Immunization of Mice with Vaccinia Virus-M2 ... - Journal of Virology

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Jan 7, 1993 - JONATHAN W. YEWDELL,3 AND BRIAN R. MURPHY'. Respiratory Viruses ...... Kutubuddin, M., J. Simons, and M. Chow. 1992. Poliovirus-.
Vol. 67, No. 7

July 1993, p. 4086-4092 0022-538X/93/074086-07$02.00/0

JOURNAL OF VIROLOGY,

Copyright X 1993, American Society for Microbiology

Immunization of Mice with Vaccinia Virus-M2 Recombinant Induces Epitope-Specific and Cross-Reactive Kd-Restricted CD8+ Cytotoxic T Cells ARUN B. KULKARNI,1* HERBERT C. MORSE III,2 JACK R. BENNINK,3 JONATHAN W. YEWDELL,3 AND BRIAN R. MURPHY' Respiratory Viruses Section, Laboratory of Infectious Diseases, 1 Laboratory of Immunopathology, 2 and Laboratory of Viral Diseases, 3 National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892 Received 7 January 1993/Accepted 14 April 1993

The M2 protein of respiratory syncytial virus (RSV) is a protective antigen in H-2d, but not H-2b or H-2k mice. None of the other RSV proteins, excluding the surface glycoproteins that induce neutralizing antibodies, is protective in mice bearing these haplotypes. Thus, the M2 protein stands alone as a nonglycoproteinprotective antigen of RSV. The M2 protein is a target for murineKd-restricted cytotoxic T lymphocytes (CTLs), and the resistance induced by infection with a vaccinia virus-RSV M2 (vac-M2) recombinant is mediated by CD8+ CTLs. Since the nonameric consensus sequence for H-2 Kd-restricted T-cell epitopes and the amino acid sequence of the M2 protein of subgroup A and B strains of RSV are known, the present study sought to identify the specific epitope(s) on the M2 protein recognized by CD8+ CTLs. This was done by examining the ability of four predicted K'-specific motif peptides present in the M2 amino acid sequence of an RSV subgroup A strain to sensitize target cells for lysis by pulmonary or splenic CTLs obtained from mice infected with RSV or vac-M2. The following observations were made. First, two of the four peptides sensitized target cells for lysis by pulmonary or splenic CTLs induced by infection with either vac-M2 or RSV. Second, one of the two peptides, namely the 82-90 (M2) peptide, sensitized targets at a very low peptide concentration (10-10 to 10-12 M). Third, cold-target competition experiments revealed that the predominant CTL population induced by infection with vac-M2 or RSV recognized the 82-90 (M2) peptide, and this CTL population appeared to recognize the 71-79 (M2) peptide in a cross-reactive manner. Fourth, CTL recognition of targets sensitized with either the 71-79 (M2) or the 82-90 (M2) peptide was K' restricted. Fifth, CTLs induced by infection with RSV subgroup A or B strains recognized the two M2 peptides. The findings suggest that the M2 protein of RSV contains an immunodominant Kd-restricted CTL epitope consisting of amino acid residues 82 to 90 (SYIGSINNI), which are shared by subgroup A and B RSVs.

CD8+ cytotoxic T lymphocytes (CTLs) make an important contribution to recovery from virus infections. It is well established that CTLs recognize processed foreign antigens as peptides, usually consisting of 8 to 9 amino acid residues presented by major histocompatibility complex (MHC) class I molecules (19, 22, 23). Peptide derivatives of endogenously synthesized proteins enter the MHC class I processing pathway and associate with newly synthesized class I molecules in the endoplasmic reticulum, whereas exogenously added synthetic peptides predominately bind to empty class I MHC molecules located on the plasma membrane (11, 24). X-ray crystallography analysis of the class I molecules HLA-A2 and HLA-Aw 68 showed a cleft on the distal tip of the protein that forms the peptide-binding site (3, 10). Class I MHC molecules are among the most polymorphic proteins in vertebrates, and peptides that bind to specific MHC class I alleles have consensus sequences in which certain amino acid residues predominate at given positions. The consensus sequence for nonameric H-2Kd-restricted T-cell epitopes possesses a Tyr residue at position 2 and a Val, Ile, Thr, Ala or Leu at position 9 (9). Entirely on the basis of this motif, bona fide K'-restricted epitopes have been identified in bacterial and viral proteins (13, 17). The CD8+ CTL response of mice to the M2 protein of respiratory syncytial *

Corresponding author.

