Primary Antibody Responses to a Well-Defined and Unique Hapten Are

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Proc. Nati. Acad. Sci. USA Vol. 83, pp. 2604-2608, April 1986 Immunology Primary antibody responses to a well-defined and unique hapten are not enhanced by preimmunization with carrier: Analysis in a viral model SHIV CHARAN GUPTA*, HANS HENGARTNER, AND ROLF M. ZINKERNAGELt Department of Experimental Pathology, Institute of Pathology, University Hospital Zlrich, 8091 Zdrich, Switzerland Communicated by A. Frey-Wyssling, December 5, 1985

ABSTRACT We used a viral model to reexamine classical been studied particularly carefully in influenza viruses experiments showing that mice previously primed with a (16-19) or vesicular stomatitis viruses (VSV, refs. 20-23), "carrier" molecule alone and then challenged with the carrier- because they are the structures used for the attachment ofthe hapten conjugate exhibited an enhanced antihapten antibody virus to cells. Antibodies against these unique determinants response. Mice were primed with live or UV-inactivated are virus-neutralizing antibodies. vesicular stomatitis virus (VSV) Indiana (bId) serotype with or The VSV glycoprotein G is the major component of the without complete Freund's adjuvant. After challenge with VSV viral coat (20). It is the only viral antigen expressed on New Jersey (NJ), these mice developed a secondary-type IgG infected cells and is the target antigen for VSV-specific response, measured by antibody binding in an ELISA, against cytotoxic T cells (20, 21, 24). It has been shown unequivo- both VSV-Ind and VSV-NJ. The same result was found for the cally that the determinant involved in virus neutralization by reciprocal experiments where mice were primed with VSV-NJ. antibody is located on G: monoclonal antibodies capable of Similarly, when mice were primed with live VSV, UV-inac- neutralizing virus only precipitate G protein (21, 22), and tivated VSV, or purified VSV glycoprotein G of Ind or NJ animals immunized with purified G are protected against serotype and later were challenged with dinitrophenyl (N2ph)- VSV and generate neutralizing antibodies (21-23). conjugated, UV-inactivated VSV or with N2ph-conjugated G In analogy to the classical hapten-carrier models, the protein of either serotype, they exhibited a secondary-type neutralizing determinant on the VSV glycoprotein would anti-N2ph antibody response as demonstrated by the binding of represent a hapten whereas the rest of the major glycopro- IgG to dinitrophenylated bovine serum albumin measured by teins or other constant viral structures represent the carrier ELISA. In contrast, when neutralizing antibody responses moiety. T-cell crossreactivity for the carrier is illustrated by were monitored, VSV-Ind-primed mice challenged with VSV- the fact that many cytotoxic T cells to VSV or influenza do NJ developed a strictly primary type of anti-VSV-NJ response not of the and vice versa. We conclude that preexistent helper T cells readily distinguish between different serotypes specific for shared carrier determinants do not improve virgin same virus type (24-29). We have analyzed the antibody B-cell responses specific for "new," unique determinants that responses in VSV-primed mice, after challenge with