Restoration of virulence in the attenuated Candid#1 vaccine virus requires reversion at both positions 168 and 427 in the envelope glycoprotein GPC

Abstract

Live-attenuated virus vaccines provide long-lived protection against viral disease but carry inherent risks of residual pathogenicity and genetic reversion. The live-attenuated Candid#1 vaccine was developed to protect Argentines against lethal infection by the Argentine hemorrhagic fever arenavirus, Junín virus. Despite its safety and efficacy in Phase III clinical study, the vaccine is not licensed in the US, in part due to concerns regarding the genetic stability of attenuation. Previous studies had identified a single F427I mutation in the transmembrane domain of the Candid#1 envelope glycoprotein GPC as the key determinant of attenuation, as well as the propensity of this mutation to revert upon passage in cell culture and neonatal mice. To ascertain the consequences of this reversion event, we introduced the I427F mutation into recombinant Candid#1 (I427F rCan) and investigated the effects in two validated small-animal models: in mice expressing the essential virus receptor (human transferrin receptor 1; huTfR1) and in the conventional guinea pig model. We report that I427F rCan displays only modest virulence in huTfR1 mice and appears attenuated in guinea pigs. Reversion at another attenuating locus in Candid#1 GPC (T168A) was also examined, and a similar pattern was observed. By contrast, virus bearing both revertant mutations (A168T+I427F rCan) approached the lethal virulence of the pathogenic Romero strain in huTfR1 mice. Virulence was less extreme in guinea pigs. Our findings suggest that genetic stabilization at both positions is required to minimize the likelihood of reversion to virulence in a second-generation Candid#1 vaccine.IMPORTANCELive-attenuated virus vaccines, such as measles/mumps/rubella and oral poliovirus, provide robust protection against disease but carry with them the risk of genetic reversion to the virulent form. Here, we analyze the genetics of reversion in the live-attenuated Candid#1 vaccine that is used to protect against Argentine hemorrhagic fever, an often-lethal disease caused by the Junín arenavirus. In two validated small-animal models, we find that restoration of virulence in recombinant Candid#1 viruses requires back-mutation at two positions specific to the Candid#1 envelope glycoprotein GPC, at positions 168 and 427. Viruses bearing only a single change showed only modest virulence. We discuss strategies to genetically harden Candid#1 GPC against these two reversion events in order to develop a safer second-generation Candid#1 vaccine virus.

Keywords: Candid#1; Junín virus; arenavirus; envelope glycoprotein; live-attenuated; molecular genetics; reversion; vaccine; virulence determinants.

Fig 1

Mutations in GPC associated with the derivation of Candid#1. (Top) Passage history and selected intermediate isolates. GP, passages in guinea pigs; MB, passages in mouse brain; FRhL, passage in fetal rhesus lung epithelium cell culture. Virulence (v) or attenuation (a) in the respective species is shown below. (Bottom) Amino-acid substitutions in Candid#1 GPC. Positions 168 in GP1 and 427 in GP2 are highlighted. This figure is adapted from Albariño et al. (26) and reproduced from York and Nunberg (29).

Fig 2

Growth and biochemical characterization of rCan revertant viruses in Vero cell culture. (A) Growth kinetics following low multiplicity of infection (MOI = 0.01) with parental rCan, A168T rCan, I427F rCan, or A168T+I427F rCan. Cell culture supernatants were harvested daily for 5 days, and infectious virus titers were determined as focus-forming units (FFUs)/milliliter. (B) Pixel areas of a representative sampling of larger foci (n = 20/rCan revertant) visualized 3 days after infection. (C) rCan and rCan revertant viruses were harvested 5 days after infection, concentrated by centrifugation, and subjected to immunoblot analysis using either the NP-directed monoclonal antibody (mAb) AG12 (left panels) or an equal mix of GP2-directed mAbs G3 and G5 (right panels). These mAbs have previously been validated (31), and full-length NP, the GP1GP2 precursor, and the mature GP2 are labeled. Typical NP degradation products are indicated as pep1 and pep2. NP forms larger than full length are ubiquitinated (unpublished). Bands were quantitated using IQTL software (GE), and chemifluorescent light units are tabulated in Table S1. The results presented are representative of three independent studies. * indicates P = 0.05–0.01, ** indicates P = 0.01–0.001, *** indicates P = 0.001–0.0001, and **** indicates P < 0.0001 compared to rCan, as determined using two-way (growth curves; panel A) or one-way (FFU area; panel B) analysis of variance (ANOVA) with Dunnett’s multiple comparisons test.

Fig 3

Pathogenesis of rCan revertant viruses in huTfR1 mice. Three-week-old mice (n  =  8–11/virus infection group) were challenged i.p. with 2 × 10⁴ CCID₅₀ of rCan, A168T rCan, I427F rCan, A168T+I427F rCan, or rRom, and (A) survival, (B) day of death, and (C) weight change were monitored for 35 days. Sham-infected controls n  =  4 were included for comparison. The day of death data represent the group means and standard deviations. The percent changes in weight represent the group means and SEM for surviving animals relative to their starting weights on the day of virus challenge. * indicates P = 0.05–0.01, ** indicates P = 0.01–0.001, and **** indicates P < 0.0001 compared to mice infected with rRom, and c indicates P = 0.001–0.0001 compared to mice infected with rCan, as determined using the Mantel–Cox log-rank test (A) or one-way ANOVA with Dunnett’s multiple comparisons test (B).

Fig 4

Pathogenesis of rCan revertant viruses in guinea pigs. Guinea pigs (n  =  6/virus infection group) were challenged i.p. with 2 × 10⁴ CCID₅₀ of rCan, A168T rCan, I427F rCan, A168T+I427F rCan, or rRom, and (A) survival, (B) weight change, and (C) body temperature were monitored for 35 days. A sham-infected control animal was included for comparison. The body temperature data represent the group means and SEM. The percent changes in weights represent the group means and SEM for surviving animals relative to their starting weights on the day of virus challenge. *** indicates P = 0.001–0.0001 compared to guinea pigs infected with rRom, as determined using the Mantel–Cox log-rank test. a indicates P < 0.05 compared to rCan, as determined using two-way ANOVA (mixed model with Geisser–Greenhouse correction) and Tukey’s multiple comparisons test.

Fig 5

Clinical disease in guinea pigs infected with rCan revertant viruses. Clinical score was recorded daily for each animal based on the presence or absence of weight loss exceeding 5% of the guinea pig’s peak weight, 1°C increase or decrease from the animal’s baseline temperature, lethargy, ruffled fur, tremors, or paralysis. The clinical score represents the sum of the disease signs scored as 0 (not present) or 1 (present). Disease severity is depicted by increased intensity of red. Gray indicates the end of monitoring/scoring due to euthanasia/death.