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. Author manuscript; available in PMC: 2014 Aug 1.
Published in final edited form as: Virology. 2013 May 16;442(2):114–121. doi: 10.1016/j.virol.2013.04.010

Pathogenesis of Lassa Fever Virus Infection: I. Susceptibility of Mice to Recombinant Lassa Gp/LCMV Chimeric Virus

Andrew M Lee a,b,*, Justin Cruite a, Megan J Welch a, Brian Sullivan a, Michael BA Oldstone a,*
PMCID: PMC3686847  NIHMSID: NIHMS472572  PMID: 23684417

Abstract

Lassa virus (LASV) is a BSL-4 restricted agent. To allow study of infection by LASV under BSL-2 conditions, we generated a recombinant virus in which the LASV glycoprotein (Gp) was placed on the backbone of lymphocytic choriomeningitis virus (LCMV) Cl13 nucleoprotein, Z and polymerase genes (rLCMV Cl13/LASV Gp). The recombinant virus displayed high tropism for dendritic cells following in vitro or in vivo infection. Inoculation of immunocompetent adults resulted in an acute infection, generation of virus-specific CD8+ T cells and clearance of the infection. Inoculation of newborn mice with rLCMV Cl13/LASV Gp resulted in a life-long persistent infection. Interestingly, adoptive transfer of rLCMV Cl13/LASV Gp immune memory cells into such persistently infected mice failed to purge virus but, in contrast, cleared virus from mice persistently infected with wt LCMV Cl13.

Keywords: Lassa Gp recombinant virus, pathogenesis, dendritic cell, immune response

INTRODUCTION

Lassa virus (LASV), the agent of a severe and often fatal hemorrhagic illness known as Lassa fever (LF), is endemic in West Africa and estimated to infect more than 300,000 individuals yearly, hospitalizing 100,000 and causing 20,000 or more deaths. LASV has entered Europe and America (Freedman and Woodall, 1999; Holmes et al., 1990; Isaacson, 2001; McCormick and Fisher-Hoch, 2002; Schmitz et al., 2002) via travelers incubating the virus. As a Category A Select Agent, LASV is a potential bioterrorist threat (Borio et al., 2002).

An early host immune response to LASV is crucial for survival (Flatz et al., 2010; Geisbert and Jahrling, 2004; McCormick and Fisher-Hoch, 2002). Infected individuals with viral titers of 9 logs/ml blood or above, fail to mount an effective innate/adaptive immune response and invariably die, whereas those with blood titer of 8 logs of virus or less do respond immunologically with a T cell response and survive (Schmitz et al., 2002). Earlier we documented that alpha-dystroglycan (α-DG) was the receptor for LASV and other Old World arenaviruses like lymphocytic choriomeningitis virus (LCMV) (Cao et al., 1998; Oldstone and Campbell, 2011; Spiropoulou et al., 2002). Further, among cells of the immune system α-DG is preferentially located on DCs (>99% of α-DG in the immune system is found on DCs) (Kunz et al., 2001; Oldstone and Campbell, 2011; Sevilla et al., 2000; Sevilla et al., 2004). DCs are a preferred target of wild-type (wt) LASV which impairs DC function (Baize et al., 2004; Baize et al., 2006; Mahanty et al., 2003; Pannetier et al., 2011).

LASV and LCMV are members of the Old World arenavirus family and contain a similar two-RNA segment organization, four viral genes and genome structure (Buchmeier et al., 2007). The glycoprotein (Gp) and nucleoprotein (NP) are encoded on the short RNA segment, while the Z matrix protein and L polymerase are encoded on the long RNA. With the recent advent of a reverse genetics for the arenaviruses (Emonet et al., 2011; Emonet et al., 2009) it is possible to construct a recombinant (r) LCMV backbone expressing the LASV glycoprotein (Gp), thereby allowing the investigation of LASV Gp binding, cell entry and replication in the context of a viral infection.

Utilizing the backbone of LCMV Armstrong (ARM) 53B, a recombinant virus expressing LASV Gp (Josiah strain) was made by Rojek and colleagues to study LASV Gp-mediated cell entry (Rojek et al., 2008). They discovered that LASV entered cells by a unique endocytic pathway distinct from the clathrin-dependent endocytosis used by pathogenic New World arenaviruses. However, this recombinant was not suitable for in vivo animal studies and thus not useful for dissecting LASV pathogenesis. For that reason we turned to use of LCMV Cl13 as the backbone for constructing rLCMV/LASV Gp.

