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J Virol. 2010 October; 84(19): 9947–9956.
Published online 2010 July 28. doi:  10.1128/JVI.00672-10
PMCID: PMC2937778

A Multivalent Vaccination Strategy for the Prevention of Old World Arenavirus Infection in Humans [down-pointing small open triangle]

Abstract

Arenaviruses cause severe human disease ranging from aseptic meningitis following lymphocytic choriomeningitis virus (LCMV) infection to hemorrhagic fever syndromes following infection with Guanarito virus (GTOV), Junin virus (JUNV), Lassa virus (LASV), Machupo virus (MACV), Sabia virus (SABV), or Whitewater Arroyo virus (WWAV). Cellular immunity, chiefly the CD8+ T-cell response, plays a critical role in providing protective immunity following infection with the Old World arenaviruses LASV and LCMV. In the current study, we evaluated whether HLA class I-restricted epitopes that are cross-reactive among pathogenic arenaviruses could be identified for the purpose of developing an epitope-based vaccination approach that would cross-protect against multiple arenaviruses. We were able to identify a panel of HLA-A*0201-restricted peptides derived from the same region of the glycoprotein precursor (GPC) of LASV (GPC spanning residues 441 to 449 [GPC441-449]), LCMV (GPC447-455), JUNV (GPC429-437), MACV (GPC444-452), GTOV (GPC427-435), and WWAV (GPC428-436) that displayed high-affinity binding to HLA-A*0201 and were recognized by CD8+ T cells in a cross-reactive manner following LCMV infection or peptide immunization of HLA-A*0201 transgenic mice. Immunization of HLA-A*0201 mice with the Old World peptide LASV GPC441-449 or LCMV GPC447-455 induced high-avidity CD8+ T-cell responses that were able to kill syngeneic target cells pulsed with either LASV GPC441-449 or LCMV GPC447-455 in vivo and provided significant protection against viral challenge with LCMV. Through this study, we have demonstrated that HLA class I-restricted, cross-reactive epitopes exist among diverse arenaviruses and that individual epitopes can be utilized as effective vaccine determinants for multiple pathogenic arenaviruses.

Arenaviruses are a family of rodent-borne viruses that are associated with severe disease in humans. Phylogenetically, the arenaviruses are organized into Old World or New World groups, with a subdivision of the New World viruses falling into three distinct lineages (A to C) (17). At least eight arenaviruses are known to cause human disease. Of the New World viruses, Junin virus (JUNV), Machupo virus (MACV), Guanarito virus (GTOV), and Sabia virus (SABV) (all members of lineage B) are etiologic agents of hemorrhagic fever syndromes in South America, while Whitewater Arroyo virus (WWAV) (lineage A) has been linked to two fatalities in North America (13, 17). Lassa virus (LASV), Lujo virus, and lymphocytic choriomeningitis virus (LCMV) represent the Old World viruses that cause disease in humans (10, 13). LASV and Lujo virus, both of which are found in Africa, can cause hemorrhagic fevers, while LCMV infection, which is endemic throughout the world, can result in aseptic meningitis in immunocompetent individuals (10, 15, 33). LCMV is also a potent teratogen responsible for outcomes such as hydrocephaly and chorioretinitis in developing fetuses (3-5, 31, 36) or high lethality in immunosuppressed patients (20). LASV, LCMV, JUNV, GTOV, and MACV are NIAID category A agents, and with the exception of LCMV, each of these viruses is also on the CDC select agent list of potential bioterrorism threat agents.

At present, vaccines for the prevention of human arenavirus disease are limited to a single live attenuated vaccine (Candid 1) for the prevention of JUNV infection. JUNV Candid 1, which is classified as an investigational new drug in the United States, was derived from the wild-type (WT) JUNV strain XJ13 through serial passage both in vivo and in vitro (22). The only antiviral agent currently utilized for treatment of a subset of arenavirus infections is ribavirin (23, 35). Passive antibody therapy has been effective in reducing mortality associated with Argentine hemorrhagic fever caused by infection with JUNV (19, 32).

Arenaviruses have an RNA genome that is encoded in an ambisense fashion and consists of two single-stranded RNA segments, the ~3-kb small (S) segment and ~7-kb large (L) segment. The arenavirus proteome consists of four proteins: the viral RNA-dependent RNA polymerase (L) and zinc binding protein, which are encoded on the L segment, and the S segment-encoded nucleoprotein (NP) and glycoprotein precursor (GPC), which is posttranslationally modified to yield a stable signal peptide, GP1, and GP2 (49). GP1 and GP2 form heterodimers that facilitate entry into permissive host cells through an interaction with either α-dystroglycan for the Old World viruses LASV and LCMV (i.e., for strain clone 13 but not strain Armstrong 53b) (16, 30) or transferrin receptor 1 for a subset of the pathogenic New World arenaviruses (1, 41, 42). GP1 is also the target of antiviral neutralizing antibodies (nAbs) (11). The NP and L protein, which are encoded in negative or antimessage sense, are the first proteins to be expressed following infection, whereas the GPC and zinc binding protein, which are encoded in positive or message sense, arise later, demonstrating the temporal control of gene expression that is inherent in arenavirus replication.

