trinitrophenyl (N3ph)-conjugated bovine gamma globulin, or heterologous ..... unprimed control mice, mice primed with unrelated vaccinia virus (data not shown) ...
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
been studied particularly carefully in influenza viruses (16-19) or vesicular stomatitis viruses (VSV, refs. 20-23), because they are the structures used for the attachment ofthe virus to cells. Antibodies against these unique determinants are virus-neutralizing antibodies. The VSV glycoprotein G is the major component of the viral coat (20). It is the only viral antigen expressed on infected cells and is the target antigen for VSV-specific cytotoxic T cells (20, 21, 24). It has been shown unequivocally that the determinant involved in virus neutralization by antibody is located on G: monoclonal antibodies capable of neutralizing virus only precipitate G protein (21, 22), and animals immunized with purified G are protected against VSV and generate neutralizing antibodies (21-23). In analogy to the classical hapten-carrier models, the neutralizing determinant on the VSV glycoprotein would represent a hapten whereas the rest of the major glycoproteins or other constant viral structures represent the carrier moiety. T-cell crossreactivity for the carrier is illustrated by the fact that many cytotoxic T cells to VSV or influenza do not readily distinguish between different serotypes of the same virus type (24-29). We have analyzed the antibody responses in VSV-primed mice, after challenge with homol-
ABSTRACT We used a viral model to reexamine classical experiments showing that mice previously primed with a "carrier" molecule alone and then challenged with the carrierhapten conjugate exhibited an enhanced antihapten antibody response. Mice were primed with live or UV-inactivated vesicular stomatitis virus (VSV) Indiana (bId) serotype with or without complete Freund's adjuvant. After challenge with VSV New Jersey (NJ), these mice developed a secondary-type IgG response, measured by antibody binding in an ELISA, against both VSV-Ind and VSV-NJ. The same result was found for the reciprocal experiments where mice were primed with VSV-NJ. Similarly, when mice were primed with live VSV, UV-inactivated VSV, or purified VSV glycoprotein G of Ind or NJ serotype and later were challenged with dinitrophenyl (N2ph)conjugated, UV-inactivated VSV or with N2ph-conjugated G protein of either serotype, they exhibited a secondary-type anti-N2ph antibody response as demonstrated by the binding of IgG to dinitrophenylated bovine serum albumin measured by ELISA. In contrast, when neutralizing antibody responses were monitored, VSV-Ind-primed mice challenged with VSVNJ developed a strictly primary type of anti-VSV-NJ response and vice versa. We conclude that preexistent helper T cells specific for shared carrier determinants do not improve virgin B-cell responses specific for "new," unique determinants that are the target for the biologically relevant neutralizing antibodies. These findings suggest that priming of B cells rather than of helper T cells may be of importance to induce protective immunity mediated by antibodies.
ogous or heterologous serotype, by (i) ELISA, revealing crossreactive antibodies binding to the whole virion or purified glycoprotein, and (it) serum neutralization assays, monitoring the type of antibody response against the unique determinants involved in neutralization. We show that mice primed with one serotype of VSV do not develop an enhanced neutralizing antibody response when challenged with a heterologous VSV, whereas antibody responses to shared determinants measured by ELISA are of secondary type.
Initiation and maintenance of an IgG antibody response against most antigens require T- and B-cell collaboration (1-5). It is thought that T cells recognize the so-called carrier determinants of an antigen, whereas B cells are thought to recognize the same or additional determinants, usually called haptens (1-6). Classical experiments have established that mice primed with the carrier part of an antigenic molecule, upon immunization with a hapten conjugated to the same carrier molecule, respond with an enhanced B-cell response against the hapten when compared with unprimed mice (7-9). Most experiments on the relative role of carrier-primed T cells in antihapten responses of primed or unprimed B cells have used artificial antigens such as 2,4-dinitrophenyl (N2ph)-conjugated keyhole limpet hemocyanin, 2,4,6trinitrophenyl (N3ph)-conjugated bovine gamma globulin, or heterologous erythrocytes (7-14). Our studies were aimed at evaluating these rules in an antiviral immune response. Viruses that belong to the same type but are subdivided according to their serotypes (species or subtype) (15) share many antigenic determinants on various structural gene products; they also possess unique determinants, usually on major viral glycoproteins. These unique determinants have
MATERIALS AND METHODS Mice. DBA/2 mice, from the Institut fir Zuchthygiene, Zurich, were used throughout these experiments. Virus. VSV-Ind (serotype Indiana) and VSV-NJ (serotype New Jersey) virus stocks were prepared in BHK-21 cells as described (30). Concentration and purification of the virus were carried out according to established procedures (21). Preparation of Purified Surface Glycoprotein G. The preparation of G protein has been described (21). In brief, purified VSV-Ind and VSV-NJ were treated with Triton X-100 at a final concentration of 2% (vol/vol). The Triton/virus mixture was stirred at room temperature for 60 min and then centrifuged at 140,000 x g at 5°C for 90 min. The supernatant Abbreviations: VSV, vesicular stomatitis virus; Ind, serotype Indiana; 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. *Present address: Department of Veterinary Microbiology, Haryana Agricultural University, Hissar, Haryana, India-125004. tTo whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 2604
Immunology: Gupta et al.