The parental LCMV ARM and its variant strain LCMV Cl13 differ from each other by six nucleotides and three amino acids of which only two, one in the Gp spike at aa 260 (ARM/Cl13: Phe/Leu) and one in the polymerase gene aa 1079 (ARM/Cl13: Lys/Glu) determine heightened entry into DCs (Gp1 Leu) and heightened replication (polymerase Glu) of LCMV Cl13 over that observed with LCMV ARM. Further, these two amino acid differences determine whether an acute viral infection is controlled by cytotoxic T (CTL) cells (ARM) or whether an inadequate generation of T cells occurs followed by their exhaustion occurs leading to viral persistence (Cl13) (Barber et al., 2006; Brooks et al., 2006; Wherry, 2011; Zajac et al., 1998). To take advantage of the enhanced replication of LCMV Cl13 in DCs to study LASV Gp-mediated pathogenesis, we used reverse genetics to construct a novel rLCMV Cl13/LASV Gp using the Cl13 backbone.

Here we report that the rLCMV Cl13/LASV Gp replicates in multiple tissues of adult mice and induces the generation of robust virus-specific CD8+ CTLs. Removal of immune cells by use of Rag2−/− mice in which lymphoid T and B cells are genetically depleted enhances viral replication and leads to persistent infection. rLCMV Cl13/LASV Gp replicates to considerable levels in plasmacytoid (p) DCs as well as conventional (c) DCs both in vitro and in vivo. Further, inoculation of newborn mice or breeding of rLCMV Cl13/LASV Gp infected mice, both called virus carrier mice, leads to a life-long persistent virus infection. Interestingly, adoptive transfer of memory T and B cells harvested from rLCMV Cl13/LASV Gp infected mouse spleens failed to clear the persistent infection of such virus carrier mice. However, similar immune memory cells could purge virus from mice persistently infected with LCMV Cl13. Hence, the rLCMV Cl13/LASV Gp provides a useful tool to study LASV pathogenesis in a small mouse model for acute and persistent infections in a BSL-2 setting.

RESULTS

Polymerase of LCMV Cl13 provides enhanced replication of LCMV when compared to the polymerase of LCMV ARM

Our first series of experiments determined the efficiency of LCMV replication comparing the polymerase of LCMV Cl13 with that of LCMV ARM. The L RNA which contains the Z gene and the polymerase gene from LCMV ARM or from LCMV Cl13 was swapped with the S RNA gene of LCMV Cl13 or LCMV ARM, respectively, as previously reported (Popkin et al., 2011; Rojek et al., 2008). Since the other gene residing on the L RNA, the Z gene and its protein, is identical between LCMV Cl13 and LCMV ARM, we were essentially evaluating the difference provided by the single amino acid mutation at aa 1079 of Glu for LCMV Cl13 compared to Lys for LCMV ARM. Utilizing cloned established murine dendritic cell lines P4H1 and 9M (Nayak, Schmedt, Sullivan, and Oldstone, manuscript in preparation 2013), we noted that reassorted virus containing the polymerase of LCMV Cl13 replicated 1.5 to 2 logs greater than did the recombinant virus containing the polymerase of LCMV ARM (Figure 1, Panels A and B). Interestingly, despite being linked to the Gp1 of the S RNA of LCMV ARM which is decidedly inferior for attachment and entry compared to Gp1 of LCMV Cl13 (Sullivan et al., 2011), the replication of S ARM/L Cl13 was equivalent to the amount of infectious virus produced by the wild-type LCMV Cl13 (S Cl13/L Cl13) (Figure 1: A,B), again revealing the greater efficiency in replication of LCMV Cl13 polymerase. These results lead us to generate and characterize a recombinant LCMV Cl13 expressing the envelope Gp of LASV Josiah strain (rLCMV Cl13/LASV Gp).

Fig. 1.

Fig. 1

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The growth of reassortant viruses in the P4H1 (A) and 9M (B) cloned DC lines is shown. Viruses containing the polymerase of LCMV Cl13 (red diamonds, green squares) replicated to 1-2 logs higher titer than those bearing the polymerase of LCMV ARM (orange circles, black triangles). Data points shown are the average of three independent experiments, and the standard deviation is plotted as error bars for each point (p < 0.01 for all data points). (C) The growth of rLCMV Cl13/LASV Gp virus (open circles) and LCMV Clone 13 (solid circles) is shown in VeroE6 cells. Cells were infected with an MOI of 0.1 of each virus, and viral titer in 100 μL supernatant was analyzed by immune focus assay. Points are mean ± standard deviation of focus-forming units per mL (FFU/mL) from three independent measurements. (D) Growth of rLCMV Cl13/LASV Gp in the P4H1 DC line is similar to that of LCMV Cl13, and two logs higher than LCMV ARM. The growth follows the pattern for LCMV viruses containing the Cl13 polymerase. (E) Immunofluorescent staining of BHK-21 cells infected with virus that was preincubated with either PBS or 100 nM soluble α-DG for 20 minutes prior to infection. Infection was done at an MOI of 0.1, and staining of fixed cells was performed 48 hours post-infection. (F) Virus titers were measured 48 hours post-infection in the supernatant of BHK-21 cells infected with virus (MOI = 0.1) that was pre-incubated with increasing concentrations of soluble α-DG. Both Clone 13 and rLCMV Cl13/LASV Gp are blocked in a dose-dependent manner by α-DG.