The cellular immune response, specifically the CD8+ T-cell response, is important for the development of protective immunity following arenavirus infection (for a review, see reference 9). This is particularly true for the pathogenic Old World arenaviruses LCMV and LASV, as high-quality antiviral nAbs typically do not form following infection with these viruses; low-titer nAbs, if formed, usually appear weeks to months following clearance of the primary infection (11, 14, 18, 21, 34). In the case of LCMV, it was recently shown that the GPC itself, not the viral backbone, is responsible for this poor nAb response (40). This observation has important implications for vaccine design, as delivery of Old World arenavirus GPCs in even the most immunogenic of vectors may not improve the kinetics or magnitude of nAb formation following immunization. Therefore, vaccine development strategies for Old World arenaviruses must necessarily focus on the induction of protective cellular immune responses. In contrast, New World arenaviruses such as JUNV and MACV are able to induce both high-quality cellular and humoral immune responses following infection (39).

A significant challenge currently facing vaccine development is pathogen heterogeneity. In the case of arenaviruses, the low incidence of disease observed for the majority of these antigenically diverse viruses combined with the low socioeconomic status of the countries where they are endemic makes it unlikely that individual vaccines will be developed for each of the eight pathogenic arenaviruses. Therefore, the development of multivalent vaccines capable of providing cross-protection against multiple pathogenic arenavirus family members is desirable. It has been well-established that CD8+ T-cell epitopes are effective vaccine determinants for arenaviruses (6, 8, 25, 29, 43, 44) and that cross-protective cellular immune responses are generated following arenavirus infection in animal models (39, 43). In the current study, we evaluated whether HLA class I-restricted epitopes that are cross-reactive among pathogenic arenavirus species could be identified for the purpose of developing an epitope-based, multivalent vaccination approach that would cross-protect against multiple arenaviruses.

MATERIALS AND METHODS

Amino acid sequence analyses.

The arenavirus open reading frames utilized in this study were those encoding LASV strain Josiah GPC (NCBI accession number NP_694870), LCMV strain Armstrong 53b GPC (NCBI accession number NP_694851), MACV strain Carvallo GPC (NCBI accession number AAN05425), JUNV strain MC2 GPC (NCBI accession number BAA00964), GTOV strain INH-95551 GPC (NCBI accession number AAN05423), SABV strain SPH114202 GPC (NCBI accession number AAC55091), WWAV strain AV9310135 GPC (NCBI accession number AAK60497), and Pichinde virus (PICV) strain Munchique GPC (NCBI accession number AAC32281).

Peptides.

Peptides (≥90% pure) were obtained from Genemed Synthesis, Inc. (South San Francisco, CA). Each peptide was purified by high-performance liquid chromatography (HPLC), and the sequence was verified by mass spectrometry. Hepatitis B virus (HBV) ENV 378 (LLPIFFCLWV) was used as an irrelevant, HLA-A*0201-restricted peptide.

MHC-peptide binding assays.

Major histocompatibility complex (MHC) molecules were purified and binding assays were performed, as previously described (47, 48). Briefly, 1 to 10 nM radiolabeled peptide was coincubated with 1 μM to 1 nM purified MHC in the presence of 1 to 3 μM human β2-microglubulin. After 2 days, binding of the radiolabeled peptide to the corresponding MHC class I molecule was determined by capturing MHC-peptide complexes on Greiner Lumitrac 600 microplates (Greiner Bio-One, Longwood, FL) coated with the W6/32 antibody and measuring bound counts per minute using a TopCount microscintillation counter (Packard Instrument Co.).

Mice.

HLA-A*0201/Kb (referred to as HLA-A*0201 throughout) transgenic mice were bred at The Scripps Research Institute (TSRI). These mice represent the F1 generation, resulting from a cross between HLA-A*0201/Kb transgenic mice (that express a chimeric gene consisting of the α1 and α2 domains of HLA-A*0201 and the α3 domain of H-2Kb) created on the C57BL/6 background and BALB/c mice (The Jackson Laboratory) (53). All studies were conducted in facilities approved by the Association for Assessment and Accreditation of Laboratory Animal Care and according to Institutional Animal Care and Use Committee-approved animal protocols.

Cells.

JA2.1 cells (human Jurkat cells that express the HLA-A*0201/Kb chimeric gene) (53) were grown as previously described (46).

Viruses.

LCMV infections were conducted using strain Armstrong 53b. This virus was expanded in BHK-21 cells, and the titers of the virus were determined on Vero E6 cells. vvLASV-GPC and vvLCMV-GPC (which express LASV strain Josiah GPC and LCMV Armstrong 53b GPC, respectively) are recombinant vaccinia viruses (rVV) that were generated on the Western Reserve (WR) background as previously described (6, 54).

Immunizations and viral challenges.