virus (RSV) is K' restricted (16), suggesting that it should be possible to identify the CTL epitope(s) in this RSV protein that induces protective immunity. Infection of BALB/c (H-2d) mice with a vaccinia virusRSV M2 (vac-M2) recombinant induced a high level of resistance to RSV challenge that was mediated by CD8+ CTLs (7). The high level of resistance induced by M2 was of particular interest for three reasons. First, of the seven RSV proteins that do not induce neutralizing antibody, namely M, M2, SH, NS1, NS2, N, and P, the M2 protein was the only protein that induced resistance in H-2" mice, and none induced resistance in H-2b or H-2c mice (12). Thus, M2 delivered by a vaccinia vector appears unusual among RSV proteins in its ability to induce a protective cellular immune response. Second, the protective immune response was mediated by primary pulmonary CD8+ CTLs, with memory CTLs contributing marginally, if at all, to the observed resistance (12). Third, the primary pulmonary CD8+ CTL activity induced by vac-M2 infection was short-lived and resembled the CTL response of mice infected with RSV (1, 12). Because of the important role that the M2 CTLs play in immunity to RSV in mice, we sought to identify the M2 epitopes recognized by CD8+ T cells and to further characterize the CTL response that they elicit. Four peptides that possessed the Kd-binding motif were present in the coding sequence of the M2 gene, and each of these was used to sensitize target cells. We found that two of the four peptides 4086

CTL EPITOPE FOR RSV M2 PROTEIN

VOL. 67, 1993 were able to sensitize target cells for lysis by CTLs, but only one possessed the ability to facilitate lysis by pulmonary and splenic CTLs at a very low concentration of

TABLE 1. RSV M2 protein-derived peptides used in this study

Kd-restricted

peptide. MATERIALS AND METHODS Viruses. The A2 strain of RSV subgroup A and the 18537 strain of subgroup B were used throughout. The virus stocks were grown in HEp-2 cells and titrated for infectivity by plaque assay in HEp-2 cells as described elsewhere (14, 18). Influenza A/PR/8/34 virus was grown in embryonated chicken eggs, and the 50% tissue culture infective dose was determined by using Madin-Darby canine kidney (MDCK) cell monolayers. Recombinant vaccinia viruses were grown and titrated by plaque assay in HEp-2 monolayers. The production and characterization of recombinant vaccinia viruses expressing the RSV M2 and parainfluenza type 3 hemagglutinin-neuraminidase (HN) proteins have been described elsewhere (15, 21). Preparation of recombinant vaccinia viruses expressing Kd and Dd. Recombinant vaccinia viruses expressing K' (vacKdWt) and Dd (vac-Dd) were constructed by inserting K' and Dd cDNA genes into pSC11 (5) modified to contain a multiple cloning site downstream of the 7.5 promoter. The ends of the Kd gene in plasmid pKCKdwt (provided by Christian Jaulin, Pasteur Institute, Paris, France) were modified from the original EcoRI (5') and XbaI (3') to SalI sites of the modified pSC11, and colonies containing the correct orientation of the Kd gene were selected for homologous recombination with vaccinia. A KpnI adapter was inserted into the PvuII site in the Dd gene contained in plasmid pDd.1 (provided by Randall Ribaudo and David Margulies, National Institute of Allergy and Infectious Diseases, Bethesda, Md.). The Dd gene was excised with Sall and KpnI and directionally inserted into the Sall and KpnI sites of the modified pSC11. The Dd gene containing the pSC11 plasmid was then inserted into vaccinia virus via homologous recombination as described previously (5). Mice and immunization. Six- to eight-week-old BALB/c (H-2d) female mice were obtained from the Frederick Cancer Research and Development Center of the National Cancer Center (Frederick, Md.). Mice were anesthetized with methoxyfluorane to permit infection with RSV or recombinant vaccinia viruses by intranasal (i.n.) administration of 106 PFU in a 0.1-ml inoculum. In some experiments, recombinant vaccinia viruses were administered intravenously (106 PFU/0.2 ml). Synthetic peptides. Peptides (kindly provided by J. Coligan, Biological Research Branch, National Institute of Allergy and Infectious Diseases) were synthesized on an ABI (Foster City, Mass.) peptide synthesizer (model 430-A) by F-moc chemistry. Peptide sequences were confirmed by high-performance liquid chromatography with a Beckman 6300 analyzer. Target cells. Target cells used in cytotoxicity assays were P815 (H-2d) mastocytoma cells, L929 (H-2") fibroblasts, MC57G (H-2b) fibroblasts, an (H-2d) BALB/c fibroblast line, or BCH4 cells that are a BALB/c fibroblast line persistently infected with the Long strain of RSV (12). These cell lines were grown in Iscove's modified Dulbecco's medium (IMDM) supplemented with 5% fetal bovine serum. BALB/c fibroblasts and P815 cells used as target cells were infected with RSV (10 PFU per cell) or influenza A/PR/8/34 virus (50 PFU per cell) for 18 h prior to the assay. Recombinant vaccinia virus-infected target cells were prepared by infect-

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in the M2 sequence

Presence of peptide sequence in RSV subgroup B A

26-34 71-79 82-90 127-135

+ + + +

Locationb Residue'