Generation and characterization of rLCMV Cl13/LASV Gp

To be able to investigate LASV at BSL-2 level in the context of a productive infection outside of BSL-4 facility, we utilized published reverse genetics technology to generate a recombinant LCMV Cl13 where the LASV Gp substituted for LCMV Cl13 Gp (Rojek et al., 2008). Briefly, BHK cells were co-transfected with plasmids pCL and pCNP, together with plasmids that allowed for Pol I-mediated intracellular synthesis of the L and S RNA species. We infected Vero and A549 cells and collected supernatants at 48, 72, and 96 hours post-infection to test for presence of replicating infectious rLCMV Cl13/LASV Gp (Figure 1: C,D). The infection kinetics of rLCMV Cl13/LASV Gp virus were similar to that of the parental LCMV Cl13. Furthermore, infecting the P4H1 DC line with rLCMV Cl13/LASV Gp resulted in a similar infection to that of Cl13 (Figure 1D), with 1.5 to 2 logs greater replication than LCMV ARM. Similar enhanced replication of 1-2 logs with viruses containing the polymerase of Cl13 over those with polymerase of ARM occurred in 9M cloned DCs (data not shown). Interestingly this enhanced replication of Cl13 and Cl13 polymerase-containing viruses, while being observed in DC lines, was not observed in VeroE6 (Figure 1C) or A549 and BHK cells (data not shown).

Alpha-dystroglycan (α-DG) has been shown to be the receptor for LASV (Cao et al., 1998; Spiropoulou et al., 2002). We next determined whether the infection with rLCMV Cl13/LASV Gp proceeded through α-DG by incubating the viruses with soluble α-DG prior to initiating infection. Previously we demonstrated that LCMV Cl13 entry into cells bearing α-DG on their surfaces was blocked in a dose-response manner by soluble α-DG (Cao et al., 1998) and virus entry was dependent on the Gp of Cl13 (Sullivan et al., 2011). Similar blunting occurred when soluble α-DG was incubated with rLCMV Cl13/LASV Gp (Figure 1E), while titers of virus produced were dependent on α-DG blockade (Figure 1F) indicating that the recombinant virus bearing LASV Gp can be used at BSL-2 level for LASV Gp-mediated binding, entry, and in vitro replication studies.

Replication of rLCMV Cl13/LASV Gp in vivo in adult mice

We next injected rLCMV Cl13/LASV Gp into adult immunocompetent H-2b C57BL/6 mice and studied viral replication in vivo. Employing a wide dose range from 2 × 105 PFU intrapertoneally (i.p.) to 2 × 106 and 4 × 106 PFU intravenously (i.v.) of rLCMV Cl13/LASV Gp, we noted limited viral replication (PFU/gm tissue) in liver, spleen, kidney, and sera (PFU/ml) (Table 1A – viral dose indicated). Tissue and serum viral titers dropped significantly by 1 to 3 logs between day 5 post-infection (PI) and day 7/8 PI with the majority of mice completely purging virus at this time. These results suggested the likelihood of a strong antiviral T-cell response limiting viral replication and clearing the acute infection. To test this possibility we utilized Rag2−/− mice on the same C57BL/6 background inoculated with 4 × 106 PFU of rLCMV Cl13/LASV Gp. As shown in Table 1A, Rag2−/− mice exhibited an increase in tissue and serum viral titers from day 5 to day 7/8, indicating that the immune response cleared the infection.

Table 1.

(A) displays viral titers and viral antigen expression in C57BL/6 immunocompetent or Rag2−/− mice on the C57BL/6 background depleted of T and B lymphocytes. Titers of virus were determined by plaque assay and the mean value for at least four individual mice infected with the virus is reported. (B) records expression of viral NP in various tissues using a monoclonal antibody against the NP (see Materials and Methods). Magnification of the pictures is 200× or 400× as indicated.