homol- are the target for the biologically relevant neutralizing anti- ogous or heterologous serotype, by (i) ELISA, revealing bodies. These findings suggest that priming of B cells rather crossreactive antibodies binding to the whole virion or than ofhelper T cells may be ofimportance to induce protective purified glycoprotein, and (it) serum neutralization assays, immunity mediated by antibodies. monitoring the type of antibody response against the unique determinants involved in neutralization. We show that mice Initiation and maintenance of an IgG antibody response primed with one serotype of VSV do not develop an en- against most antigens require T- and B-cell collaboration hanced neutralizing antibody response when challenged with (1-5). It is thought that T cells recognize the so-called carrier a heterologous VSV, whereas antibody responses to shared determinants of an antigen, whereas B cells are thought to determinants measured by ELISA are of secondary type. recognize the same or additional determinants, usually called haptens (1-6). Classical experiments have established that MATERIALS AND METHODS mice primed with the carrier part of an antigenic molecule, upon immunization with a hapten conjugated to the same Mice. DBA/2 mice, from the Institut fir Zuchthygiene, carrier molecule, respond with an enhanced B-cell response Zurich, were used throughout these experiments. against the hapten when compared with unprimed mice (7-9). Virus. VSV-Ind (serotype Indiana) and VSV-NJ (serotype Most experiments on the relative role of carrier-primed T New Jersey) virus stocks were prepared in BHK-21 cells as cells in antihapten responses of primed or unprimed B cells described (30). Concentration and purification of the virus have used artificial antigens such as 2,4-dinitrophenyl were carried out according to established procedures (21). (N2ph)-conjugated keyhole limpet hemocyanin, 2,4,6- Preparation of Purified Surface Glycoprotein G. The prep- trinitrophenyl (N3ph)-conjugated bovine gamma globulin, or aration ofG protein has been described (21). In brief, purified heterologous erythrocytes (7-14). Our studies were aimed at VSV-Ind and VSV-NJ were treated with Triton X-100 at a evaluating these rules in an antiviral immune response. final concentration of2% (vol/vol). The Triton/virus mixture Viruses that belong to the same type but are subdivided was stirred at room temperature for 60 min and then centri- according to their serotypes (species or subtype) (15) share fuged at 140,000 x g at 5°C for 90 min. The supernatant many antigenic determinants on various structural gene products; they also possess unique determinants, usually on Abbreviations: VSV, vesicular stomatitis virus; Ind, serotype Indi- major viral glycoproteins. These unique determinants have ana; NJ, serotype New Jersey; G, major viral coat glycoprotein; CFA, complete Freund's adjuvant; pfu, plaque-forming unit(s); N2ph, 2,4-dinitrophenyl; N3ph, 2,4,6-trinitrophenyl. The publication costs of this article were defrayed in part by page charge *Present address: Department of Veterinary Microbiology, Haryana payment. This article must therefore be hereby marked "advertisement" Agricultural University, Hissar, Haryana, India-125004. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed.

