INTRODUCTION

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The humoral (antibody) and cellular (T-lymphocyte) responses represent two distinct pathways by which the immune system recognizes and combats viral infections (reviewed in reference 30). Antibodies primarily recognize viruses or viral antigens circulating in the blood and other fluids, whereas T lymphocytes interact with viral antigens in the form of processed peptides bound to host cells and presented in a groove between the two -helices of the major histocompatibility complex (MHC) glycoproteins (3). Cytotoxic T lymphocytes (CTLs), most of which bear the CD8 molecule, recognize and interact with infected cells that express viral antigen (peptide) presented by MHC class I molecules. Observations of humans and mice with genetic or acquired deficiencies in antibody or T-cell production and experimentally manipulated animals clearly show that control of most acute viral infections is critically dependent on MHC class I-restricted CD8 CTLs (reviewed in reference 30). However, immune clearance of persistent viral infections, in addition to dependence on CD8 CTLs, also requires the participation of CD4 T cells (2, 8, 12, 16, 29).

CTL responses to infection with the arenaviruses lymphocytic choriomeningitis virus (LCMV) and, likely, Lassa fever virus (LFV) essentially rely on cell-mediated CD8 CTL responses (reviewed in references 6, 29, 30, and 34). Features of interactions between arenavirus peptides and MHC class I molecules have been delineated molecularly and structurally by biochemical characterization of the naturally presented peptides (10, 11, 13, 19, 32, 33) and analysis of CTLs as viral escape variants (9, 14, 15, 22). Studies with these and other viruses whose peptides were isolated by elution from MHC class I molecules led to the identification of allele-specific anchor residues within the peptide sequence (reviewed in reference 23). Similarly, the impact of structural factors at nonanchoring residues in peptide-MHC interactions has been identified by using single amino acid mutations of viral gene products (9, 10).

The nucleoprotein (NP) sequence of LCMV consisting of amino acids (aa) 118 to 126 (NP 118-126) (RPQASGVYM) was found to be L^d restricted and to constitute >97% of the total clonal or total bulk primary CTL responses (32). A single injection of recombinant vaccinia virus (VV)-LCMV NP vaccine incorporating this L^d epitope provided complete protection for H-2^d mice later challenged intracerebrally with an ordinarily lethal dose of virus (18, 19). Further, study of this CTL epitope indicated that one-third of mice from nine established haplotypes (H-2^d, H-2^u, and H-2^q) possessed MHC class I glycoproteins capable of presenting the LCMV NP 118-126 peptide for recognition and lysis by virus-specific CTLs (19). This concept was further strengthened when incorporation of this virus-specific NP CTL epitope into a recombinant VV vaccine and administration of a single dose protected mice of these three haplotypes from an ordinarily lethal challenge of virus. This outcome indicated the presence of a common epitope among disparate MHC class I types and suggested the possibility of developing an effective CTL vaccine for outbred populations, such as humans (31). To further explore this possibility, we studied the NP CTL epitope (117/118)-126 for its representation among other arenaviruses. We noted that both the Old World (LVF, Mopeia) and New World (Machupo, Sabia, Junin, Pichinde, Oliveros, and Tacaribe) viruses had 22 to 78% homology with the LCMV NP 118-126 epitope. Sequence analysis of the NP (117/118)-126 among the arenaviruses combined with site-specific amino acid exchanges revealed the requirement for peptide binding to MHC class I L^d molecules and the requirement for subsequent cross-recognition of these MHC-restricted peptides by LCMV-specific L^d-restricted CD8⁺ CTLs. Of the nine arenaviruses studied, the NP 118-126 peptide from four, LCMV, LFV, Mopeia virus, and Sabia virus, bound at high affinity to L^d MHC class I molecules. Binding of the Sabia virus peptide NP 118-126 to L^d indicated that a Ser (Sabia virus aa 119) would substitute for the known position 2 (P2) MHC anchor residue Pro (aa 119: LCMV, Mopeia virus, LFV) and that Ala in P3 might serve as an ancillary MHC anchor. The three Old World arenaviruses, LCMV, LFV, and Mopeia virus, but not the New World virus Sabia virus, showed functional CTL cross-reactivity when their peptides were used to coat MHC-matched target cells. Single-amino-acid exchanges between LCMV and Sabia virus indicated that an amino acid change of Gly at P5 (aa 122) for a Ser or Ala aborted cross-recognition by anti-LCMV CD8⁺ CTLs. Hence, a recombinant, plasmid or subunit viral vaccine including a common NP CTL epitope could offer immune protection from diseases caused by Old World but not New World arenaviruses.

