A Vaccine Platform against Arenaviruses Based on a Recombinant Hyperattenuated Mopeia Virus Expressing Heterologous Glycoproteins

Abstract

Several Old World and New World arenaviruses are responsible for severe endemic and epidemic hemorrhagic fevers, whereas other members of the Arenaviridae family are nonpathogenic. To date, no approved vaccines, antivirals, or specific treatments are available, except for Junín virus. However, protection of nonhuman primates against Lassa fever virus (LASV) is possible through the inoculation of the closely related but nonpathogenic Mopeia virus (MOPV) before challenge with LASV. We reasoned that this virus, modified by using reverse genetics, would represent the basis for the generation of a vaccine platform against LASV and other pathogenic arenaviruses. After showing evidence of exoribonuclease (ExoN) activity in NP of MOPV, we found that this activity was essential for multiplication in antigen-presenting cells. The introduction of multiple mutations in the ExoN site of MOPV NP generated a hyperattenuated strain (MOPV_ExoN6b) that is (i) genetically stable over passages, (ii) has increased immunogenic properties compared to those of MOPV, and (iii) still promotes a strong type I interferon (IFN) response. MOPV_ExoN6b was further modified to harbor the envelope glycoproteins of heterologous pathogenic arenaviruses, such as LASV or Lujo, Machupo, Guanarito, Chapare, or Sabia virus in order to broaden specific antigenicity while preserving the hyperattenuated characteristics of the parental strain. Our MOPV-based vaccine candidate for LASV, MOPEVAC_LASV, was used in a one-shot immunization assay in nonhuman primates and fully protected them from a lethal challenge with LASV. Thus, our hyperattenuated strain of MOPV constitutes a promising new live-attenuated vaccine platform to immunize against several, if not all, pathogenic arenaviruses.IMPORTANCE Arenaviruses are emerging pathogens transmitted to humans by rodents and responsible for endemic and epidemic hemorrhagic fevers of global concern. Nonspecific symptoms associated with the onset of infection make these viruses difficult to distinguish from other endemic pathogens. Moreover, the unavailability of rapid diagnosis in the field delays the identification of the virus and early care for treatment and favors spreading. The vaccination of exposed populations would be of great help to decrease morbidity and human-to-human transmission. Using reverse genetics, we generated a vaccine platform for pathogenic arenaviruses based on a modified and hyperattenuated strain of the nonpathogenic Mopeia virus and showed that the Lassa virus candidate fully protected nonhuman primates from a lethal challenge. These results showed that a rationally designed recombinant MOPV-based vaccine is safe, immunogenic, and efficacious in nonhuman primates.

Keywords: Lassa fever; arenavirus; innate immunity; live-vector vaccines; viral hemorrhagic fevers.

FIG 1

MOPV NP has ExoN activity required for efficient multiplication. (A) In vitro degradation of a 5′-γ-³²P-labeled dsRNA probe by the recombinant C-terminal domain of WT or D390A/G393A mutant MOPV NP. Purified ExoN domains and substrate probes were incubated with 5 mM MnCl₂ for up to 30 min. The WT ExoN domain was incubated with EDTA (10 mM) as a positive control for the abrogation of ExoN activity. T, time. (B) Inhibition of SeV-induced IRF3 activation by ExoN activity of MOPV NP. HEK293T cells were transfected with plasmids encoding WT or ExoN mutant NP of MOPV or LASV along with a plasmid with FF-Luc under the control of an IRF3 response element (p55CIB-Luc) for 24 h prior to infection with SeV. At 24 h postinfection, cells were lysed, and Luc activities were measured by using the Dual-Glo luciferase assay (Promega). FF-Luc activity was normalized to that of renilla Luc. Both levels of luciferase activity are expressed as arbitrary units, and the results are expressed as the means ± standard errors of the means of data from three independent experiments performed in triplicate. ***, P < 0.001. The vertical lines indicate where the original blots were spliced together. NI, noninfected. (C) Reverse genetics for MOPV. Transfection of mouse Pol-I-driven expression plasmids for the S (with the WT or D390A/G393A mutant NP ORF) and WT L segments with plasmids encoding WT NP and L-polymerase (Lpol) ORFs of MOPV allowed the rescue of rec-MOPV_WT and rec-MOPV_ExoN. The replication kinetics of passage 2 recombinant viruses were compared to those of nat-MOPV_WT in Vero E6 cells infected at an MOI of 0.001. Supernatants were collected and titrated. Results are expressed as FFU per milliliter. Plaque phenotypes for the three viruses harvested 72 h after infection are shown.