NYFEWPPHA

EYALGVVGV SYIGSINNI VYNTVISYI

+

-c + +

Peptide

designation in

used this study

26-34 (M2) 71-79 (M2) 82-90 (M2) 127-135 (M2)

a Four nonamer peptides that conform to the H-2Kd-binding motif (9) were found in the M2 protein of RSV subgroups A and B. Peptides were aligned according to their Y residues. NP (147-155), a K-restricted motif (TYQRTRALV) from the nucleoprotein of influenza A/PR/8/34 virus (4), which was used as a control in the present study, was designated 147-155 (NP). Open reading frame 2 of the M2 protein of RSV-A2 also contains an additional Kd-binding motif at positions 13 to 21 (KYPCSITSI) (6). b Amino acids are numbered according to their sequence of the M2 protein in open reading frame 1 (6). c The subgroup B sequence differs from the subgroup A sequence at one position (underlined): EYALGIVGV (6).

ing these cells with 10 PFU per cell of virus for 3 h prior to the assay. Cytotoxicity assay. Cytotoxicity assays were performed as described previously (12). Briefly, P815 target cells labelled with 51Cr were incubated in IMDM supplemented with 5% fetal bovine serum and 2-mercaptoethanol (5 x 10-4 M) (IMDM+) for 90 min at 37°C in different concentrations of peptides and then washed four times at room temperature. Primary in vivo effector cells were prepared from lungs of infected mice as detailed elsewhere (12). In some experiments, effector cells were obtained from the spleens of mice infected intravenously with vac-M2 6 days previously. The level of cytotoxicity activity is expressed as the percent specific lysis. Cold-target competition. P815 target cells (104 per well in 50 ,ul) were labelled with 51Cr and peptides as described above. Unlabelled P815 cells were incubated with peptide for 1 h, washed twice with IMDM+, and added in 50-,ul aliquots per well to achieve ratios of one, two, four, or eight times the number of labelled target cells. Primary vac-M2immune T cells (6 x 105/100 ,ul) were added to each well, and the assay was run for 6 h. Specific lysis of the hot targets in the presence of competing cold targets was calculated as the percent lysis of the hot targets alone. RESULTS Virus specificity of H-2Kd binding peptides. The M2 protein which consists of 194 amino acid residues contains four nonapeptides which have tyrosine at position 2 and either Leu, Ile, Val, or Ala at the C terminus (Table 1). These peptides were used to sensitize P815 cells for lysis by lymphocytes derived from lungs or spleens of RSV- or vac-M2-infected mice. Initial experiments sought to determine conditions necessary for sensitizing target cells by using peptides at a concentration of 10-5 M. A nonameric Kd-restricted peptide, a known CTL epitope (4), derived from the nucleoprotein (NP) of influenza A/PR/8/34 virus was used as a control. Of the four peptides studied, the peptides consisting of amino acid residues 71 to 79 and 82 to 90, referred to as the 71-79 (M2) and 82-90 (M2) peptides, respectively, consistently sensitized the targets more efficiently than peptide 26-34 (M2) or 127-135 (M2) (data not

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J. VIROL.

KULKARNI ET AL.

TABLE 2. M2-specific, H-2Kd motif peptides sensitize targets for lysis by H-2 compatible pulmonary and splenic effector cells from mice immunized with vac-M2a Percent specific lysis with pulmonary effector cells from mice infected with:

Target1 P815 P815 P815 P815 P815 P815 MC-57G BCH4e

added Peptides to

26-34 (M2) 71-79 (M2) 82-90 (M2) 127-135 (M2) 147-155 (NP)d

82-90 (M2)

60:1c

20:1

6:1

16 40 55 8 6 8 0 48

5 22 26 4 3 0 0 18

3 12 8 2 0 0 0 17

Vac-HN

Vac-M2

Vac-HN

Vac-M2

targets

Percent specific lysis with splenic effector cells from mice infected with:

60:1

20:1

6:1

100:1

30:1

10:1

100:1

30:1

10:1

3 6 1 4 3 0 0

0 2 1 4 1 0 0 0

0 1 0 2 1 0 0 0

21 61 63 10 7 0 0 60

7 31 28 7 4 0 0 28

0 12 10 3 2 0 0 3

1 5 1 0 0 0 0 0

0 2 0 6 5 0 0 0

0 0 0 3 2 0 0 0

0

a Mice were immunized with vac-M2 or vac-HN (5 x 106PFU/0.05 ml i.n. for pulmonary effector cells or 5 x 106 PFU/0.2 ml intravenously for splenic effector cells), and the effector cells were obtained on day 6. b P815 (H-2d) and MC-57G targets were incubated independently with 50 ,ul of peptides (10-5 M) and 51Cr (10 mCi/ml) for 90 min and washed three times with IMDM+ before being used. c Effector cell to target cell ratio. d Control peptide derived from the NP of influenza A/PR/8/34 virus. e BCH4 cells, BALB/c fibroblast line persistently infected with the Long strain of RSV. An unsensitized BALB/c fibroblast line had