	MATERIALS AND METHODS

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Mouse strains, cell lines, and virus stocks. BALB/WEHI or BALB/cByJ (H-2^d) and C57BL/6 (H-2^b) mice were obtained from the breeding colony at The Scripps Research Institute and used when 6 to 10 weeks of age. BALB/cl 7 (H-2^d), MC57 (H-2^b), and human T2 cells transfected with H-2 L^d (T2-L^d) were used in CTL and binding experiments. Cells were grown in RPMI 1640 (BALB/cl 7, MC57) or Iscove's modified Dulbecco's medium (T2-L^d) containing 8% bovine serum and L-glutamine as described previously (10, 11, 13-15, 20, 32, 33). Gentamicin (400 µg/ml) was added to Iscove's modified Dulbecco's medium to maintain selection of T2-L^d cells. The LCMV Armstrong clone 53b (LCMV ARM) was used at an MOI of 1 to infect cells. The origin, cloning, sequencing, plaque purification, and quantification of LCMV on Vero cells, expression of viral NP or glycoprotein (GP), and their identification with NP- or GP-specific monoclonal antibodies have been described previously (10, 14, 15, 32). Details describing the construction and use of recombinant VV expressing full-length LCMV ARM GP, NP, or NP epitope aa 118 to 127 have been published (14, 15, 31, 32).

Peptides. Peptides were synthesized by the solid-phase method using the N-9-fluorenyl(methylcarbonyl) (Fmoc) chemistry, purified by high-pressure liquid chromatography on a RP300-C8 reverse-phase column (Brownlee lab), and identified by fast atom bombardment or electrospray mass spectrometry (10, 20).

MHC binding studies. An MHC stabilization assay was performed. RMAS cells (D^b K^b) transfected with L^d molecules and P815 (H-2^d) were used to measure L^d stabilization in the presence of increasing peptide concentrations, using the monoclonal antibody HB27, 28-14-8S (anti-D^b, anti-L^d), Y3 (anti-K^b) or SF1-1.1.1 (anti-K^d) staining followed by flow cytometry analysis, as previously described (10, 20; M. B. A. Oldstone, J. E. Gairin, H. Mazarguil, F. Laval, V. C. Asensio, I. L. Campbell, S. J. DeArmond, B. Coon, and D. Homann, unpublished data.). The peptides were considered MHC binders when displaying a peptide concentration giving half of the maximal stabilization effect (SC₅₀) affinity values of 50 µM or lower.

Computer analysis. Molecular modeling of the interaction between H-2 L^d and viral peptides was performed using Insite (Biosym Technology, San Diego, Calif.). The calculated binding motif for L^d and crystallographic studies on this molecule are published (1, 26).

Generation and detection of CTLs. Primary LCMV-specific CTLs were generated by priming mice intraperitoneally with 10⁵ PFU of LCMV ARM. Spleens from such mice were removed 7 days after inoculation, and single-cell suspensions of spleens (free from erythrocytes) were prepared in complete RPMI 1640 media as described previously (14, 15, 29, 31, 32). These cells were then tested for their ability to lyse virus-infected or peptide-coated targets as reported previously (14, 15, 24, 29, 31, 32). The L^d-restricted CTL clone HD-8 has been described, and its specificities have been recorded (32).