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Low viral titers in C57BL/6 mice precluded the ability to detect viral antigens in most tissues while, in contrast, infection of Rag2−/− mice yielded significant expression of viral antigen in tissues (Table 1B). Of interest was the location of viral antigen at day 5 to the marginal zone of the white pulp of the spleen. This anatomical location of recombinant LCMV containing the LASV Gp is identical to that observed earlier (Sevilla et al., 2004; Smelt et al., 2001) which was dependent on the Gp of LCMV Cl13. Thus, early during infection LASV Gp mimics LCMV Cl13 Gp on entry in the marginal zone and into white pulp DCs (Table 1B, lower left panel) which is dependent on expression of αDG on DCs (Oldstone and Campbell, 2011; Sevilla et al., 2000; Sevilla et al., 2004; Smelt et al., 2001; Sullivan et al., 2011).

Dendritic cells (DCs) are permissive to rLCMV Cl/LASV Gp infection

We next infected groups of four Rag2−/− mice with rLCMV Cl13/LASV Gp and focused on rLCMV Cl13/LASV Gp infection of DCs. Five and eight days post-infection, both plasmacytoid DCs obtained from the bone marrow and conventional DCs from the spleen were assayed for expression of viral antigen using a monoclonal antibody that detected NP viral antigen and flow cytometry. As shown in Figure 2, 29% to 32% of DCs expressed rLCMV Cl13/LASV Gp virus during acute infection at day 8. Replication of infectious rLCMV Cl13/LASV Gp from isolated DCs was observed by infectious center assay (data not shown).

Fig. 2.

Fig. 2

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Infection of dendritic cells in vivo by rLCMV Cl13/LASV Gp and by Cl13. Bone marrow or spleen was harvested from C57BL/6 mice at day 5 and day 8 post infection and analyzed for viral antigen in (A) pDCs (CD11blo, CD11c+, B220+) and (B) cDCs (CD11b+, CD11c+) by intracellular staining for NP antigen and flow cytometry. As the figure shows, rLCMV Cl13/LASV Gp and Cl13 viruses can be found in equivalent amounts in both DC subsets on day 5, and increase significantly by day 8 post-infection. Percentages shows are mean ± standard error of the mean for three independent experiments, with two representative experiments shown.

rLCMV Cl13/LASV Gp generates virus-specific MHC-restricted CTL in adult immunocompetent mice

We next measured the generation and efficiency of virus-specific MHC-restricted CTLs. To determine MHC-restriction, we infected syngeneic H-2b MC57 and allogeneic Balb Cl7 H-2d targets with an MOI of 1 of rLCMV Cl13/LASV Gp. We first insured that these target cells were infected by and express rLCMV Cl13/LASV Gp by immunofluorescent staining for the viral NP (Figure 3A). We then performed a 51Cr release assay using splenic lymphocytes from C57BL/6 mice primed 7 days earlier with 1 × 105 PFU of rLCMV Cl13/LASV Gp i.p. The results showed, in a dose response manner using different ratios of effector to target cells, that a robust virus-specific MHC-restricted CTL response was generated (Figure 3B). We then asked what epitopes of rLCMV/LASV Gp were being recognized by virus-specific MHC-restricted CTL. As shown in Figure 3C, CTLs generated recognize the H-2b immunodominant NP aa 396-404 peptide coating the H-2b target but not coating the H-2d target cells, as expected. The LCMV NP epitope is expressed in both rLCMV/LASV Gp and LCMV and thus CTLs generated against either of these two viruses recognize NP protein aa 396-404. The Gp amino acid sequence between LASV Josiah and LCMV ARM is only 61.5% homologous and LCMV ARM and LCMV Cl13 share the exact same Gp CTL epitopes. As shown in Figure 3C, the complete LCMV Gp expressed using a vaccinia virus (VV) promoter (Whitton et al., 1988b) is not recognized by CTL generated against rLCMV/LASV Gp indicating that LASV Gp epitope(s) lie in the 38.5% non-homologous amino acid sequence between Gp of LASV and that of LCMV Cl13. As expected CTLs generated against either LCMV Cl13/LASV Gp or VV expressing whole LCMV NP recognized the NP epitope 396-404.

Fig. 3.