FIG 2

ExoN mutant MOPV is a potent activator of MP and the type I IFN response. MP were infected with nat-MOPV_WT, rec-MOPV_WT, or rec-MOPV_ExoN for 1 h; washed twice; and cultured in complete MP medium. (A) The supernatants of MP infected at an MOI of 0.1 were collected after infection, and every 24 h for 4 days, and titrated. (B) MP infected at an MOI of 1 were detached 48 h after infection; saturated with human IgG; surface stained with antibodies to CD40, CD80, and CD86 before final fixation in PBS–1% PFA; and analyzed by flow cytometry (BD Biosciences). **, P < 0.01; ***, P < 0.001. (C) RT-qPCR analysis of IFN-α1, -α2, and -β; TNF-α; and CXCL10 mRNA levels in mock-infected MP and MP infected at an MOI of 1, 24 h after infection. The expression levels of all genes were normalized to GAPDH mRNA levels. The final results are expressed as fold induction relative to GAPDH. The results represent the means ± standard errors of the means of data gathered from four different donors.

FIG 3

Reinforcement of the mutated ExoN site of MOPV NP. (A) Schematic representation of the DEDDH domain of MOPV NP and nomenclature of the introduced mutations. The capacities of WT and mutated NP to inhibit the IRF3-dependent expression of FF-Luc activity in HEK293T cells infected by SeV were measured and normalized as described in the legend of Fig. 1B. The results obtained are shown as the means ± standard errors of the means of data from three independent experiments performed in triplicate. (B) Reverse genetics for the NP mutant viruses MOPV_Exo3, MOPV_Exo5, and MOPV_ExoN6b and comparison of growth kinetics with those of MOPV_WT and MOPV_ExoN in Vero E6 cells. Infected-cell supernatants were collected and titrated as described in the legend of Fig. 1D. (C to F) MP were infected with MOPV_WT, MOPV_ExoN, and MOPV_ExoN6b for 1 h; washed twice; and cultured in complete MP medium. (C and D) The supernatants of MP infected at an MOI of 0.1 were collected and titrated (C), and NP RNA was quantified from total RNA extracted from infected cells by using an in-house RT-qPCR assay (D). (E) MP infected at an MOI of 1 were stained 48 h after infection for cell surface expression of the CD40, CD80, and CD86 activation markers before analysis by flow cytometry. (F) RT-qPCR quantification of IFN-α1, -α2, and -β mRNA levels in mock-infected MP and MP infected at an MOI of 1, 24 h after infection, normalized to GAPDH mRNA levels, as described in the legend of Fig. 2C. The results represent the means ± standard errors of the means of data collected from three different donors. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 4

GPC of LASV and MOPV are not involved in the type I IFN response. (A) Schematic representation of the GPC-deleted version of the LASV or MOPV S segments used to generate the chimeric viruses MOPV_{GPC LASV} and LASV_{GPC MOPV}. (B) Growth kinetics of the recombinant viruses MOPV_WT, MOPV_{GPC LASV}, LASV_WT, and LASV_{GPC MOPV} in Vero E6 cells infected at an MOI of 0.01. Supernatants of infected cells were titrated as described above. (C) RT-qPCR quantification of IFN-α1, -α2, and -β mRNA levels in mock-infected MP and MP infected at an MOI of 1, 24 h after infection, normalized to GAPDH mRNA levels. Results represent the means ± standard errors of the means of data collected from three different donors.

FIG 5

Generation of a vaccine strain platform for arenaviruses based on attenuated recombinant MOPV. (A) Reverse-genetics strategy for the generation of MOPV_ExoN6b expressing GPC of the pathogenic arenaviruses LASV, LUJV, MACV, GUAV, CHAV, and SABV. Passage 2 viruses were used to infect Vero E6 cells at an MOI of 0.01, and supernatants were collected and titrated. A representative example of the plaque phenotype of passage 2 viruses is shown. (B) Multiple passages of MOPV_WT, MOPV_ExoN6b, MOPV_{ExoN6b GPC LASV}, MOPV_{ExoN6b GPC LUJV}, and MOPV_{ExoN6b GPC MACV} in Vero E6 cells. De novo stocks of all five viruses, from passage 1 to passage 10, were used to infect cells for 4 days at an MOI of 0.01.

FIG 6

The MOPEVAC_LAS strain is an effective activator of MP and the type I IFN response. MP were infected with MOPV_WT, MOPV_ExoN6b, or MOPV_{ExoN6b GPC LASV} for 1 h, washed twice, and cultured in complete MP medium. (A) The supernatants of MP infected at an MOI of 0.1 were collected after infection for 4 days and titrated as described in Materials and Methods. (B) Total RNA was extracted from the cells, and NP RNA levels were quantified by using an in-house RT-qPCR assay. (C) Cell surface expression of CD40, CD80, and CD86 in mock-infected MP and MP infected at an MOI of 1, 48 h after infection. (D) Total RNA in MP infected at an MOI of 1 were collected 24 h after infection and DNase treated, and mRNA levels of IFN-α1, -α2, and -β were quantified after oligo(dT) cDNA-driven RT and primer/probe PCR quantification (Applied Biosystems). The expression levels of all genes were normalized to GAPDH mRNA levels. The final results are expressed as fold induction relative to GAPDH. (E) Quantification of IFN-α2 levels in supernatants of infected MP (MOI of 0.1) harvested 24 h postinfection by an ELISA. For panels C to E, results represent the means ± standard errors of the means of data collected from three different donors. **, P < 0.01; ***, P < 0.001.