To evaluate peptide immunogenicity, CD8+ T-cell avidity, endogenous processing of peptides from native LASV or LCMV antigens, or in vivo cytotoxic T-lymphocyte (CTL) killing, HLA-A*0201 mice (8 to 14 weeks old) were inoculated subcutaneously (s.c.) at the base of the tail with a mixture of CD8+ T-cell peptides (50 μg of each peptide per mouse) and the helper T-cell peptides human lambda repressor 12 (YLEDARRLKAIYEKKK), chicken ovalbumin 323 (ISQAVHAAHAEINE), and HBV core 128 (TPPAYRPPNAPIL) (46.7 μg of each peptide per mouse) that had been emulsified 1:1 in incomplete Freund adjuvant (IFA). For intraperitoneal (i.p.) challenge studies, mice were immunized with peptide-IFA emulsions and challenged at 14 days postimmunization via i.p. inoculation with 2 × 105 PFU of LCMV. At 4 days postchallenge, spleens were harvested for CD8+ T-cell purification and LCMV titer determination. To determine viral titer, a portion of each spleen was homogenized and serial 10-fold dilutions of these homogenates were plated on Vero E6 cells and overlaid with 1.4% agarose-Dulbecco modified Eagle medium. Cells were fixed, and plaques were counted at 4 days postinoculation.

ELISPOT assay.

Enzyme-linked immunospot (ELISPOT) assays were performed as previously described (50). Briefly, 4 × 105 splenic CD8+ T cells (isolated by anti-CD8-coated magnetic beads [Miltenyi Biotec, Auburn, CA]) were cultured with 1 × 105 peptide-pulsed or rVV-infected JA2.1 target cells. Target cells were pulsed by incubating them with peptide for at least 2 h at room temperature, followed by three washes to remove free peptide. For rVV infections, JA2.1 cells were infected (multiplicity of infection of 10) 18 h prior to the assay. Effector and target cells were incubated in flat-bottom 96-well nitrocellulose plates (Immobilon-P membrane; Millipore) precoated with 50 μl/well of anti-gamma interferon (anti-IFN-γ) monoclonal antibody (MAb) (10 μg/ml Mabtech AN18). After 16 to 20 h, plates were washed and wells were incubated with 100 μl biotinylated anti-IFN-γ MAb (1 μg/ml Mabtech R4-6A2) for 2 h. After additional washing, spots were developed by sequential incubation with Vectastain ABC peroxidase (Vector Laboratories) and 3-amino-9-ethyl carbazole solution (Sigma-Aldrich) and counted by computer-assisted image analysis (Zeiss KS ELISPOT reader).

Each assay was performed in 3 replicate wells, and the experimental values were expressed as the mean spots/106 CD8+ T cells ± standard deviations (SD) for each peptide. Responses of CD8+ T cells derived from peptide-immunized mice against JA2.1 cells pulsed with irrelevant peptide (HBV ENV 378) or infected with irrelevant VV constructs were measured to establish background values.

In vivo cytotoxicity assay.

Splenocytes from HLA-A*0201 mice were labeled with 0.3 μM or 0.06 μM CFSE (5,6-carboxy-fluorescein diacetate succinimidyl ester; Molecular Probes). CFSEhi cells were pulsed with 1 μg/ml of LASV GPC spanning positions 441 to 449 (GPC441-449), LCMV GPC447-455, MACV GPC444-452/ JUNV GPC429-437, GTOV GPC427-435, or WWAV GPC428-436, while CFSElo cells were pulsed with 1 μg/ml of the irrelevant peptide HBV ENV 378. Following extensive washing, equal numbers of CFSEhi and CFSElo cells were mixed and delivered (~8 × 106 total cells per mouse) via intravenous (i.v.) injection to syngeneic HLA-A*0201 mice that had been (i) immunized 10 days earlier either with peptide or adjuvant alone or (ii) infected via i.p. inoculation with 2 × 105 PFU of LCMV Armstrong 53b or were naïve. After 18 h, CFSE-labeled cells were identified from the spleens of recipient mice by flow cytometry. The percent killing was determined as follows: 100 −{[(% of immunizing peptide-pulsed cells in peptide-immunized or LCMV-infected mice/% of irrelevant peptide-pulsed cells in peptide-immunized or LCMV-infected mice)/(% of immunizing peptide-pulsed cells in adjuvant-immunized or naïve mice/% of irrelevant peptide-pulsed cells in adjuvant-immunized or naïve mice)] × 100}. Cell staining was analyzed by flow cytometry at the TSRI core facility by using a BD Biosciences FACSCalibur flow cytometer and CellQuest software.

Statistical analyses.

To evaluate whether a CD8+ T-cell response to peptide-pulsed or rVV-infected target cells was statistically significant, we utilized the Student t test to compare mean IFN-γ spots generated by CD8+ T cells in response to JA2.1 cells pulsed with peptide (relevant versus irrelevant) and those infected with an rVV construct (relevant versus irrelevant). To determine whether immunization with a given epitope led to viral titer reduction in the spleen following i.p. challenge, we used the Student t test.