Fig. 3

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(A) Mouse fibroblasts (MC57, H-2b and Balb cl7, H-2d) were infected with an MOI of 0.1 of rLCMV Cl13/LASV Gp, and were fixed and stained for intracellular viral NP at the indicated times post-infection. Virus grows equally well in both H-2b and H-2d fibroblasts rendering them excellent target cells. (B) 51Cr release assays done with varying effector:target ratios for both rLCMV Cl13/LASV Gp and LCMV Clone 13. H-2b targets infected with recombinant virus were killed equally by splenocytes from either recombinant or Clone-13 infected mice, while Cl13 infected targets were killed more efficiently by Cl13 memory splenocytes than recombinant infected memory cells. There was no killing of allogeneic H-2d targets, as expected. (C) 51Cr release assays with targets infected with either Cl13, recombinant virus, or vaccinia viruses (VV) encoding LCMV NP, Gp, or the immunodominant NP396-404 epitope. Splenocytes infected with recombinant virus killed targets displaying all the immunogens except the LCMV Gp, while Cl13 splenocytes were able to kill all the targets, as expected. LASV Gp and Cl13 Gp have 61% homology at the amino acid level, indicating that CTL to LASV Gp likely recognized LASV Gp epitope(s) differing from Cl13 Gp epitopes. P0 refers to primary inoculation. (D) Expression of pro-inflammatory cytokines IFN-γ and TNF-α in CD8+ T-cells infected with either Cl13 or rLCMV Cl13/LASV Gp. Intracellular cytokine staining of splenocytes showed similar levels of both IFN-γ and TNF-α expression between Cl13 and rLCMV Cl13/LASV Gp. Percentages are shown as mean ± standard error of the mean for two independent experiments.

The last experiment in this series used tetramers to NP 396-404 to determine the efficiency of the generated CTLs in terms of the number and amount of inflammatory cytokines released. As shown in Figure 3D, CTLs released interferon-γ, IL-2, and TNF equivalently when generated against either rLCMV/LASV Gp or the parental LCMV.

rLCMV Cl13/LASV Gp injected into newborn mice results into a life-long persistent virus carrier model

Inoculation of newborn mice on H-2b (C57BL/6) or H-2d (Balb) backgrounds with 1 × 104 to 1 × 105 PFU of recombinant virus intracranially resulted in a lethality of over 85%, while in contrast inoculation of parental LCMV caused a lethality of <20%. We then inoculated H-2q (SWR/J) mice with rLCMV Cl13/LASV Gp and had a survival rate of >75%. Such H-2q virus-inoculated mice carried virus throughout their life. When sacrificed at 42 days PI, analysis of tissues indicated viral titers of 105 to 106.5 in spleen, kidney, brain, lung, heart, liver, and sera. Tissues harvested from these organs, fixed in 4% paraformaldehyde, sectioned and stained with monoclonal antibody to viral NP antigen revealed widespread evidence of viral replication in these tissues similar to pictures in Table 1B and titers in Table 1A.

We have previously shown that adoptive transfer of MHC-restricted immune memory cells harvested from the spleen were able to clear virus from life-long virus carriers whose infection was initiated similarly at birth with either LCMV ARM or LCMV Cl13 (Berger et al., 2000; Oldstone et al., 1986; Tishon et al., 1993). In the last series of experiments, we tested whether immune memory cells generated to rLCMV Cl13/LASV Gp that had demonstrated robust virus-specific MHC-restricted CTL activity (Figure 3) were able to clear rLCMV Cl13/LASV Gp virus carrier mice. For these experiments we infected groups of 9 newborn C57BL/6 mice each with 1 × 103 PFU of either LCMV Cl13 or rLCMV Cl13/LASV Gp i.c. The lower dose of virus delivered i.c. resulted in increased survival of the mice following infection. All mice developed a life-long persistent infection as judged by presence of 104.5 to 105 logs of virus in their sera. When such persistently infected mice were 30 days old, they each received 2 × 107 immune memory splenocytes i.v. harvested at day 42 post-infection from C57BL/6 mice immunized with rLCMV Cl13/LASV Gp. Unexpectedly, as shown in Figure 4, rLCMV Cl13/LASV Gp persistently infected mice failed to clear virus when receiving adoptive transferred CTLs generated against rLCMV Cl13/LASV. Virus persisted as long as 160 days post-transfer (last time-point analyzed). In contrast, similar CTLs generated from rLCMV Cl13/LASV successfully purged virus from all LCMV Cl13 virus carriers by 30 days post-transfer of CTL.

Fig. 4.

Fig. 4

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Clearance of viral infection by adoptive transfer of memory splenocytes. Donor B6 mice were infected with 2 × 105 PFU i.p. of either Clone 13 or rLCMV Cl13/LASV Gp virus, and splenocytes were harvested at day 42 post-infection. After red blood cell lysis, splenocytes were counted and 2 × 107 total memory splenocytes were injected i.p. into B6 mice infected at birth with either Clone 13 or rLCMV Cl13/LASV Gp. Memory splenocytes were matched to the respective viruses; i.e. Clone 13 memory was injected into Clone 13 carrier mice. Serum viral titers were measured in the mice at the indicated timepoints by plaque assay. As the graph shows, titers of Clone 13 virus are significantly reduced (by 2.5 logs) in the mice by memory splenocytes at day 14 and totally purged by day 60 post-transfer (black circles). In contrast, the recombinant rLCMV Cl13/LASV Gp remains in the mouse at high titer beyond day 160 post-transfer (white circles).

