nization with an unbound lymphocytic choriomeningitis virus peptide also induced a virus-specific CTL response, provided the peptide was dissolved in ...
Proc. Natl. Acad. Sci. USA Vol. 88, pp. 2283-2287, March 1991 Medical Sciences
Protection against lethal Sendai virus infection by in vivo priming of virus-specific cytotoxic T lymphocytes with a free synthetic peptide (immunologic memory/vaccination)
W. MARTIN KAST*t¶, LAURENT Rouxt, JOSEPH CURREN*, HENDRIKA J. J. BLOM*, ARIE C. VOORDOUW*, ROB H. MELOEN§, DANIEL KOLAKOFSKYt, AND CORNELIS J. M. MELIEF* *Division of Immunology, The Netherlands Cancer Institute, Antoni van Leeuwenhoek Huis, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands;
*University of Geneva, 9 Avenue de Champel, 1211 Geneva, Switzerland; and §Central Veterinary Institute, P.O. Box 65, 8200 AB Lelystad, The Netherlands Communicated by Stanley G. Nathenson, December 17, 1990
ABSTRACT The only peptide of Sendai virus that is recognized by cytotoxic T lymphocytes (CTL) in B6 mice was found with (g) the use of recombinant vaccinia virus constructs containing separate genes of Sendai virus and (d) a set of overlapping peptides completely spanning the identified nudeoprotein (NP) gene product. This immunodominant NP peptide is recognized by Sendai virus-specific CTL that are known to have therapeutic effects in vivo. By subcutaneous i'mmunization, this peptide induced Sendai virus and NP peptide-specific CTL memory responses in vivo. Most importantly, mice that had been immunizd with this peptide were protected against a lethal virus dose, indicating that viral peptides can be used as antiviral T-cell vaccines. The induction of T-cell memory by free peptide immunization potentially has wide applicability in biology and medicine, including protection against infectious disease.
Antiviral cytotoxic T lymphocytes (CTL) recognize short peptides derived from viral proteins when bound to major histocompatibility complex class I molecules (1). Such virus-specific CTL are crucially important in the resistance against many virus infections, including lymphocytic choriomeningitis virus (2, 3), influenza virus (4-6), herpes simplex virus type 1 (7), and Sendai virus (8). The elicitation of virus-specific CTL responses is best achieved in vivo by immunization with attenuated virus, but this bears a certain risk of causing disease. Therefore, means are sought to induce virus-specific CTL responses in vivo with synthetic viral antigens. Recently it was demonstrated that immunization with short synthetic peptides of the influenza virus nucleoprotein (NP) can induce virus-specific CTL to the priming peptide and to the virus when these peptides are chemically modified and linked to a lipid component. Free peptides, however, failed to do so (9). Furthermore, immunization with an unbound lymphocytic choriomeningitis virus peptide also induced a virus-specific CTL response, provided the peptide was dissolved in incomplete Freund's adjuvant (IFA) (10). However, these studies did not show whether peptide vaccination induced protection against subsequent virus challenge. Since we have demonstrated that cloned Sendai virus-specific CTL can cure mice that are lethally infected with this virus (8), we evaluated whether protection from lethal virus infection can be achieved by vaccination with the peptide recognized by these cloned CTL. The Sendai virus NP was identified as the protein containing the peptide recognized by these CTL clones with the use of vaccinia virus recombinants expressing different genes of Sendai virus. Furthermore, a single peptide recognized by 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.
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these clones was identified by screening with a set of overlapping peptides covering the complete amino acid sequence of the NP. Vaccination with this peptide, known to be recognized by CTL clones that are effective in vivo, led to successful induction of CTL memory associated with in vivo protection against a lethal dose of virulent Sendai virus that was introduced by the natural respiratory route of infection.