Proc. Natl. Acad. Sci. USA 83 (1986)
Switzerland) at 40C overnight. The plates were then washed five times with phosphate-buffered saline containing 0.05% Tween 20 (PBS/Tween). Sera diluted in PBS/Tween (100 ,uI per well) were incubated in the antigen-coated plates for 2 hr at room temperature. Plates were again washed five times with PBS/Tween and then were incubated with goat antimouse IgG, labeled with horseradish peroxidase (Tago 4143, Burlingame, CA) and diluted in PBS/Tween (100 Al per well), for 2 hr at room temperature. The plates were washed again in PBS/Tween, and 200 ,ul of substrate [2 mg of 2,2'-azinobis(3-ethylbenzthiazolinesulfonate) (Boehringer Mannheim; ref. 6) in 20 ml of 0.1 M NaH2PO4 (pH 4.0) and 15 Al of 30%6 H2021 was added per well. The optical density at 405 nm was measured with a micro-ELISA reader (Virion AG, Ruschlikon, Switzerland). For the determination of anti-N2ph antibodies, 1.0 jug of coupled bovine serum albumin (BSA-N2ph, a gift from H. Binz, Institute of Virology and Immunology, University of Zilrich) was used per well of a microwell plate. Before adding the second antibody conjugate, the wells were pretreated with 1.0% normal goat serum for 30 min at room temperature; otherwise the same protocol was used as described for the ELISA to determine anti-VSV antibody titers.
containing G was checked for purity by polyacrylamide gel electrophoresis (20). Immunization. For primary or secondary immunization with live virus, 106 plaque-forming units (pfu) of VSV-Ind or VSV-NJ were given i.v. Virus inactivated by UV light was injected i.p. at a dose of 108 pfu, emulsified in complete Freund's adjuvant (CFA). The optimal doses of VSV or VSV glycoprotein coupled with N2ph were predetermined by titration in vivo; for primary immunization, 20 jig of antigen was injected with CFA i.p., and for secondary immunizations, the same amount was given i.v. without CFA. Coupling of VSV-Ind and VSV-NJ with N2ph. Dinitrophenylated VSV-Ind (Ind-N2ph) and VSV-NJ (NJ-N2ph) were prepared according to standard methods (31). In brief, virus purified on sucrose gradients was mixed with sodium 2,4dinitrobenzenesulfonate at pH 8.0. The mixture was stirred overnight, protected from light and at room temperature. The mixture was centrifuged at 5,000 x g to remove insoluble material and then the hapten-modified virus was resedimented. To wash off excess sodium dinitrobenzenesulfonate, the virus was suspended in Hanks' balanced salts solution (BSS) and resedimented three times. The sedimented dinitrophenylated virus was collected in 500 jul of BSS and the optimal dilution for the immunization ofmice was determined in a preliminary experiment in vivo. Coupling of VSV-Ind Glycoprotein and VSV-NJ Glycoprotein with N2ph. Reaction of VSV-Ind and VSV-NJ G proteins with N2ph was carried out essentially as described for the whole virus (31), except that the free N2ph was removed by filtration through Amicon YM10 filters (Amicon). The optimal amounts of G(Ind)-N2ph and G(NJ)-N2ph for the immunization of mice were predetermined in vivo. Serum Neutralization Test. The protocol for the test has been described (20). In brief, serial 2-fold dilutions of heatinactivated sera were mixed with equal volumes of virus containing 50 pfu in 100 ,ul and incubated at 370C for 90 min. Dilution of the serum resulting in 50%6 reduction of plaques on Vero monolayers grown in a 24-well plate (Costar 3024) was doubled to compensate for the addition of an equal volume of virus in the serum/virus mixture. To determine IgG titer, serum was pretreated with 0.05 M 2-mercaptoethanol (32). Enzyme-Linked Iminunosorbent Assay (ELISA). The procedure adopted was a modification of the assay described by Voller et al. (33). For the determination of anti-VSV antibodies, the optimal dilution of the purified virus in 100 Al of coating buffer (pH 9.6) was adsorbed to flat-bottomed 96-well polystyrene type F microwell plates (Petra Plastic, Inotech, A
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RESULTS Primary Antibody Responses Against VSV. After primary immunization with VSV-Ind and VSV-NJ, serotype-specific IgM antibodies (2-mercaptoethanol-sensitive serum neutralization antibody) were detectable on day 2 and reached maximal levels on day 6 (Fig. 1 A and D). IgM antibody responses dropped below a detectable level by about 3 weeks (data not shown). IgG antibodies (2-mercaptoethanol-resistant serum neutralization antibody) were first detected on day 6 or 7, reached maximal levels around day 10-12 (Fig. 1 A and D), and persisted for several weeks. At 22 weeks after primary immunization with 106 pfu, IgG serum neutralization antibody titers on the order of 1280-2560 were usually observed. The kinetics of anti-VSV IgG responses assessed by ELISA were found to be comparable to that of antibody responses measured by serum neutralization (Fig. 2). Secondary Antibody Responses Against VSV. After secondary immunization with the homologous virus, anti-VSV serum neutralization IgG antibody responses had increased 4-fold from prechallenge values by day 4 and reached peak titers (usually about 80,000) after day 6 (Fig. 1 C and F). Similarly, IgG titers assessed in ELISA increased rapidly
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5,120 1,280 320 80
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