DISCUSSION

Here we report the generation of a recombinant LCMV expressing the Gp of LASV and make four observations. First, using the LCMV backbone, the optimal recombinant virus contains the polymerase gene of LCMV Cl13 which enhances its replication in DCs (Figure 1D), and this rLCMV Cl13/LASV Gp virus shows tropism and replication for DCs in vivo (Figure 2). Second, the rLCMV Cl13/LASV Gp replicates in mouse and generates a robust CTL response that clears the viral infection (Figure 3, Table 1). Third, although the CTLs generated against rLCMV Cl13/LASV Gp are competent in cleansing virus from an acute infection, they are ineffectual upon adoptive transfer in terminating a persistent infection initiated by rLCMV Cl13/LASV Gp (Figure 4). Lastly the rLCMV Cl13/LASV Gp can be used at BSL2 for both in vitro and in vivo studies of the role of LASV Gp in LASV pathogenesis.

Generating genetic reassortant viruses between LCMV ARM and LCMV Cl13 indicated the advantage of Cl13 polymerase over that of ARM polymerase for enhanced replication in DCs (Figure 1A, B). This led us to construct a recombinant virus bearing LASV Gp that included the LCMV Cl13 polymerase. The rLCMV Cl13/LASV Gp demonstrated a dependency of LASV Gp for binding to α-DG and for cell entry as soluble α-DG was able to blunt infection by a LCMV Cl13/LASV Gp virus in a dose-dependent fashion. Interesting was the observation of a difference in viral replication between LASV Gp recombinant using Cl13 backbone over ARM backbone with infection of DCs but not with infection of several cell lines including Vero, A549 and BHK cells.

Injection of rLCMV Cl13/LASV Gp into C57BL/6 mice led to an initial systemic infection that was curtailed by a robust H-2b MHC-restricted CTL response. Analysis of this response indicated CTLs were generated against LCMV Cl13 NP as well as LASV Gp amino acid sequences. The Gp sequences recognized by CTLs generated to rLCMV Cl13/LASV Gp are distinct from LCMV H-2b restricted immunodominant Gp aa 33-41, Gp aa 276-286 and subdominant Gp aa 92-101 and Gp aa 118-125, as the full length LCMV Gp containing these sequences was not recognized by CTL made to the recombinant virus bearing LASV Gp when expressed on target cells using vaccinia virus vectors. The LASV epitopes restricted by MHC H-2b recognized by CTLs generated to LASV Gp must reside in the 38.5% of the LASV Gp sequence that is different from the LCMV Cl13 or ARM Gp. These differences do not comprise any predominant region of the Gp, but are scattered throughout the sequence. As such, it is difficult to determine which specific sequences within the LASV Gp elicit the CTL responses seen here. Earlier genetic studies of LCMV genes showed that CTL responses to LCMV were preferentially generated to the Gp and NP with no detectable response to the Z protein and only one epitope was noted in the polymerase protein (Riviere et al., 1986; Whitton et al., 1988a; Whitton et al., 1988b). As such, LASV Gp H-2b restricted CTL are likely responsible for the additional level of CTL response to the recombinant LASV noted over the observed CTL response to NP 396-404 alone. Further, the observed generation of a vigorous antiviral immune response to LASV Gp suggests that the rLCMV Cl13/LASV Gp might be evaluated as a potential vaccine candidate against LASV infection.

Persistent lifelong infection can be induced in mice infected either at birth or in utero with rLCMV Cl13/LASV Gp. This mimics the naturally occurring LASV persistent infection of most rodents in LASV endemic areas. Unexpectedly, adoptive transfer of MHC-restricted immune memory CTLs generated against rLCMV Cl13/LASV Gp was unable to clear a persistent infection induced by the recombinant virus. However, MHC-restricted immune memory CTLs generated to LCMV Cl13 were able to clear the persistent infection due to rLCMV Cl13/LASV Gp. These results suggest something unique in the environment of mice infected with the LASV recombinant virus that prevents clearance of persistent viral infection. Previous studies utilizing LCMV in which the immunodominant H-2b restricted CTL epitope for NP 396-404 was knocked out (Lewicki et al., 1995a; Lewicki et al., 1995b) or in which NP 396-404 specific CTLs were adoptively transferred into mice indicated the NP 396-404 CTLs were themselves sufficient to terminate the persistent virus infections. Our analysis of the functional activity of CTLs generated against NP 396-404 using either rLCMV Cl13/LASV Gp or CL13 showed that these CTLs were equally capable of lysing infected targets and expressing the pro-inflammatory cytokines TNFα and IFN-γ (Figure 3 C,D). Thus, it is unclear why the CTLs generated by recombinant virus infection were ineffectual in in vivo clearance. Perhaps either an environmental difference in the persistently infected mouse, akin to our recent report in the TLR7−/− mouse upon LCMV infection (Walsh et al., 2012), or a requirement for other immune effector cells differentially elicited by the two viruses is involved.

