General Molecular Strategy for Development of Arenavirus Live-Attenuated Vaccines

Abstract

Hemorrhagic fever arenaviruses (HFA) pose important public health problems in regions where they are endemic. Thus, Lassa virus (LASV) infects several hundred thousand individuals yearly in West Africa, causing a large number of Lassa fever cases associated with high morbidity and mortality. Concerns about human-pathogenic arenaviruses are exacerbated because of the lack of FDA-licensed arenavirus vaccines and because current antiarenaviral therapy is limited to an off-label use of ribavirin that is only partially effective. The Mopeia virus (MOPV)/LASV reassortant (ML29) is a LASV candidate live-attenuated vaccine (LAV) that has shown promising results in animal models. Nevertheless, the mechanism of ML29 attenuation remains unknown, which raises concerns about the phenotypic stability of ML29 in response to additional mutations. Development of LAVs based on well-defined molecular mechanisms of attenuation will represent a major step in combatting HFA. We used the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) to develop a general molecular strategy for arenavirus attenuation. Our approach involved replacement of the noncoding intergenic region (IGR) of the L genome segment with the IGR of the S genome segment to generate a recombinant LCMV, rLCMV(IGR/S-S), that was highly attenuated in vivo but induced protection against a lethal challenge with wild-type LCMV. Attenuation of rLCMV(IGR/S-S) was associated with a stable reorganization of the control of viral gene expression. This strategy can facilitate the rapid development of LAVs with the antigenic composition of the parental HFA and a mechanism of attenuation that minimizes concerns about increased virulence that could be caused by genetic changes in the LAV. IMPORTANCEHemorrhagic fever arenaviruses (HFA) cause high morbidity and mortality, and pose important public health problems in the regions where they are endemic. Implementation of live-attenuated vaccines (LAV) will represent a major step in combatting HFA. Here we have used the prototypic arenavirus LCMV to document a general molecular strategy for arenavirus attenuation that can facilitate the rapid development of safe and effective, as well as stable, LAV to combat HFA. Hemorrhagic fever arenaviruses (HFA) pose important public health problems in the regions where they are endemic (1-3). Thus, Lassa virus (LASV) infects several hundred thousand individuals yearly in West Africa, resulting in a high number of Lassa fever (LF) cases associated with high morbidity and significant mortality (4). Notably, increased traveling has led to the importation of LF cases into metropolitan areas around the globe where the virus is not endemic (5, 6). Moreover, regions of LASV endemicity are expanding (4), and the association of the recently identified arenavirus Lujo with a recent outbreak of HF in southern Africa (7,8) has raised concerns about the emergence of novel HFA. In addition, evidence indicates that the worldwide-distributed prototypic arenavirus lymphocytic choriomeningitis vi...

The High Degree of Sequence Plasticity of the Arenavirus Noncoding Intergenic Region (IGR) Enables the Use of a Nonviral Universal Synthetic IGR To Attenuate Arenaviruses

“…The IGR is necessary for the production of infectious VLPs (26), but the efficiency of production of infectious VLPs appears to be mainly determined by 3= and 5= untranslated regions (UTRs) within each genome segment (21). The sequence of Slas is very different from that of the LCMV S-IGR, but the production of LCMV infectious VLPs was minimally affected when Slas was substituted for the LCMV S-IGR.…”

“…We have previously shown that rLCMV(IGR/ S-S) has significantly impaired growth in IFN-I-competent A549 cells and induces a high level of reporter gene expression driven by the ISRE promoter (21). Compared to the fitness of the rLCMV WT, all rLCMVs containing an altered L-IGR exhibited significantly reduced fitness in A549 cells (Fig.…”

“…We used an A549 cell line (A549/ISRE-Fluc) that expresses firefly luciferase (Fluc) via interferon (IFN)-stimulated response elements (ISREs) (21). We seeded A549/ISRE-Fluc cells (2.5 ϫ 10 4 cells/96-well plate) cultured in DMEM containing 10% FBS, 2 mM L-glutamine, 100 mg/ml streptomycin, 100 U/ml penicillin, and 10 g/ml puromycin at 37°C in 5% CO 2 at 20 h prior to infection with rLCMVs (MOI ϭ 0.1) or treatment for 17 h with universal alpha IFN (IFN-␣) at the concentrations indicated in the corresponding figures.…”

“…The IGR plays a critical role in the control of viral gene expression at the posttranscriptional level (21). Replacement of the WT S-IGR with Ssyn could therefore affect viral gene expressions in infected cells, which may result in lower levels of NP expression and, subsequently, compromise the ability to inhibit type I interferon (IFN-I) induction (31).…”

“…The L-segment RNA encodes the viral RNA-dependent RNA polymerase (L) and the matrix RING finger protein Z (19,20). We have documented that the sequences of the IGRs of the L and S segments (L-IGR and S-IGR, respectively) have distinct functional roles in virus multiplication, and we harnessed that knowledge to implement a general molecular strategy for arenavirus attenuation (21). Our approach involved replacement of the IGR of the L genome segment by the IGR of the S genome segment to generate a recombinant LCMV (rLCMV), rLCM-V(IGR/S-S), that was highly attenuated in vivo but able to induce protection against a lethal challenge with wild-type (WT) LCMV.…”