2604 Downloaded by guest on September 25, 2021 Immunology: Gupta et al. Proc. Natl. Acad. Sci. USA 83 (1986) 2605 containing G was checked for purity by polyacrylamide gel Switzerland) at 40C overnight. The plates were then washed electrophoresis (20). five times with phosphate-buffered saline containing 0.05% Immunization. For primary or secondary immunization Tween 20 (PBS/Tween). Sera diluted in PBS/Tween (100 ,uI with live virus, 106 plaque-forming units (pfu) of VSV-Ind or per well) were incubated in the antigen-coated plates for 2 hr VSV-NJ were given i.v. Virus inactivated by UV light was at room temperature. Plates were again washed five times injected i.p. at a dose of 108 pfu, emulsified in complete with PBS/Tween and then were incubated with goat anti- Freund's adjuvant (CFA). The optimal doses ofVSV or VSV mouse IgG, labeled with horseradish peroxidase (Tago 4143, glycoprotein coupled with N2ph were predetermined by Burlingame, CA) and diluted in PBS/Tween (100 Al per well), titration in vivo; for primary immunization, 20 jig of antigen for 2 hr at room temperature. The plates were washed again was injected with CFA i.p., and for secondary immuniza- in PBS/Tween, and 200 ,ul of substrate [2 mg of 2,2'-azino- tions, the same amount was given i.v. without CFA. bis(3-ethylbenzthiazolinesulfonate) (Boehringer Mannheim; Coupling of VSV-Ind and VSV-NJ with N2ph. Dinitrophen- ref. 6) in 20 ml of 0.1 M NaH2PO4 (pH 4.0) and 15 Al of 30%6 ylated VSV-Ind (Ind-N2ph) and VSV-NJ (NJ-N2ph) were H2021 was added per well. The optical density at 405 nm prepared according to standard methods (31). In brief, virus was measured with a micro-ELISA reader (Virion AG, purified on sucrose gradients was mixed with sodium 2,4- Ruschlikon, Switzerland). dinitrobenzenesulfonate at pH 8.0. The mixture was stirred For the determination of anti-N2ph antibodies, 1.0 jug of overnight, protected from light and at room temperature. The coupled bovine serum albumin (BSA-N2ph, a gift from H. mixture was centrifuged at 5,000 x g to remove insoluble Binz, Institute of Virology and Immunology, University of material and then the hapten-modified virus was resediment- Zilrich) was used per well ofa microwell plate. Before adding ed. To wash off excess sodium dinitrobenzenesulfonate, the the second antibody conjugate, the wells were pretreated virus was suspended in Hanks' balanced salts solution (BSS) with 1.0% normal goat serum for 30 min at room temperature; and resedimented three times. The sedimented dinitro- otherwise the same protocol was used as described for the phenylated virus was collected in 500 jul of BSS and the ELISA to determine anti-VSV antibody titers. optimal dilution for the immunization ofmice was determined in a preliminary experiment in vivo. RESULTS Coupling of VSV-Ind Glycoprotein and VSV-NJ Glycopro- tein with N2ph. Reaction ofVSV-Ind and VSV-NJ G proteins Primary Antibody Responses Against VSV. After primary with N2ph was carried out essentially as described for the immunization with VSV-Ind and VSV-NJ, serotype-specific whole virus (31), except that the free N2ph was removed by IgM antibodies (2-mercaptoethanol-sensitive serum neutral- filtration through Amicon YM10 filters (Amicon). The opti- ization antibody) were detectable on day 2 and reached mal amounts of G(Ind)-N2ph and G(NJ)-N2ph for the immu- maximal levels on day 6 (Fig. 1 A and D). IgM antibody nization of mice were predetermined in vivo. responses dropped below a detectable level by about 3 weeks Serum Neutralization Test. The protocol for the test has (data not shown). IgG antibodies (2-mercaptoethanol-re- been described (20). In brief, serial 2-fold dilutions of heat- sistant serum neutralization antibody) were first detected on inactivated sera were mixed with equal volumes of virus day 6 or 7, reached maximal levels around day 10-12 (Fig. 1 containing 50 pfu in 100 ,ul and incubated at 370C for 90 min. A and D), and persisted for several weeks. At 22 weeks after Dilution ofthe serum resulting in 50%6 reduction ofplaques on primary immunization with 106 pfu, IgG serum neutralization Vero monolayers grown in a 24-well plate (Costar 3024) was antibody titers on the order of 1280-2560 were usually doubled to compensate for the addition ofan equal volume of observed. The kinetics of anti-VSV IgG responses assessed virus in the serum/virus mixture. To determine IgG titer, by ELISA were found to be comparable to that of antibody serum was pretreated with 0.05 M 2-mercaptoethanol (32). responses measured by serum neutralization (Fig. 2). Enzyme-Linked Iminunosorbent Assay (ELISA). The pro- Secondary Antibody Responses Against VSV. After second- cedure adopted was a modification ofthe assay described by ary immunization with the homologous virus, anti-VSV Voller et al. (33). For the determination of anti-VSV anti- serum neutralization IgG antibody responses had increased bodies, the optimal dilution of the purified virus in 100 Al of 4-fold from prechallenge values by day 4 and reached peak coating buffer (pH 9.6) was adsorbed to flat-bottomed 96-well titers (usually about 80,000) after day 6 (Fig. 1 C and F). polystyrene type F microwell plates (Petra Plastic, Inotech, Similarly, IgG titers assessed in ELISA increased rapidly