RESULTS

Identification of candidate HLA-A*0201-restricted epitopes that are cross-reactive among arenavirus family members.

The primary goal of our study was to determine whether highly conserved, cross-reactive human CD8+ T-cell epitopes exist among pathogenic arenaviruses and, if so, to determine whether they could be successfully utilized as multivalent vaccine determinants to provide cross-protection against heterologous arenavirus infection. In previous studies, we had identified HLA-A*0201-restricted epitopes derived from Old World arenaviruses LCMV (GPC447-455) and LASV (GPC42-50 and GPC60-68) that were protective against viral challenge following peptide immunization of HLA-A*0201 mice (6, 8). To evaluate whether these protective epitope sequences would be conserved among diverse arenaviruses and therefore serve as candidates for multivalent vaccination, we conducted sequence alignments comparing each linear epitope sequence to its corresponding region in other Old World and New World arenavirus family members. None of the epitopes examined shared 100% sequence homology with other arenaviruses (Table (Table11 and data not shown). We did, however, identify a panel of peptide sequences corresponding to LCMV GPC447-455 that differed from this epitope by 1 to 4 amino acids and contained permissive HLA-A2 supertype B- and F-pocket anchor residues at positions 2 and 9, respectively (Table (Table1).1). The highest degree of sequence conservation was found between LCMV GPC447-455 and LASV GPC441-449, where a single, conservative Val-to-Ile substitution was observed at position 3. Greater diversity in primary sequence (range, 2 to 4 amino acid substitutions) was found between LCMV GPC447-455 and the corresponding peptides found in the New World arenaviruses.

TABLE 1.
Summary of candidate cross-reactive epitopes from pathogenic arenaviruses, including sequence comparison and binding affinity to common HLA-A2 supertype alleles

Next, to examine the breadth of HLA coverage afforded by this panel of arenavirus peptides, we measured the binding affinities of these peptides to common HLA-A2 supertype alleles (HLA-A*0201, -A*0202, -A*0203, -A*0206, and -A*6802). To determine binding affinity, each peptide was mixed with a radioactively labeled control peptide and coincubated with purified MHC as described in Materials and Methods. High-affinity binding (defined here as a 50% inhibitory concentration [IC50] less than or equal to 100 nM) to 3 or more HLA-A2 supertype alleles was observed for each peptide (Table (Table1).1). Binding affinity to purified HLA-A*0201 ranged from 10 nM for PICV GPC455-463 to 326 nM for JUNV GPC429-437/MACV GPC429-437. These data indicate that this set of arenavirus peptides has the potential to provide broad population coverage.

CD8+ T cells from HLA-A*0201 mice infected with LCMV cross-reactively kill APC pulsed with Old World and New World arenavirus peptides.

Next, we wished to determine whether the candidate peptides we had identified from pathogenic arenaviruses (LASV GPC441-449, LCMV GPC447-455, MACV GPC444-452, JUNV GPC429-437, GTOV GPC427-435, and WWAV GPC428-436) would be cross-reactively recognized by CD8+ T cells in the context of an arenavirus infection. To do so, we infected HLA-A*0201 mice with 2 × 105 PFU of LCMV strain Armstrong by i.p. inoculation and 7 days later screened these animals for in vivo CTL killing of syngeneic, CFSE-labeled target cells that had been pulsed with an arenavirus peptide (CFSEhi) or an irrelevant peptide (CFSElo). We observed significant levels of specific killing against antigen-presenting cells (APC) pulsed with the homologous LCMV peptide GPC447-455 (range, 70 to 95%) as well as targets pulsed with heterologous arenavirus peptides, including LASV GPC441-449 (range, 96 to 97%), GTOV GPC427-435 (range, 60 to 86%), and WWAV GPC428-436 (range, 52 to 73%) (Fig. (Fig.1).1). The JUNV GPC429-437/MACV GPC429-437 peptide, which had the weakest HLA-A*0201 binding affinity (326 nM) (Table (Table1),1), did not induce significant killing. Collectively, the results of this experiment demonstrate that most of the peptides in the set have potential diagnostic value as they can be recognized following infection with a heterologous arenavirus. Cross-reactive killing of APC pulsed with PICV GPC455-463 was not assessed in this experiment, as PICV has not been shown to cause naturally occurring human disease (12). While laboratory-acquired infections have been documented, they have not been linked with any definite illness.

FIG. 1.
Cross-reactive in vivo killing of peptide-pulsed target cells in LCMV-infected HLA-A*0201 mice. HLA-A*0201 mice were inoculated by i.p. injection with 2 × 105 PFU of LCMV strain Armstrong 53b or were uninfected (control). Seven ...

Immunization of HLA-A*0201 mice with arenavirus epitopes induces cross-reactive CD8+ T-cell responses to heterologous peptides.

