandwich ELISA, it is necessary to select pairs of Mabs that are able to bind concurrently to the same antigen molecule because the epitopes they recognize are sufficiently distant on the surface of the antigen. BIAcore is particularly well-suited for the rapid mapping of viral epitopes and for identifying which combinations of capturing and detector Mabs will give the best results in sandwich assays (Ffigerstam et al., 1990; Saunal and Van Regenmortel, 1995a,b; Tarrab et al., 1995; Tosser et al., 1994). In constrast to the situation in other epitope mapping techniques, the absence of binding activity when the antibody is used in one or the

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other sandwich position is directly apparent from the sensorgram (F/igerstam et al., 1990). The ability of certain Mabs to induce a conformational change in the antigen upon binding to it can be detected by the altered reactivity of a second Mab. The conformational change can lead to the disappearance of an epitope recognized by the second M a b or to the induction of a new conformational epitope not present before the binding of the first M a b (Dubs et al., 1992; Saunal and Van Regenmortel, 1995a,b).

5. Selection of antigenic probes Synthetic peptides are used extensively as probes for detecting viral antibodies produced during infection and for raising antipeptide antibodies capable of detecting viral antigens in infected tissues (Leinikki et al., 1993). BIAcore has been used extensively for locating continuous epitopes in viral proteins, using synthetic peptides either as immobilized ligands on the sensor chip or as inhibitors of the reaction between the antigen and specific antibodies (Altschuh et al., 1992; F/igerstam et al., 1990; Kantrong et al., 1995; Schellekens et al., 1994; Zeder-Lutz et al., 1993b). Once the epitope has been located on a particular peptide fragment, the contribution of individual residues to the antigenic activity can be assessed by analysing peptide analogues presenting single substitutions (Zeder-Lutz et al., 1993b) or deletions (Zvi et al., 1995). Several laboratories have used biosensor technology in an attempt to optimize the peptide probes used in HIV diagnosis. Various linear and cyclic peptides, corresponding to immunodominant epitopes of the gpl20 and glM1 proteins of HIV, have been studied using peptides in various formats, either as immobilized peptides, as free inhibitor peptides or as conjugated peptides (Lucey et al., 1993; Mani et al., 1994; RichaletS6cordel et al., 1994a,b; VanCott et al., 1992, 1995). In one particular study, monoclonal antibodies and rabbit polyclonal antibodies were raised against a cyclic peptide representing a chimeric sequence of a consensus V3 loop of H I V - I gpl20.

When tested by ELISA and with the BIAcore, the antibodies cross-reacted extensively with the V3 regions of different HIV-1 strains and recognized the cyclic form of the homologous peptide better than its linear form. This is illustrated in Fig. 1. There was an excellent correlation between ELISA titers and BIAcore data, indicating that biosensor data are useful for selecting peptides to be used in diagnostic solid-phase assays (RichaletS6cordel et al., 1994a,b). V3 peptides of different H I V - I types were used to inhibit the binding of antibodies to the chimeric immobilized peptide. In this way, peptide residues critical for antibody recognition could be identified. The chimeric peptide was also a good antigenic probe for detecting antibodies in the sera of patients infected with various HIV-1 strains (Richalet-S6cordel et al., 1994a). By determining the dissociation rate constants of monoclonal antibodies directed to the chimeric peptide with respect to increasingly distant V3 peptides, it was found that certain

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Fig. 1. Inhibition of binding in the BIAcore of rabbit antiserum elicited against a chimeric and cyclic HIV V3 peptide to the peptide immunogen, by increasing amounts of different V3 peptides, The sequence of the chimeric V3 peptide is a consensus sequence derived from 245 different HIV-I isolates ( LaRosa et al.. 1990). A total of 153 RU of the chimeric, cyclic V3 peptide was immobilizedon the dextran matrix. The rabbit antiserum, diluted 1/1000, was first mixed with various amounts of peptide inhibitors (range 10- 3 _102 #M) before being injected (30~1) at a flow rate of 3/~l/min over the matrix. The V3-MN and the V3-SF2 sequences share 12 and 7 residues, at the top of the loop, with the consensus sequence respectively. The ability of these peptides to compete with the binding of antibodies to the immobilized cyclic peptide is proportional to the extent of homology found in the V3 tip region (Richalet-S~cordel et al., 1994a).