Lastly, the infection of pDCs or cDCs by LASV Gp-bearing recombinant virus in vitro and in vivo should allow a biochemical analysis of virus-DC interactions mediated by the LASV Gp. This is important as DCs are one of the primary cells infected by wild type LASV and LASV infection of human DCs has been demonstrated to alter DC function (Baize et al., 2004; Baize et al., 2006; Mahanty et al., 2003; Pannetier et al., 2011). Incorporation of the LASV Gp into the LCMV Clone 13 backbone enabled us to replicate the tropism of LASV for DCs without having to incorporate any of the other LASV transcriptional proteins, mitigating the biosafety concerns inherent in LASV work. It is important to note that the overall immune responses against wild-type LASV in infected individuals will likely vary from the immune responses against rLCMV Cl13/LASV Gp shown here owing to the differences between the other LASV and LCMV proteins (NP, Z, and polymerase). However, the rLCMV Cl13/LASV Gp virus will serve as a useful tool for uncovering mechanisms of cell binding, entry, infection and immune subversion initiated by the Gp of LASV in DCs in a BSL-2 environment rather than the highly restrictive BSL-4 environment required by work with the wild-type LASV.

MATERIALS AND METHODS

Cell lines and reagents

BHK-21 cells were grown in Dulbecco’s Modified Essential Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 100 μg/mL penicillin-streptomycin (Gibco), 2mM L-glutamine (Gibco), 0.24% glucose (w/v), and 7% tryptose phosphate broth solution (Sigma). VeroE6 cells were propagated in Eagle’s minimal essential medium (MEM) supplemented with 7% FBS, 100 μg/mL penicillin-streptomycin, and 2mM L-glutamine. MC57 and BALB/c17 cells were propagated in DMEM supplemented with 10% FBS, 100 μg/mL penicillin-streptomycin, and 2mM L-glutamine. Dendritic cell lines P4H1 and 9M were a gift from Bishnu Nayak (Genomics Institute of the Novartis Foundation) and were propagated in RPMI 1640 medium supplemented with 10% FBS, 100 μg/mL penicillin-streptomycin, 2 mM L-glutamine, 50 μM β-mercaptoethanol, 1% (v/v) sodium pyruvate, and 20 μg/mL GM-CSF (Peprotech).

Mouse strains

C57BL/6 and Balb/c mice were obtained from the Rodent Breeding Colony at The Scripps Research Institute. SWR/J mice were purchased from The Jackson Laboratory (Bar Harbor, Maine).

Virus strains and stocks

All recombinant viruses used were generated using reverse genetics technology as described previously (Emonet et al., 2011; Emonet et al., 2009). To produce rLCMV Cl13/LASV Gp virus, cloning of the LASV Josiah strain Gp was performed as described previously for rLCMV ARM/LASV Gp (Rojek et al., 2008). Viral stocks were generated by passage in BHK-21 cells by infecting at low multiplicity of infection (MOI = 0.01) for 48 hours and collecting virus-containing culture supernatant followed by cell debris centrifugation at 2000× g for 10 min. at 4 °C. Viral titers were assessed by immune focus assay (Urata et al., 2010) or by plaque assay (Sullivan et al., 2011). Recombinant vaccinia viruses expressing the LCMV nucleoprotein or glycoprotein used in chromium release assays were generated as described previously (Whitton et al., 1988b).

Virus infections

For cell lines, viral infections were done by adding virus to the cell culture at a multiplicity of infection (MOI) of 0.1 for 1 hour. Virus was then removed, cells washed, and fresh media was added to the culture. Viral titers were measured in the supernatants at various times post-infection. For in vivo studies, mice were injected either intraperitoneally (i.p.) with a low dose (non-immunosuppressive) or intravenously (i.v.) with a high dose (immunosuppressive) of virus as indicated. Viral titers were assessed in sera and tissues at the indicated timepoints post-infection as above. For intracranial (i.c.) infection, virus was diluted to the required dose in 30 μL final volume, and 30 μL was administered to anesthetized mice by injection at the midline of the skull using a stepper syringe with a 30 gauge needle and an attached depth stopper.