81,920 A B 20,480I ,D0 I- 5,120 W0._ 1,280 4) 320 . laW-i 80 NJ-Ind Ind-Ind 0 <80: / .0 ._

D F D- FIG. 1. Anti-VSV-Ind (A-C) and anti-VSV-NJ be1 81,920 (D-F) serum neutralizing antibody responses in mice. 20,480 - Cr *, 2-mercaptoethanol-resistant titers; c, total antibody 5,120 titers. (A andD) Anti-VSV serum neutralizing antibody a) 1,280 kinetics in unprimed mice. (B andE) Anti-VSV kinetics in mice primed 22 weeks earlier with VSV-NJ (B) or 320 VSV-Ind (E) and challenged with the heterologous 80 0-/NJ .NJ-NJ serotype. (C and F) Anti-VSV kinetics in mice primed <80 E - 22 weeks earlier with VSV-Ind (C) or VSV-NJ (F) and 2 4 16 8 10 12 14 2 4 6 8 10 12 14 2 4 6 8 10 12 14 challenged with the homologous serotype. (Antibody Bleeding intervals, days titers before challenge at 22 weeks were 1280.) Downloaded by guest on September 25, 2021 2606 Immunology: Gupta et A Proc. Natl. Acad. Sci. USA 83 (1986)

A Ind-Ind B NJ-NJ 81,920 -/ U------Ind-NJ I " NJ-Ind a 20,480 0-Ind w------~ I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 11 ;% 5,120 // A" I ., ,,a"'~-NI FIG. 2. (A) Kinetics of anti-VSV-Ind IgG anti- 4 "I, body responses, detected by ELISA, in mice 1,280 a II primed with VSV-Ind 22 weeks before challenge II II with homologous (v) or heterologous (v) VSV, V) 320 NJ compared with a primary anti-VSV-Indresponse- Ind II (A). (B) Kinetics of anti-VSV-NJ IgG antibody 80 I II responses, detected by ELISA, in mice primed with <80 ------Id VSV-NJ 22 weeks before challenge with, homolo- D- 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14 gous (o) or heterologous (v) VSV, compared wmth a Time after infection, days primary anti-VSV-NJ response (o). after secondary immunization and peaked at 6-8 days (Fig. were found on virus-coated plates and on plates coated with 2). purified VSV glycoprotein G; this suggested that many ofthe Effect of Priming on Serum Neutralization Antibody Re- antibodies against whole virus were directed against the sponses Against Heterologous VSV. Serum neutralization major VSV glycoprotein (data not shown). antibody kinetics of anti-VSV-Ind and anti-VSV-NJ were Effects ofPriming with Homologous or Heterologous VSVon measured in mice that had been primed with heterologous N2ph- or VSV-Specific IgG Responses Against VSV-N2ph. In (Fig. 1 B and E) or homologous (Fig. 1 C and F) serotype 22 order to evaluate the capacity of viral antigens to mediate a weeks earlier; unprimed mice served as controls. The kinet- carrier function comparable to that described for classical ics of IgM and IgG serum neutralization antibodies in carriers such as keyhole limpet hemocyanin or bovine gamma unprimed control mice, mice primed with unrelated vaccinia globulin or albumin, mice were primed with VSVInd or virus (data not shown) or mice primed with the heterologous VSV-NJ in CFA and 3 weeks later were injected with serotype followed identical patterns; they were typical for UV-inactivated Ind-N2ph or NJ-N2ph. primary responses, with IgM titers being high on days 4-6, Mice primed with VSV, upon reimmunization with dini- whereas IgG titers only rose after day 6. Only mice primed trophenylated VSV, showed enhanced secondary responses with the homologous serotype showed a typical secondary against N2ph, with titers around 10,000 (Fig. 3, first 4 groups) type of response. as compared to very weak responses (titer 4100) in unprimed Effect of Priming on IgG Responses Measured by ELISA. mice (Fig. 3, last 2 groups). Moreover, the anti-N2ph re- VSY-Ind-primed mice made a secondary type of anti-VSV- sponses were enhanced irrespective of the VSV. serotype Ind antibody response upon challenge with either VSV-Ind or used for priming; this is indicative of common determinants VSV-NJ, when tested by ELISA (Fig. 2A); similarly, VSV- between the two serotypes that serve as carrier determinants. NJ-primed mice showed secondary-type anti-VSV-NJ anti- However, antibodies against the unique determinant(s) mea- body responses when VSV-NJ or VSV-Ind was used for the sured by the serum neutralization test did not show enhanced second infection (Fig. 2B). Thus, it is possible to readily or accelerated responses in mice primed with a heterologous cross-prime with VSV of the heterologous serotypes for a serotype (see Fig. 3, anti-VSV SN titers), confirming the secondary-type IgG antibody response. results obtained with live virus (Fig. 1). To further evaluate It was important to evaluate that the crossreactive anti- whether shared determinants expressed on the major glyco- bodies induced by live VSV and detected in ELISA were protein G could serve as carrier determinants for helper T directed against the same surface glycoprotein that also cells, mice were primed with live (data not shown) or carries the determinant involved in the antibody-dependent UV-inactivated whole virus (Fig. 4A) or purified G protein neutralization. Antibody titers in sera from mice primed with (Fig. 4B) and later were injected with purified G-N2ph of VSV-Ind and given second injections with the homologous or homologous or heterologous serotype. Mice preimmunized heterologous serotype were measured by ELISA on plates with whole virus ofeither serotype showed a comparable titer coated with purified VSV-Ind or VSV-Ind glycoprotein G. of N2ph-specific IgG response irrespective of which G-N2ph Comparable patterns of antibody titers and of their kinetics serotype was usedforthe second immunization. Afterchallenge