P.M. Richalet-Skcordel et al./ Clinical and Diagnostic Virology 5 (1996) 111-119

residues of the V3 loop (Lys 312 and Thr 326) were particularly important for recognition. Recently, retro-inverso peptides which contain N H - C O bonds instead of C O - N H peptide bonds and which are more resistant to proteolysis than L-peptides, have been shown to be useful for mimicking viral epitopes (Benkirane et al., 1995; Guichard et al., 1994; Muller et al., 1995). The binding properties of such peptidomimetics can be easily quantified with BIAcore. A retro-inverso peptide of the immunodominant loop 141-159 of the VP1 protein of foot-and-mouth disease virus, when used as immunogen, led to higher serum antibody titers which appeared earlier after the start of immunization and lasted longer than those obtained with L-peptides. Analysis with the BIAcore showed that antibodies to retro-inverso peptides cross-reacted strongly with L-peptides and with virus particles, while antisera to VPI protein and to virions cross-reacted strongly with the retro-inverso peptides. In view of their improved stability and high level of antigenic mimicry with viral epitopes, retro-inverso peptidomimetics should have considerable potential in viral immunology (Benkirane et al., 1995; Muller et al., 1995).

6. Diagnosis of hepatitis C virus infection Second and third generation ELISA diagnostic assays for hepatitis C virus (HCV) are based on the use of recombinant proteins and synthetic peptides containing epitopes of structural and non-structural viral proteins. The combined use of these antigenic probes in ELISA improves the early detection of HCV antibodies (Huang et al., 1993; Kotwal, 1993) although additional tests such as RIBA (recombinant immunoblot assay) are needed to achieve a level of 98% sensitivity (Filice et al., 1993). Only a few peptides corresponding to envelope proteins of HCV have been used in ELISA so far. Peptides P1 and P2 corresponding to regions 210-223 and 315-327 of the E1 protein of HCV (genotype II/lb; isolate IND8) were shown to be recognized in ELISA by 45% and 92% of sera of infected individuals respectively (Ray et al., 1994).

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Peptide PI is located in one of the two variable regions of E1 protein situated in residues 210-221 and 230-237. These two regions are flanked by two Cys residues respectively at positions 207 and 226, and positions 229 and 238. Peptide P2, on the other hand, is located in a conserved region of the E1 protein which may explain its superiority as an antigenic probe for detecting HCV antibodies. Since the P1 peptide is flanked by two Cys residues in the E1 protein, we tested the hypothesis that it might be better recognized by HCV antibodies when constrained as a cyclic peptide compared to its activity as a linear peptide. Three peptides corresponding to residues 209--222, 21022i and 315-327 of the E1 protein of HCV II 1/b were synthesized by the procedures described by Briand et al. (1989) and biotinylated at their N-terminal position. Peptide 209-222 was cyclized as described by Richalet-S6cordel et al., 1994a. The capacity of the various peptides to be recognized by 30 human sera found to be positive for HCV antibodies by RIBA (HCV 3.0 Simple Addition Verification (SAVe), Ortho Diagnostic System, Emeryville, CA, USA) were tested with BIAcore. Ten normal sera were used as control. The biotinylated peptides were bound to streptavidin immobilized on sensor chips. Typical sensorgrams obtained with one HCV-positive and one HCV-negative human serum are shown in Fig. 2. No binding was observed when the normal control sera were allowed to react with the sensor chip containing immobilized cyclic peptide 209222. The RIBA-positive serum gave a 2.5-fold higher resonance signal with the cyclic 209-222 peptide than with the 210-221 linear peptide. Furthermore, 87% of the RIBA-positive sera recognized the cyclic peptide whereas only 40% recognized the linear peptide. In addition, the linear P2 peptide (residues 315-327) was recognized by 90% of the same sera. When BIAcore results obtained with the P2 peptide and the cyclic P1 peptide were combined, a total of 93% of the sera were found to contain HCV antibodies. It should be noted, however, that the sera that were tested are not representative of the usual HCV-positive human sera. They were selected for the present

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7. Application in vaccine studies

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Fig. 2. Typical sensorgrams obtained when normal serum (sensorgram 1) and RIBA-positive serum (sensorgrams 2 and 3) were injected over HCV peptides immobilized on the dextran matrix. Phase (a) corresponds to the injection of 30/~1 of serum (diluted 1/100). Phase (b) corresponds to the injection of buffer for 2 min. The dissociation of antibodies is visualized by the RU signal decreasing with time. The amount of bound antibodies (RU) was obtained 10 s after the beginning of buffer injection. Phase (c) corresponds to the regeneration phase with 6 /~l of HCI 0.IN. Sensorgrams I and 3 were obtained when 188 RU of the cyclic HCV E2 peptide (residues 209 222) was immobilized on the dextran matrix via amine groups. Sensorgram 2 was obtained when 195 RU of the linear HCV E2 peptide (residues 210-.221) was immobilized on the dextran matrix via amine groups. A flow rate of 3/zl/min was used during all phases.