Generation of persistently infected mouse and adoptive immune memory T-cell transfer experiments

To generate persistently infected mice, newborn mice (< 24 hours old) were injected with 1 × 103 PFU of virus intracranially (i.c.) in 30 μL. Mice and virus titers were weaned at 28 days of age and virus titers checked in blood obtained from retroorbital bleeding to ensure mice carried infectious virus. Such virus-carrying mice received immune splenocytes harvested from adult C57BL/6J mice infected with 2 × 105 PFU of virus i.p. Virus carrier mice received 2 × 107 memory splenocytes i.p. in 200 μL PBS. Serum was isolated from these mice by retroorbital bleeding at the indicated days post transfer and assayed for viral titer.

51Chromium release assay

Erythrocyte-depleted single lymphocyte suspensions of cells obtained from splenocytes of C57BL/6J (H-2b) mice infected with either LCMV Cl13 or rLCMV Cl13/LASV Gp were used in 51Cr release assays. Briefly, MHC-haplotype matched MC57 (H-2b) and unmatched Balb/cl7 (H-2d) cell lines were either infected with virus (MOI = 1) or coated with peptide corresponding to immunodominant LCMV epitopes for 24 hours as indicated in the experiments. Cells were subsequently labeled with 51Cr for 1 hour at 37 °C/5% CO2 and excess unincorporated label was washed away. The labeled cells were incubated with effector lymphocytes taken from the infected mice at day 7 post-infection at indicated effector:target ratios in triplicate for 5 hours, and cell lysis was quantified by assaying the supernatant for 51Cr using a gamma counter as reported (Sullivan et al., 2011; Walsh et al., 2012; Welch et al., 2012). Values are expressed as percent lysis, with an untreated control serving as no lysis and an NP-40 incubated control serving as 100% lysis. For assays using recombinant vaccinia viruses expressing the LCMV NP or GP, target cells were infected with an MOI of 3.

Flow cytometric analysis

All flow cytometric analysis was performed on an LSR-II flow cytometer (BD Biosciences). Briefly, cells were stained for surface markers in PBS/1% FBS/0.1% sodium azide buffer prior to any intracellular staining. Intracellular staining was done using the BD Cytofix/Cytoperm kit to simultaneously fix and permeabilize the cells. Cells were incubated with Fc block prior to antibody staining to reduce background staining from Fc receptors. Antibody staining and FACS analysis on DCs were performed as previously reported (Sullivan et al., 2011; Walsh et al., 2012; Welch et al., 2012).

Immunofluorescence staining of tissues and cells

Immunofluorescence staining of tissues was performed by collecting tissues and snap-freezing them on dry ice in embedding medium (OCT, Tissue-Tek). Tissues were cut into 6-μm sections using a Leica cryomicrotome and fixed with 4% paraformaldehyde. After fixation, sections were blocked with 10% fetal bovine serum in PBS, and then stained overnight at 4°C with a 1:200 dilution of mouse monoclonal antibody against the LCMV NP. Tissues were washed and incubated for 1 hour at 4°C with a 1:200 solution of Alexa Fluor 488-conjugated anti-mouse IgG antibody (Life Technologies), and were then washed and mounted using VectaShield mounting medium (Vector Laboratories). Sections were imaged using a Zeiss Axiovert S100 immunofluorescence microscope fitted with an Axiocam color digital camera and 20× and 40× objectives.

For immunofluorescence staining of cells, cells were washed to remove tissue culture medium and fixed in 4% paraformaldehyde for 10 minutes at room temperature. The cells were then blocked with PBS/1% FBS/0.1% saponin buffer, and incubated overnight at 4°C with a 1:200 solution of anti-LCMV NP antibody. Cells were washed three times with PBS/1% FBS/0.1% saponin and incubated for 1 hour at 4°C with a 1:200 solution of Alexa Fluor 488-coupled anti-mouse IgG antibody. Cells were washed again and imaged using a 10× objective on the Zeiss Axiovert immunofluorescence microscope.

HIGHLIGHTS.

  • A recombinant LCMV expressing the Gp of Lassa virus is generated.

  • Lassa Gp/LCMV chimeric virus replicates in mouse dendritic cells in vivo.

  • Chimeric virus generates robust CTL responses that clear acute infection.

  • Immune memory against chimeric virus fails to clear a persistently infected host.

  • Chimeric virus should help uncover mechanisms of Lassa infection in BSL-2 studies.

ACKNOWLEDGMENTS

This is publication number 21976 from the Department of Immunology and Microbial Science at The Scripps Research Institute in La Jolla, CA. We thank Dr. Sebastien Emonet and Ms. Nhi Ngo for technical advice and assistance with rescuing the recombinant virus, and Dr. Juan Carlos de la Torre for his donation of the LCMV genome plasmids. This work was supported by U.S. Public Health Grants (AI055540, AI009484) to M.B.A.O. and an NIH Kirschstein National Research Service Award (AI072994) to A.M.L.

Footnotes

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