FIG. 3. Capacity ofVSV-Ind Anti-VSV SN titers Anti-VSV ELISA titers Anti-N2ph titers -and VSV-NJ viral antigens to a F,& function as carrier molecules. Immunization >R8 -I Mice were primed with 108 pfu of v v 4. lb V ____-___ UV-inactivated VSV-Ind or Primary Secondary VSV-NJ in CFA and injected i.v. 3 weeks later with Ind-N2pH Ind (UV)/CFA Ind-N2ph I or NJ-N2ph (20 pg) in Hanks' S~~~~~~~~~~~~~~~~~~~~~~~~~~balanced salts solution (BSS). 2-~yj Sera were obtained on days 4 Ind (UV)/CFA NJ-N2ph and 8 after secondary immunization. Anti-VSV-Ind IgG anti- NJ (UV)/CFA Ind-N2ph body titers (i) and anti-VSV-NJ IgG antibody titers (in) were detected by serum neutralization NJ (UV)/CFA NJ-N2ph (SN) test and ELISA, and anti- were d 1010 N2ph titers (m) measured Ind-N2ph by ELISA. Titers on day 4 are shown by hatched or stippled

- NJ-N2ph [ bars, and increased titers on day 8, by extended open bars. Downloaded by guest on September 25, 2021 Immunology: Gupta et al. Proc. Natl. Acad. Sci. USA 83 (1986) 2607 with G-N2ph, anti-VSV responses detected by ELISA cross- Ind-N2ph (or vice versa) the anti-N2ph response was vastly reacted in all combinations tested, confirming the results ob- enhanced. These latter results are compatible with the tained with live or UV-inactivated VSV (Figs. 2 and 3). Titers findings that carrier-primed mice, guineapigs, or rabbits (7-9) peaked somewhat earlier in mice primed with the whole virus respond with an enhanced antibody response against the (Fig. 4A) than in those primed with G protein (Fig. 4B). This hapten when challenged with a hapten-carrier conjugate. may be a result ofdiffering amounts ofantigen available and/or A trivial reason that might explain our results is that a difference in antigen presentation. Anti-VSV serum neutral- antibodies against carrier or haptenic parts of VSV G block ization IgG antibody responses induced after challenge with the secondary response. This form ofinhibition of secondary purified G-N2ph of the heterologous serotype were again of antibody responses (10-12, 34) has been found when re- primary type. sponses were tested in the same animal instead of by In some groups of mice that had been primed with a adoptive-transfer experiments (34). However, this explana- preparation of one VSV type and subsequently were chal- tion is not likely to apply to the results obtained with VSV lenged with the heterologous VSV type, the serum neutral- because only neutralizing antibodies can prevent productive ization titer against the priming VSV occasionally rose to infection or antigen expression on host cells (16, 17). variable extents during the second challenge; examples are in Our results may reflect limitations imposed on T cells, B Fig. 4 A and B and Fig. 3 in the second group, with a rise in cells, or antigen presentation. First, from the T-cell point of anti-Ind serum neutralization titer. This phenomenon is view, a trivial reason for not seeing