	RESULTS AND DISCUSSION

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The L^d MHC CD8⁺ CTL-restricted peptide sequence was remarkably similar among diverse arenaviruses. The L^d MHC molecule bound viral peptides via an MHC anchor of proline (P) at P2 and a methionine (M), phenylalanine (F), leucine (L), or isoleucine (I) at P9 or P10 according to elution, alignment, and immunochemical studies (23). Others (9) employing limited single-amino-acid substitution analysis reported that replacing the authentic P at P2 with an alanine (A) or arginine (R), S at P5 with asparagine (N), or M at P9 with a lysine (K) or L diminished or abolished the capacity of NP peptide to increase cell surface L^d expression and to induce L^d stabilization in L cells (H-2^k) manipulated to express L^d molecules. In addition, crystallographic analysis of the L^d MHC molecule suggested that P3 may also act as an auxiliary MHC anchor with P4 and P8 forming contacts for the CD8⁺-T-cell receptor (1, 26). With the L^d-restricted LCMV ARM immunodominant CTL epitope RPQASGVYM (32) used as a guide, the corresponding NP sequences of several Old World and New World arenaviruses were obtained. As shown in Table 1, NP 118-126 of the Old World LFVs Nigeria (G) and Josiah (J) as well as Mopeia virus shared from 6 to 7 aa with the LCMV ARM peptide. These viruses bore homology to the MHC binding anchor of a P at P2 and an M at P9 but not the suggested P3 auxiliary MHC anchor LCMV ARM (Q), LFV (G and J), and Mopeia (L) residues. As anticipated, L^d-expressing cells bound tightly at concentrations of 4 µM for Mopeia virus, 20 µM for LFV (G), and 5 µM for LFV (J) (Table 1; Fig. 1). In contrast, the corresponding aligned NP sequence from the New World arenaviruses had less homology to the LCMV prototype, i.e., 2 to 4 aa were shared with the 9 aa residues of LCMV ARM. Further, as shown in Table 1, none of the New World arenaviruses, Junin, Machupo, Sabia, Pichinde, Oliveros, or Tacaribe, had a P in P2 or a Q or L in P3, although Junin, Machupo, Sabia, and Tacaribe viruses had the correct amino acids to serve as an MHC anchor in P9. Therefore, it was surprising that Sabia bound well to the L^d molecules (30 µM [Table 1 and Fig. 1], and in a separate experiment, 10 µM [Table 2]). Sequence comparison showed that Machupo virus, a nonbinder (affinity of good binders is less than 50 µM), and Sabia virus, a good binder, shared serine (S) in P2 but differed with respect to amino acids in P3; Sabia virus had an alanine (A), and Machupo virus had a glycine (G). Further, Sabia virus, like the Old World viruses, had an arginine (R) in P1, whereas Machupo virus had a glutamic acid (E) in that position. To evaluate the role of P at P2 and E at P1 in binding to L^d, we substituted E for R in LCMV ARM at P1 and R for E in Machupo virus at the same amino acid residue. Table 2 shows that these substitutions did not change the high binding efficiency of wild-type (wt) LCMV ARM NP 118-126 (parental sequence) or mutant LCMV ARM NP 118-126 (E 118) to L^d or reverse the low or absent binding of wt Machupo NP 117-125 or mutant Machupo NP 117-125 (R 117) to L^d. From these results, we conclude that amino acid E in P1 does not interfere with binding to the L^d molecule if P is at P2. The binding of Sabia to L^d suggests that an S in P2 with the appropriate flanking amino acid could serve as an ancillary MHC anchor for P2.

View larger version (30K): [in this window] [in a new window]   FIG. 1.   Data for the MHC stabilization assay using target cells expressing Ld molecules. Tenfold dilutions of various arenavirus peptides, monoclonal antibody to Ld, and fluorescence-activated cell sorting were used. LCMV, LFV, Mopeia virus, and Sabia virus NP peptides aa 117/118 to 126 bind to Ld. Data were confirmed in two repeated studies. See Materials and Methods and Table 1 for details.

L^d-restricted LCMV ARM CD8⁺ CTLs lyse target cells coated with related NP peptides from diverse Arenaviridae. For the next series of experiments, we inoculated BALB H-2^d mice with LCMV ARM to generate primary day 7 anti-LCMV ARM CTLs. The lytic capacity of these CTLs was then tested against ⁵¹Cr-labeled target cells coated with various concentrations of related NP peptides from Old World and New World arenaviruses. As expected (Table 3), LCMV ARM day 7 CTLs in a virus-specific MHC-restricted manner killed target cells expressing the relevant Old World peptides from Mopeia, LFV (G), and LFV (J) viruses. For confirmation, a well-characterized L^d-restricted CTL clone (HD-8) specific for LCMV ARM NP 118-126 (32) was used at effector-to-target ratios of 5:1 and 1:1 (5:1 shown in Table 3). Since these CTLs also lysed L^d targets coated with peptides from Old World arenaviruses, CTLs generated specifically to LCMV could cross-react and kill targets of Old World Arenaviridae. In contrast to these findings, Sabia virus peptide sequence NP 118-126, which bound robustly to L^d, was not recognized by LCMV ARM CTLs (Table 3), indicating a lack of cross-reactivity by LCMV to the CTL peptide epitope of the New World Sabia virus.

View larger version (19K): [in this window] [in a new window]   FIG. 2.   Intracellular cytokine assay done on lymphocytes obtained from BALB mice 7 days after infection with LCMV. Lymphocytes were incubated with various arenavirus NP peptides (see Table 1), recombinant interleukin-2, and brefeldin A and stained for both surface CD8 molecules and intracellular IFN- and TNF- by FACS. Only LFV and Mopeia virus NP peptides consisting of aa 118 to 126 cross-react at the CD8+-T-cell level with LCMV CD8+ CTLs. Data displayed are the mean values of triplicate samples, and each bar represents 1 SD. Data are representative of two assays. See Materials and Methods and references 2 and 24 for details.