MATERIALS AND METHODS Mice. B6 mice were bred at The Netherlands Cancer Institute under specific pathogen-free conditions. They were
used for in vivo experiments when about 8 wk old. In vivo experiments were carried out at the Central Laboratory ofthe Netherlands Red Cross Blood Transfusion Service (Amsterdam, The Netherlands). Viruses. Virulent Sendai virus strain p3193 was a gift from J. C. Parker (Microbiological Associates). This virus was propagated in the lungs of 129/J mice. The titer was 3.2 x 106 tissue culture median infectious dose (TCID50) per ml. Nonvirulent Sendai virus, lot 40340087, was obtained from Flow Laboratories. This virus has been propagated in pathogenfree eggs and does not cause cytopathic effects on rhesus monkey kidney cells (8). The titer was 104 hemagglutination units/ml. Virus Challenge. Mice were inoculated intranasally with 20 ,ul of diluted virulent Sendai virus at a median lethal dose of 2 LD50 (= 300 TCID50) (8, 11). Infected mice were observed for disease or death up to day 40. They were housed in a low-pressure isolator. Immunization. Mice were primed by one i.v. injection of 100,ug offree synthetic peptide or by one s.c. injection of 100 pg of free synthetic peptide dissolved in phosphate-buffered saline or dissolved in IFA or by one i.p. injection of 102 hemagglutination units of nonvirulent Sendai virus and used between 4 and 6 wk after the immunization. In Viro Generation of Sendai Virus-Specific Bulk CTL. The method to generate Sendai virus-specific CTL has been described (12) and was performed with slight modifications. In brief, responder spleen cells (5 x 106) from in vivo primed mice were cocultured with irradiated (25 Gy) spleen cells (5 x 106) in the presence of 10 uM Sendai virus NP-(321-336) peptide (fragment from amino acids 321 to amino acid 336) in 2 ml of culture medium for 5 days at 37°C in humidified air containing 5% CO2. For the preparation of Sendai virusAbbreviations: TCID50, tissue culture median infectious dose; NP, nucleoprotein; MEC, mouse embryo cell; IFA, incomplete Freund's adjuvant; CTL, cytotoxic T lymphocytes. tPresent address: Department of Immunohematology and Blood Bank, Academic Hospital Leiden Rijnsburgerweg 10, 2333 AA Leiden, The Netherlands. tTo whom reprint requests should be sent at the present address.
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infected stimulator cells, 108 spleen cells in 2 ml of culture medium were incubated with 6 x 102 hemagglutination units of nonvirulent Sendai virus for 1 hr at 370C and then were washed three times. The culture medium consisted of Iscove's modified Dulbecco's medium (Flow Laboratories) supplemented with 10% fetal calf serum, penicillin (100 international units/ml), kanamycin (100 jug/ml), and 2-mercaptoethanol (20 AM). When indicated, 2 units of recombinant interleukin 2 (rIL-2) (= 10 international'units) (Eurocetus, Amsterdam) was added to the culture. Cloned CTL. The Sendai virus-specific CTL clone Tc5 was used in this study. The establishment, culture, function, major histocompatibility complex restriction specificity (H-2Kb), and phenotype (CD4- CD8+) of this clone have been described (8). Cell-Mediated Cytotoxicity Assay. Various numbers of effector cells were added to 2 x 103 Na25lCrO4 (51Cr)-labeled target cells in 0.2 ml of culture medium in 96-well U-bottom plates and were incubated for 6 hr at 370C in humidified air containing 5% CO2. After incubation, the supernatant was collected. The percentage of specific 51Cr release was calculated by the formula: % specific lysis = cpm experimental well - cpm background 51Cr release cpm 2% Triton X-100 release - cpm background 51Cr release
Background (medium) release was always less than 30% of maximal release. The SD of triplicate wells was always less than 5% of the specific 51Cr release. Virus-infected target cells were prepared as described above for stimulator cells, except that 107 cells were incubated with 10 hemagglutination units of virus in 100 ,ul of medium. When peptides were used to label uninfected target cells, they were present during the whole assay at a concentration of 50 ,M when the peptide was made in bulk amounts, or 0.4 jig of peptide was added per well when the peptide was made in the "pepscan" method (see below). When targets were infected with vaccinia virus recombinant, 5 x 106 cells were incubated with 10 plaqueforming units per cell in 5 ml of culture medium for 14 hr at 37°C in