enhanced neutralizing known as "original antigenic sin" and has been studied antibody responses despite previous priming with a carrier extensively in the influenza model (reviewed in refs. 16 and may be that the postulated crossreactive helper T cells do not 17). exist. The existence ofcytotoxic T cells crossreactive against VSV-Ind- and VSV-NJ-infected targets (26) suggests that DISCUSSION such helper T cells may, however, be present. Crossreactive T help is also suggested by the efficient acceleration and Our experiments show that priming of helper T cells specific increase of antibody responses to N2ph or to VSV or VSV G for VSV glycoprotein G did not enhance virus-specific B-cell protein in a secondary immunization with heterologous VSV responses to the serologically unique determinants on VSV G or G protein (Figs. 3 and 4), which shows that VSV is no that are involved in virus neutralization. This result does not exception to the rule established for N2ph-conjugated clas- agree with the previously established rule that carrier-primed sical soluble carrier antigens such as keyhole limpet helper T cells accelerate and enhance primary IgG responses hemocyanin. to hapten epitopes on the same carrier structure (7-9). We One might argue that large molecules may induce several found that antibody responses against VSV-Ind in mice subpopulations of helper T cells, each of which can collab- primed previously with VSV-NJ (or vice versa) were en- orate with B cells specific for an epitope that is rather close hanced and of secondary type when antibodies were meas- to the one recognized by the helper T cells but cannot help B ured by their capacity to bind to virus or to purified VSV G. cells specific for more distant epitopes. Thomas et al. (35) Also, when mice primed with VSV-NJ were challenged with have shown that at least two sets of helper T cells for

Anti-VSV SN titers Anti-VSV ELISA titers Anti-N2ph titers

CNNF~ -O-v aO ! C' ° D <4 .4 - ° - -4a & V Immunization . 0 Primaruy Secondary A Ind (UV)/CFA G(Ind)-N2ph

Ind (UV)/CFA G(NJ)-N2ph t ....

NJ (UV)/CFA G(Ind)-N2ph

NJ (UV)/CFA G(NJ)-N2ph

G(Ind)/CFA G(Ind)-N2ph-]

G(Ind)/CFA G(NJ)-N2ph Z0

G(NJ)/CFA G(Ind)-N2ph j FIG. 4. The effect ofpreimmunizing G(NJ)/CFA G(NJ)-N2ph mice with whole virus or G protein on IgG responses to challenge injections with N2ph coupled to G protein of I homologous or heterologous VSV. Mice CBSS/CFA G(Ind)-N2ph I _I. j- -j were primed with 108 pfu of UV-inac- I tivated virus in CFA i.p. (A), 20 ,ug ofG BSS/CFA G(NJ)-N2ph protein in CFA i.p. (B), or balanced salts solution (BSS) in CFA or were unprimed (C). Six weeks later, the mice were given a secondary injection i.v., G(Ind)-N2ph IY 1 i containing 20 ,ug of G-N2ph from ho- M__ mologous or heterologous serotype. G(NJ)-N2ph -E For symbols see legend to Fig. 3. Downloaded by guest on September 25, 2021 2608 Immunology: Gupta et al. Proc. Natl. Acad. Sci. USA 83 (1986)

