The major determinant of attenuation in mice of the Candid1 vaccine for Argentine hemorrhagic fever is located in the G2 glycoprotein transmembrane domain

Abstract

Candid1, a live-attenuated Junin virus vaccine strain, was developed during the early 1980s to control Argentine hemorrhagic fever, a severe and frequently fatal human disease. Six amino acid substitutions were found to be unique to this vaccine strain, and their role in virulence attenuation in mice was analyzed using a series of recombinant viruses. Our results indicate that Candid1 is attenuated in mice through a single amino acid substitution in the transmembrane domain of the G2 glycoprotein. This work provides insight into the molecular mechanisms of attenuation of the only arenavirus vaccine currently available.

Fig. 1.

(A) Development of the Candid1 vaccine. This live-attenuated vaccine virus was originated from human clinical material and was passaged twice in guinea pigs (GP2) and 44 times in mouse (MB44), followed by clonal selection and stock amplification through 19 passages in fetal rhesus monkey lung cells (FRhL19). Virulence in mice and guinea pigs is indicated on the right side of the figure. (B) Sequence comparisons of Candid1 and its parental strains. For XJ-derived viruses (XJ13, XJ17, XJ34, XJ39, and XJ48), total RNA was purified from mouse brain lysates and used for reverse transcriptase PCR (RT-PCR) amplifications and sequencing. For Candid1, total RNA extracted from an original vial of the vaccine was used as input material. Amino acid positions (aa.pos.) correspond to each individual viral protein: GPC, N, and L. No mutations were found in Z or in nontranslated regions. XJ48 was derived from the Candid1 parental strain XJ44 and had a sequence identical to that of XJ39. GenBank accession numbers are listed for each strain.

Fig. 2.

(A) Schematic of plasmids used to generate fully infectious viruses. Recombinant viruses were generated in BSRT7/5 cells and passaged twice in VeroE6 cells as previously described (3, 4). The indicated combinations of plasmids were used to rescue the parental strains, S and L reassortant viruses, and viruses carrying gene replacements. The sequences of the parental strains correspond to GenBank accession no. HQ126700, HQ126701, FJ805379, and JN200801. (B) Virulence testing in the mouse model. Recombinant viruses were assayed by intracranially injecting 500 TCID₅₀/mouse into 14-day-old mice. Infected mice (10 animals/group) were examined for clinical signs and mortality for 28 days postinfection. (C) hTfR1 receptor usage by JUNV Candid1. 293T cells were incubated for 30 min with 200 nM nonspecific mouse IgG2a (Southern Biotech) or anti-hTfR1 (clone M-A712; BD Biosciences) antibody. After treatment, cells were infected with 0.5 PFU/cell of JUNV Candid1. Twenty hours later, the cell monolayer was fixed and analyzed by immunofluorescence assay (IFA) using an anti-JUNV rabbit serum and an anti-rabbit Alexa Fluor 488 secondary antibody (Invitrogen).

Fig. 3.

(A) Schematic of plasmids used to generate fully infectious viruses. Recombinant viruses were generated as described before (3, 4). The indicated combinations of plasmids were used to rescue the parental strains, viruses carrying G1 and G2 replacements, and viruses carrying single-amino-acid changes. (B) Virulence assays in the mouse model were performed as explained in the legend for Fig. 2.

Fig. 4.

(A) Sequence alignment of the critical G2 regions of JUNV and LASV. Amino acid changes were introduced into the TMD or the CT region of LASV GPC to mimic the equivalent location within Candid1. (B) Western blot analysis of the glycoprotein content of HIV-based pseudotypes. (C) 293T cells were infected with HIV-LASV pseudotypes, and luciferase activity was measured in the supernatant collected 3 days postinfection. RLU, relative luciferase units; error bars indicate standard deviations. (D) Sequence comparison of recombinant LASVs. Amino acid positions correspond to GPC. No mutations were found in rLASV or rLASV_CT virus during passage in VeroE6 cells. The rLASV_TMD mutant virus acquired spontaneous mutations after the second passage in VeroE6 cells. (E) Growth kinetics of recombinant LASV. Growth curves were generated by infecting VeroE6 cells with 0.1 PFU/cell of recombinant LASVs. Supernatants were collected at 24-h intervals, and virus titers were determined with a TCID₅₀ assay. rLASV, wild type; rLASV_CT, CT mutant; rLASV_TMD, TMD mutant; rLASV_TMD(rev), TMD revertant.