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.