anti-N2ph responses could be demonstrated in an anti- important message of this study is that preexisting T-cell influenza hemagglutinin-specific response: one set specific help, even in great excess, does not enhance and accelerate for a common part, the other for the unique determinants. IgG responses of virgin B cells to epitopes such as virus- However, this result does not prove that these two helper- neutralizing determinants. T-cell sets help B cells specific for the same respective These findings not only modify an established rule but are domains of the molecule. of practical relevance: B cells specific for unique epitopes Our results were the same whether immunization was with cannot be helped to generate a greater primary IgG response replicating virus or with nonreplicating UV-inactivated virus, more rapidly by primed helper T cells than by helper T cells or alternatively with UV-inactivated VSV or purified G induced during a primary response. Therefore, the aim of a proteins in CFA. Therefore, the differences observed cannot vaccine must be to prime B cells against the neutralizing have been caused by different kinetics or specificities of determinant to increase numbers and/or inducibility of mem- helper T cells induced in the presence or absence of CFA. It ory B cells. This is the most efficient if not the only way to was important to exclude this possibility, since most classical induce prospective immunity mediated by antibodies. If experiments establishing enhanced antihapten response after priming for T help has no beneficial effect on IgG kinetics, the priming with a carrier molecule have been done by priming T use of a standard carrier for synthetic viral epitopes may not cells with antigens emulsified in adjuvants (1, 13, 14). improve chances of obtaining antibody titers more quickly. A second explanation for our findings is that the B-cell response to neutralizing determinants is very restricted or 1. Katz, D. H. & Benacerraf, B. (1972) Adv. Immunol. 15, 1-94. 2. Miller, J. F. A. P. & Mitchell, G. F. (1969) Transplant. Rev. 1, 3-42. selective when compared with classical haptens. Three major 3. Mitchison, N. A. (1971) Eur. J. Immunol. 1, 18-27. points should be considered. (i) Classical haptenic epitopes 4. Rajewski, K., Schirrmacher, V., Nase, S. & Jerne, N. K. (1969) J. Exp. used as antigens may not be truly "new" to the B cells of the Med. 129, 1131-1142. responding host; N3ph, N2ph, and arsonate are small epitopes 5. Miller, J. F. A. P. (1971) in 2nd International Convocation ofImmunol- & to a ogists, Buffalo, N.Y., eds. Cohen, S., Cudkowicz, G. McCluskey, that fit with varying affinities wide variety of B-cell R. T. (Karger, Basel), pp. 2-12. receptors ofeither truly unprimed B cells or B cells that have 6. Landsteiner, K. (1962) The Specificity ofSerological Reactions (Dover, been previously primed by crossreactive antigens. (it) This New York), Rev. Ed. great variability of responding B cells is probably increased 7. Katz, D. H., Paul, W. E., Goidl, E. A. & Benacerraf, B. (1970) J. Exp. Med. 132, 261-282. by the fact that N2ph or N3ph may modify NH2 groups in an 8. Tada, T. & Okumura, K. (1971) J. Immunol. 107, 1137-1145. undefined distribution, generating great numbers of different 9. Kontiainen, S. (1971) Eur. J. Immunol. 1, 276-280. antigenic specificities on carrier proteins; the coupling den- 10. Ashley, H. & Ovary, Z. (1965) Proc. Soc. Exp. Biol. Med. 119, 311-314. sity used to characterize such modified carriers is a very 11. Levine, B. B. (1967) J. Immunol. 99, 1173-1177. 12. Rittenberg, M. B. & Amkraut, A. A. (1966) J. Immunol. 97, 421-430. unprecise measure for the antigenic quality. Therefore, such 13. Schierman, L. W. & McBride, R. A. (1967) Science 156, 658-659. haptens give the illusion of expressing precisely defined 14. Coe, J. E. & Salvin, S. B. (1964) J. Immunol. 