To determine whether the identified peptides could induce cross-reactive CD8+ T-cell responses in vivo and thus be used as vaccine candidates, groups of HLA-A*0201 mice were immunized with individual candidate peptides via s.c. inoculation as described in Materials and Methods. CD8+ T cells were isolated at 11 to 14 days postimmunization and exposed to JA2.1 target cells that had been pulsed with decreasing serial 20-fold doses (range, 1 × 10−5 to 6.25 × 10−11 M) of each candidate peptide in an ex vivo IFN-γ ELISPOT assay. A given dose was considered immunogenic if it induced IFN-γ spot formation that was significantly higher than the observed response to an identical dose of an irrelevant, HLA-A*0201-restricted peptide.

Following immunization, high-avidity CD8+ T-cell responses (endpoint reactivity range, 1 × 10−9 to 6.25 × 10−11 M) were observed against each immunizing peptide, with the exception of JUNV GPC429-437/MACV GPC429-437 (endpoint, 1 × 10−5 M) (Fig. (Fig.22 A to F). Binding affinity for HLA-A*0201 correlated with CD8+ T-cell avidity as immunizing peptides that bound HLA-A*0201 with IC50s of 10 to 49 nM induced high-avidity CD8+ T-cell responses (endpoint, 1 × 10−9 M or less), while the JUNV GPC429-437/MACV GPC429-437 peptide that bound at 326 nM led to a low-avidity response (endpoint, 1 × 10−5 M).

FIG. 2.
Immunization of HLA-A*0201 mice with arenavirus epitopes induces cross-reactive CD8+ T-cell responses to heterologous peptides. HLA-A*0201 mice were immunized s.c. with either LASV GPC441-449 (A), LCMV GPC447-455 (B), JUNV GPC ...

Variable patterns of CD8+ T-cell cross-reactivity were observed following peptide immunization. For example, following immunization with the Old World peptides LASV GPC441-449 and LCMV GPC447-455, nearly identical high-avidity CD8+ T-cell responses to both the immunizing peptide and the corresponding heterologous Old World peptide were observed in each case (Fig. 2A and B). Detectable responses were also observed against a subset of the New World peptides in these animals, including PICV GPC455-463, JUNV GPC429-437/MACV GPC429-437, GTOV GPC427-435, and WWAV GPC428-436 following LCMV GPC447-455 immunization or PICV GPC455-463 and MACV GPC444-452/JUNV GPC429-437 following LASV GPC441-449 immunization. With that said, responses against the New World peptides were of low avidity (range, 1 × 10−5 to 2.5 × 10−8) and relatively low frequency.

Following immunization with the New World peptides GTOV GPC427-435, WWAV GPC428-436, and PICV GPC455-463, high-avidity CD8+ T-cell responses were observed against a subset of the New World peptides in each case while cross-reactive responses to the Old World peptides, while detectable, were of lower avidity and frequency (Fig. 2D to F). For example, following immunization with GTOV GPC427-435, the MACV GPC444-452/JUNV GPC429-437 and WWAV GPC428-436 peptides were recognized by CD8+ T-cell responses with frequencies and avidities similar to those seen against the immunizing GTOV GPC427-435 peptide, while PICV GPC455-463 was not recognized (Fig. (Fig.2D).2D). The LASV GPC441-449 and LCMV GPC447-455 peptides were recognized in these animals, but the responses to these peptides were of lower magnitude and avidity (endpoint reactivity, 2.5 × 10−8 M). WWAV GPC428-436-primed CD8+ T cells recognized each of the heterologous peptides screened, but IFN-γ spot formation was ~10-fold lower than that of the immunizing peptide, and only the GTOV GPC427-435 and PICV GPC455-463 peptides induced significant responses at 1 × 10−9 M (Fig. (Fig.2E).2E). Finally, following PICV GPC455-463 immunization, heterologous peptides induced low-avidity CD8+ T-cell responses (endpoint range, 1 × 10−5 to 2.5 × 10−8 M) of relatively low magnitude.

Cross-reactive CD8+ T-cell recognition of naturally processed peptides from native LASV GPC or LCMV GPC expressed in HLA-A*0201-restricted human APC.

Because the strongest cross-reactive CD8+ T-cell responses were observed among the Old World epitopes LASV GPC441-449 and LCMV GPC447-455 following peptide immunization (Fig. (Fig.2),2), we focused on addressing the suitability of these two peptides as vaccine candidates for the remainder of the study. One important consideration was whether these peptides could be processed from their native viral antigens by human APC and presented to CD8+ T cells that had been primed with either LASV GPC441-449 or LCMV GPC447-455. To express native arenavirus GPC proteins within human APC, we infected JA2.1 cells with rVV encoding the full-length LASV GPC (vvLASV-GPC) or LCMV GPC (vvLCMV-GPC) (see Materials and Methods). To assess epitope processing and CD8+ T-cell recognition of presented peptides, splenic CD8+ T cells from HLA-A*0201 mice that had previously been immunized with LASV GPC441-449 or LCMV GPC447-455 were screened for IFN-γ secretion in response to JA2.1 cells that had been either pulsed with peptide (irrelevant A*0201-restricted peptide, LASV GPC441-449, or LCMV GPC447-455) or infected with VV (vvWT, vvLASV-GPC, or vvLCMV-GPC) (Fig. (Fig.3).3). Both LASV GPC441-449-primed (Fig. (Fig.3A)3A) and LCMV GPC447-455-primed (Fig. (Fig.3B)3B) CD8+ T cells generated significant levels of IFN-γ spots following exposure to JA2.1 cells that expressed the homologous and heterologous GPC proteins, respectively, confirming that cross-reactive recognition of naturally processed peptides occurs in the context of human APC.