study because they contained high antibody titres to each of the core, NS3, NS4 and NS5 antigens when tested by the Ortho Diagnostic System. Our results suggest that when the cyclic peptide 209 222 is immobilized on the sensor chip via a biotin moiety, it retains a constrained conformation which is better recognized by HCV antibodies than the linear form. This finding, which is similar to the situation found with the V3 loop of HIV-l (Richalet-S6cordel et al., 1994a), needs to be confirmed with a larger panel of sera from HCV-infected individuals. It will also be important to test sera obtained soon after sero-conversion. These preliminary observations, however, demonstrate the value of the biosensor technology for rapidly assessing the suitability of an antigenic peptide probe in viral diagnosis.

There is little understanding, at present, of the molecular basis of virus neutralization by antibodies. Several authors have tried to establish a relationship between the affinity and neutralization capacity of Mabs specific for viral antigens. In some cases, it was found that antibodies neutralized viral infectivity in proportion to their affinity (Langedijk et al., 1991; Roost et al., 1995) while in others, no such relationship was found (Brown et al., 1990). Zolla-Pazner et al. (1992) showed that there was a relationship between the affinity of human Mabs directed to the V3 loop of HIV-I (when these were tested against the corresponding peptide or gpl20 antigen) and their capacity to neutralize the virus. Subsequently, VanCott et al. (1994) using the BIAcore showed that this relationship was due to corresponding changes in the dissociation rate constant rather than in the association rate constant of the antibodies. Since similar binding kinetic profiles were obtained with synthetic peptides and with purified recombinant gpl20 protein, it was easier to screen many diverse isolates with peptides rather than with recombinant proteins. The influence of amino acid substitutions within the V3 loop on subsequent Mab binding could be ascertained by observing alterations in dissociation rates (Richalet-S6cordel et al., 1994b; VanCott et al., 1994). The biosensor technique, therefore, facilitates the screening and selection of antibodies that are likely to possess the most potent HIV-1 neutralizing potential. BIAcore has also been used to study the antibody affinity maturation after vaccination of volunteers with subunit influenza vaccine (Cox et al., 1993). Average affinities of antibodies in human sera collected from 2 to 60 days after vaccination were measured and an appreciable increase in affinity was observed after 14 days. Recently, VanCott et al. (1995) measured the binding to oligomeric gpl60 protein of antibodies appearing early during acute HIV-infection. They found that HIV-I seroconversion could be detected earlier with the oligo gpl60 probe than by many of the commercial ELISA screening kits. Detection of serum binding to oligo gpl60 by BIAeore was

P.M. Richalet-S~cordel et al. / Clinical and Diagnostic Virology 5 (1996) 111-119

usually m o r e sensitive than by E L I S A which m a y be due to enhanced preservation o f native oligo g p l 6 0 structure within the biosensor matrix (VanC o t t et al., 1995). The authors are also currently examining if this oligomeric g p l 6 0 protein, when used as an immunogen, is able to induce an i m m u n e response with increased HIV-1 cross-reactivity and neutralization capacity.

8. Conclusion The introduction o f biosensor instruments in 1990 heralded a new dimension in biochemical analysis. This new technology allows binding interactions between viral antigens and antibodies to be measured with unparalleled ease without labelling the reagents. Artefactual changes in binding properties that often result from the labelling process are thus avoided. The biosensor instruments m a k e it possible to determine which molecules interact, h o w quickly and h o w strongly they interact, what their t o p o g r a p h i c distribution looks like, what the binding stoichiometry is and whether c o n f o r m a t i o n a l changes occur u p o n binding. The binding can be expressed not only in terms o f equilibrium affinity constants but also in terms o f on and off kinetic rates. The impact o f residue substitutions on epitope activity can be measured in terms o f altered binding kinetics and useful information on the t h e r m o d y n a m i c s o f the binding interaction can thus be obtained (Kelley, 1994). Since there is a g o o d correlation between B I A c o r e and E L I S A data, the biosensor technology will be increasingly used for developing reagents intended for viral diagnosis.

Acknowledgements We thank P r o f A. G o u d e a u , H6pital Bretonneau, Tours, France, for the generous gift o f h u m a n sera. W e are indebted to Fabienne Holtzmann, Neosystem S.A., Strasbourg, France, for advice on peptide cyclization. This w o r k was partially funded by a grant to M H V V R f r o m A R C , Paris.

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