humidified air containing 5% CO2 and were washed three times. Synthesis of Peptides. Peptides were synthesized by two methods. The first method (pepscan) has been described (13, 14) and was used with modifications as described (15). In brief, synthesis took place on polyethylene rods onto which polymers of polyacrylic acid had been formed by radiation grafting. To permit the specific detachment of peptides after synthesis, a tripeptide sequence (Asp-Pro-Gly) was cosynthesized between the peptide and the activated polyethylene rods. After deprotection and removal on noncovalently bound impurities, acid cleavage of Asp-Pro bonds was performed by heating in 0.2 ml of 70% formic acid for 20 hr at 37°C. The solvent was removed by lyophilization. The second method to generate bulk amounts of peptides has been described (16) and was performed with slight modifications. In brief, peptides were synthesized with COOH-terminal ends on a Biosearch 9500 peptide synthesizer with the use of 4-methylbenzhydrylamine resin. Construction of the Recombinant Vacciniia Viruses. The assembly of genes encoding the P/C, HN, and F Sendai virus proteins (see Results) has been described (17, 18). The gene encoding the matrix protein M was pieced together from two overlapping clones: (i) clone S1-2 (19), containing most of the 5' end sequence of the M gene and extending into the adjacent P gene; and (ii) clone 3'/2' [obtained with the same cloning procedure as described in ref. 18], containing the exact 3' end flanked by a Kpn I site. S1-2 was cleaved at its 5' end at the Sma I site (nucleotide 1815 of the P gene) and recombined with 3'/2' at the unique HindIII site of the M gene (nucleotide
Proc. Natl. Acad Sci. USA 88 (1991)
854). A full-length clone of the gene encoding NP was produced by the cloning procedure described by Vidal et al. (18). The genes encoding M, HN, F, and NP were cloned in plasmid pSP64 and that encoding P/C was cloned in plasmid pSP65. The individual genes were then transferred to the insertion vector pGS62 (kindly provided by G. L. Smith, Cambridge University), and the vaccinia recombinants were prepared exactly as described (17) for the P/C gene.
RESULTS Protein Specificity of Sendai Vius-Specific B6 Bulk and Cloned CTL. Bulk and cloned Sendai virus-specific CTL were assayed on a panel of target cells expressing different viral proteins. The target cells were mouse embryo cells (MEC) infected with different recombinant vaccinia virus constructs containing individual Sendai virus genes. Both the Sendai virus-specific bulk and cloned CTL recognize Sendai virus-infected MEC, whereas uninfected or vaccinia virusinfected MEC were not recognized, indicating Sendai virus specificity (Table 1). On assaying MEC that were infected with the different recombinant vaccinia virus constructs, we noted that the infected cells expressing the Sendai virus fusion (F) protein, hemagglutinin neuraminidase (HN), matrix (M) protein, and polymerase (P/C) protein were not recognized. Only MEC that expressed the Sendai virus NP were recognized by both the bulk and cloned CTL. This indicates that the major reaction of Sendai virus-specific CTL of B6 mice is directed against the NP of this virus. Specificity of Sendai Virus-Specific Bulk and Cloned CTL Assayed with Overlapping Peptides. To identify the NP pepTable 1. Protein specificity of Sendai virus-specific B6 bulk and cloned CTL 51Cr release by effector cells, %t Agent infecting Bulk CTL Tc5 51Cr-labeled target cells E/T* 40 Sendai virus 16 36 8 56 4 30 5 16 4 8 0 2 4 8 16 Vaccinia virus 9 8 4 8 2 5 16 Vaccinia-SV/F 5 8 3 2 4 6 16 Vaccinia-SV/HN 3 8 2 1 4 7 16 Vaccinia-SV/M 3 8 2 3 4 5 16 Vaccinia-SV/P/C 6 8
Vaccinia-SV/NP
4 16 8 4
4 42 38 29
3 40
B6 target cells were 51Cr-labeled MEC (2 x 103). Infection was carried out as described in Materials and Methods. Target cells
infected by each separate vaccinia virus construct have been shown to express the appropriate Sendai virus (SV) protein as tested by
[3S]methionine labeling followed by specific immunoprecipitation (L.R., J.C., and D.K., data not shown).
*Effector-totarget cell ratio. tMean percentage of specific 51Cr release of three experiments.
Medical Sciences: Kast et al. Table 2. Specificity of Sendai virus-specific bulk and cloned CTL assayed with overlapping peptides 51Cr release by Sendai NP fragment effector cells, %* Addition AA sequence nos. Bulk CTL TcS None 45 32 None 3 2 + 1-12 2 1 + 2-13 3 2 + n-n+11