FIG. 3.
CD8+ T-cell recognition of naturally processed peptides from native LASV GPC or LCMV GPC expressed in HLA-A*0201-restricted human APC. HLA-A*0201 mice were immunized with either LASV GPC441-449 (A) or LCMV GPC447-445 (B) as indicated ...

Cross-reactive in vivo CTL killing following peptide immunization.

We next wished to evaluate whether peptide immunization of HLA-A*0201 mice with LCMV GPC447-455 or LASV GPC441-449 would induce CD8+ T cells capable of cross-reactively killing peptide-pulsed target cells in vivo. Mice were immunized with peptide (LCMV GPC447-455 or LASV GPC441-449) or adjuvant alone (control), and 10 days later, CFSE-labeled target cells that had been pulsed with either LCMV GPC447-455 or LASV GPC441-449 (CFSEhi) were combined with equal numbers of target cells pulsed with an HLA-A*0201-restricted, irrelevant peptide (CFSElo) and delivered via i.v. injection. The results of this experiment are displayed in Fig. Fig.4.4. Each of the peptides was able to induce specific killing of target cells pulsed with either the immunizing peptide or the corresponding heterologous peptide in each case. Following immunization with LCMV GPC447-455, the magnitude of killing against the immunizing peptide ranged from 16.0 to 46.0% while cross-reactive killing against targets pulsed with LASV GPC441-449 ranged from 30.3 to 48.0%. Following immunization with LASV GPC441-449, specific killing against the immunizing peptide ranged from 23.8 to 58.7% while cross-reactive killing against targets pulsed with LCMV GPC447-455 ranged from 34.0 to 39.4%. Thus, peptide immunization could elicit CD8+ T cells capable of cross-reactive killing.

FIG. 4.
Heterologous in vivo killing of LASV GPC441-449- or LCMV GPC447-455-pulsed target cells in peptide-immunized HLA-A*0201 mice. HLA-A*0201 mice were immunized with LASV GPC441-449, LCMV GPC447-455, or adjuvant alone (control). Ten days later, ...

HLA-A*0201 mice immunized with LASV GPC441-449 are protected from heterologous LCMV challenge.

Having previously demonstrated that immunization of HLA-A*0201 mice with LCMV GPC447-455 could provide protection against LCMV challenge (8), we wished to determine whether immunization with LASV GPC441-449 could provide cross-protection against a heterologous LCMV challenge. Figure Figure55 A outlines the logistics of the challenge experiment. Groups of HLA-A*0201 mice (n = 5 per group) were immunized with LASV GPC441-449, LCMV GPC447-455, or adjuvant alone (control group). LCMV GPC447-455 served as a positive control for this experiment. On day 14 postimmunization, mice were challenged with 2 × 105 PFU of LCMV via i.p. inoculation. Spleens were harvested from each mouse on day 4 postchallenge and screened for LCMV titer (Fig. (Fig.5B)5B) and epitope-specific CD8+ T cells (Fig. (Fig.5C).5C). Each of the control animals (adjuvant alone) displayed evidence of high-titer LCMV replication following challenge (Fig. (Fig.5B).5B). As expected, prior immunization with LCMV GPC447-455 led to a significant decrease (85%; P = 0.004) in mean viral titer compared to that in control animals. We also observed a significant reduction in mean viral titer following immunization with the heterologous peptide LASV GPC441-449 (92% reduction; P = 0.005).

FIG. 5.
Peptide immunization of HLA-A*0201 mice with LASV GPC441-449 provides heterologous protection against a subsequent challenge with LCMV. The logistics of the challenge experiment are outlined in panel A. HLA-A*0201 mice (n = 5 per ...

Protection correlates with expansion of epitope-specific CD8+ T cells.

We next wished to determine whether an expansion of CD8+ T cells specific for LCMV GPC447-455 and/or LASV GPC441-449 would correlate with the protection observed in Fig. Fig.5B.5B. To do so, for each challenge group (LCMV GPC447-455-immunized, LASV GPC441-449-immunized, or adjuvant only-immunized mice), we measured the frequency of splenic CD8+ T cells specific for LCMV GPC447-455, LASV GPC441-449, or an irrelevant HLA-A*0201-restricted peptide in an IFN-γ ELISPOT assay. Four days after viral challenge, mice that had previously been immunized with LCMV GPC447-455 or LASV GPC441-449 showed a clear expansion of CD8+ T cells that were specific for either of these peptides in each case (Fig. (Fig.5C).5C). In both of these peptide-immunized groups, approximately equal numbers of IFN-γ spots were formed against LCMV GPC447-455 and LASV GPC441-449.