93, 495-510. epitopes, but in fact they may create tens to hundreds of 15. Davis, N. F., Dulbecco, T., Eisen, H. N. & Ginsberg, H. S. (1980) antigenically different, even noncrossreacting, determinants Microbiology (Harper & Row, Philadelphia), p. 1121. 16. Fazekas De St. Groth, -S. (1967) Cold Spring Harbor Symp. Quant. Biol. (or haptens). (iii) Possible heterogeneity of antigens is further 32, 525-536. increased by the use of CFA (36). 17. Fazekas De St. Groth, S. (1981) in Immunol. Methods, eds. Steinberg, An aspect to analyze is the apparently special case of the C. M. & Lefkovits I. (Karger, Basel), pp. 155-168. antigenic determinants involved in virus neutralization. The 18. Wilson, I. A., Skehel, J. J. & Wiley, D. C. (1981) Nature (London) 289, 373-378. readout for antibodies specific for either N2ph, N3ph, eryth- 19. Palese, P. & Young, J. P. (1982) Science 215, 1468-1474. rocytes, or heterologous proteins such as bovine gamma 20. Wagner, R. R., Snyder, R. M. & Yamazaki, S. (1970) J. Virol. 5, globulin or lysozyme usually is the binding of antibody. To 548-558. single out unique specificities has been difficult, as discussed 21. Kelley, J. M., Emerson, S. V. & Wagner, R. R. (1972) J. Virol. 10, a test 1231-1235. above. Obviously, virus neutralization yields precise 22. Dietzschold, B., Schneider, L. & Cox, J. H. (1974) J. Virol. 14, 1-7. for specificity, which makes virus a unique tool in immunol- 23. Volk, W. A., Snyder, R. M., Benjamin, D. C. & Wagner, R. R. (1982) J. ogy. The tight packaging of the virus coat glycoproteins Virol. 42, 220-227. bearing the neutralizing determinant probably plays a key 24. Hale, A. H., Ruebush, M. J. & McGee, M. P. (1981) Antiviral Res. 1, determinants are accessible to 63-70. role, because only neutralizing 25. Lefrangois, L. & Lyles, D. S. (1982) Virology 121, 157-167. antibodies (16); thus, antibodies with specificities for com- 26. Zinkernagel, R. M. & Rosenthal, K. L. (1981) Immunol. Rev. 58, mon determinants cannot bind and neutralize virus. One may 131-155. argue that virus-neutralizing determinants that function as 27. Effros, R. B., Doherty, P. C., Gerhard, W. & Bennink, J. (1977) J. Exp. Med. 145, 557-569. antigens in a general immune response are the exception 28. Reiss, C. S. & Burakoff, S. J. (1981) J. Exp. Med. 154, 541-546. rather than the rule. However, there is no doubt that immune 29. Anders, F. M., Katz, J. M., Jackson, D. C. & White, D. 0. (1981) J. responses are generally aimed at defending vertebrate hosts Immunol. 127, 669-672. against infectious agents. Most virus glycoproteins involved 30. Charan, S. & Zinkernagel, R. M. (1985) J. Immunol., in press. in attachment to cells have been forced to develop variability 31. Good, A. H., Wofsy, L., Henry, C. & Kimura, J. (1980) in Selected Methods in Cellular Immunology, eds. Mishell, B. B. & Shilgi, S. M. between members of the same virus type or between the (Freeman and Company, San Francisco), pp. 343-350. various virus types and virus families to achieve minimal 32. Scott, D. W. & Gershon, R. K. (1970) Clin. Exp. Immunol. 6, 313-316. crossreactivity and maximal uniqueness of these determi- 33. Voller, A., Bidwell, D. & Bartlett, A. (1980) in Manual of Clinical nants in order to escape neutralization by preexisting anti- Immunology, eds. Rose, N. R. & Friedman, H. (Am. Soc. Microbiol., bodies. Washington, DC), pp. 359-371. 34. Hamaoka, T., Takatsu, K. & Kitagawa, M. (1973) Immunology 24, In conclusion, the rule that carrier priming enhances IgG 409-424. responses by B cells only holds when B cells have been 35. Thomas, D. B., Hackett, C. J. & Askonas, B. A. (1982) Immunology 47, primed previously by crossreacting epitopes and when anti- 429-436. body titers are measured by antibody-binding assays. The 36. Freund, J. (1951) Am. J. Clin. Pathol. 21, 645-656. 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