DISCUSSION

Arenaviruses are a family of rodent-borne viruses that cause significant human disease throughout the world. Currently, no FDA-approved vaccines exist for the eight antigenically diverse species of arenavirus that are known to cause human disease. Therefore, it is a priority to develop vaccines for the prevention of human disease caused by these pathogens. Considering the large number of arenavirus species that need to be targeted, it would be highly desirable to develop multivalent vaccines that could provide cross-protection against multiple arenavirus species. Over the past several years, our research team has focused on the development of such multivalent vaccines. Our approach has been to capitalize on the known importance of the cellular immune response for providing protective immunity and accordingly to develop epitope-based vaccines capable of inducing protective CD8+ T-cell responses specific to multiple arenaviruses (7). We have tested two strategies to induce multivalent protection: inclusion of protective HLA-restricted epitopes corresponding to each of several targeted arenaviruses in a single vaccine (28) and, alternatively, to reduce the complexity of the vaccine, inclusion of highly conserved, cross-reactive epitopes, each of which has the capacity to induce cross-protection against multiple arenaviruses. In the present study, we have formally demonstrated that highly conserved, cross-reactive human CD8+ T-cell epitopes exist among pathogenic arenaviruses and that they can be successfully utilized as multivalent vaccine determinants to provide cross-protection against heterologous arenavirus infection.

Our finding of heterologous immunity following immunization with an HLA-restricted epitope extends observations initially made by Peters et al. when they demonstrated, in a guinea pig model of arenavirus infection, that heterologous immunity could be induced between several species of Old World arenaviruses (39). In that study, prior infection of guinea pigs with LCMV or another Old World arenavirus, Mopeia virus (MOPV), provided complete protection against a subsequent challenge with a lethal dose of LASV. Likewise, previous infection with LASV provided protection against subsequent challenge with LCMV. In each case, nAbs specific for the heterologous challenge viruses were not detectable at the time of challenge or viral clearance. Furthermore, adoptive transfer experiments formally demonstrated that splenocytes, but not plasma, were responsible for this heterologous immunity. Finally, lymphocytes from these animals were able to kill syngeneic target cells that had been infected with either the homologous priming virus or the heterologous challenge virus. Similarly, heterologous immunity has also been observed in nonhuman primate models of arenavirus infection. For example, previous infection of nonhuman primates with MOPV provided heterologous protection against a subsequent challenge with LASV despite an absence of nAb to the heterologous challenge virus (39). While the absence of nAb in the above-mentioned studies strongly suggests that cell-mediated immunity is responsible for providing protective heterologous immunity, it was not formally shown. More recently, however, Rodriguez-Carreno et al. demonstrated that immunization of BALB/c mice with a DNA vaccine encoding a CD8+ T-cell epitope derived from LASV NP was sufficient to confer heterologous protection against LCMV challenge (43). Likewise, in the present study, we show for the first time that an HLA-restricted epitope derived from LASV can induce heterologous protection against LCMV challenge in HLA transgenic mice. These findings confirm that, regardless of the host species' MHC background, CD8+ T-cell responses directed against an epitope that is conserved among arenaviruses is sufficient to provide robust heterologous immunity to antigenically diverse arenavirus species.

One potential concern in this study was whether our approach to multivalent vaccination, namely, immunization with a CD8+ T-cell epitope derived from LCMV or LASV, would lead to “original antigenic sin” with regard to the resulting CD8+ T-cell response to the heterologous peptide in each case (either the LASV or LCMV peptide, respectively). The epitopes used as vaccine determinants, LCMV GPC447-445 and LASV GPC441-449, differ from one another by a single, conservative amino acid substitution at position 3 (Table (Table1).1). Klenerman and Zinkernagel had previously demonstrated that original antigenic sin can occur with CD8+ T-cell responses in the murine model of LCMV infection (26). Specifically, they found that C57BL/6 mice primed with LCMV strain WE generated a CD8+ T-cell response to GPC33-41 but that upon secondary challenge with a WE variant containing a mutant GPC33-41 epitope (differing from the wild type [WT] by a single, conservative amino acid substitution at position 3), the CD8+ T-cell response was directed against the WT epitope and not the variant epitope. Inversely, a primary infection with the WE variant followed by a secondary challenge with WT WE led to CD8+ T-cell responses that were equal between the WT and mutant epitopes. This same phenomenon was also observed for the H-2d-restricted peptide, NP118-126. Based on these observations, Klenerman and Zinkernagel concluded that the CD8+ T-cell response was determined by the original peptide presented and that cross-reactivity was asymmetrical (26). Therefore, the order in which epitopes are seen can potentially determine the breadth of the response to heterologous peptides that share high levels of amino acid sequence identity. In our study, we clearly show that original antigenic sin does not occur with respect to the peptides LCMV GPC447-445 and LASV GPC441-449 as immunization with either peptide leads to virtually identical CD8+ T-cell responses to both the immunizing and heterologous peptides in each case (Fig. (Fig.22 to to4).4). Likewise, in the challenge assay, previous immunization with LASV GPC441-449 followed by heterologous LCMV challenge led to equivalent CD8+ T-cell responses against LCMV GPC447-445 and LASV GPC441-449 (Fig. (Fig.5).5). With that said, immunization of HLA-A*0201 mice with certain New World peptides in our set did not always result in equal CD8+ T-cell responses to both the immunizing peptide and heterologous peptide(s). For example, while immunization with GTOV GPC427-435 led to a CD8+ T-cell response that was similar in both magnitude and avidity to those of both GTOV GPC427-435 and WWAV GPC428-436, immunization with WWAV GPC428-436 resulted in a response where the magnitude of CD8+ T cells recognizing WWAV GPC428-436 was approximately 10-fold higher than the number of CD8+ T cells recognizing GTOV GPC427-435. Therefore, it is important to verify, for each cohort of cross-reactive epitopes, that original antigenic sin is not impacting immunity to the targeted, heterologous epitopes.

In the present study, we focused on one particular set of epitopes that are conserved among pathogenic arenaviruses to provide proof of principle that such HLA class I-restricted epitopes exist in nature and that they can be used to induce cross-protective immunity to heterologous arenavirus challenge. However, in our ongoing efforts to identify HLA-restricted epitopes from pathogenic arenaviruses, we have found additional HLA-A2 and HLA-A3 supertype-restricted epitopes that are either (i) completely homologous in primary sequence among one or more pathogenic arenaviruses (n = 1) or (ii) highly conserved (differing in primary sequence from one virus to another by 1 to 2 amino acid residues) and cross-reactive among several pathogenic arenaviruses (n = 4) (28). In addition to our findings, ter Meulen et al. have shown that CD4+ T cells from patients previously exposed to LASV are able to cross-reactively recognize epitopes derived from MOPV (51). There are also many examples of HLA-restricted epitopes that are conserved/cross-reactive among multiple species of viruses, including viruses within the Hantavirus genus (24, 52) and Flaviviridae family (37, 38, 55). Therefore, our strategy for multivalent vaccination may be applicable beyond the arenavirus family. Indeed, members of our team have recently shown that HLA-restricted CD4+ T-cell epitopes that are conserved among pathogenic influenza virus strains exist and that they too are protective (2).

In addition to having utility as vaccine determinants, the cross-reactive epitopes identified in this study also have value as diagnostic reagents. As we demonstrate in Fig. Fig.1,1, following an experimental infection with LCMV in HLA transgenic mice, peptides derived not only from the corresponding Old World virus LASV but also from the New World viruses GTOV and WWAV are recognized via an in vivo cytotoxicity assay. Sets of peptides such as this would be valuable tools for monitoring at-risk populations for arenavirus exposure, investigating the role of T cells in arenavirus pathogenesis in humans, and evaluating the capacity of new candidate vaccines to effectively induce cell-mediated immunity. One important consideration regarding our study is the fact that there is an incomplete overlap in the epitopes recognized by HLA transgenic mice and humans (27). Therefore, it will be important to verify that the epitopes identified in the current study can indeed be recognized in HLA-A*0201-restricted humans following exposure to pathogenic arenaviruses.

In conclusion, we have demonstrated that HLA-restricted, cross-reactive epitopes exist among pathogenic arenaviruses and that individual epitopes can be utilized as effective vaccine determinants for multiple pathogenic arenaviruses. Because the epitopes in this study are HLA-A2 supertype restricted, they are capable of providing broad population coverage (~43% of the population, regardless of ethnicity) (45). Inclusion of cross-protective epitopes such as these in multivalent vaccine formulations will allow for a reduction in the complexity of the resulting vaccine. While the current study provides proof of concept for the proposed multivalent vaccination strategy, future studies to advance this approach will focus on the identification of other cross-protective epitopes that correspond to additional HLA supertype families and arenaviruses to increase coverage of both HLA backgrounds and pathogenic arenavirus species.

Acknowledgments

We thank Renaud Burrer, Claire Crimi, Howard Grey, Marie-France del Guercio, Carla Oseroff, Valerie Pasquetto, Bjoern Peters, David Seiber, and Joey P. C. Ting for helpful comments and/or technical assistance. We are grateful to Bernard Moss for providing the reagents needed to generate rVV.

This work was supported by National Institutes of Health grants AI50840 to M.J.B., AI065359 RCE to J.B. and M.J.B., AI074862 to J.K.W., AI27028 and AI077607 to J.L.W., and P20RR021905, T32 AI07354, and F32 AI056827 to J.B. and contract N01-AI-40023 to A.S.

No conflict of interest exists among the authors.

Footnotes

[down-pointing small open triangle]Published ahead of print on 28 July 2010.

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