An attenuated Machupo virus with a disrupted L-segment intergenic region protects guinea pigs against lethal Guanarito virus infection

Abstract

Machupo virus (MACV) is a New World (NW) arenavirus and causative agent of Bolivian hemorrhagic fever (HF). Here, we identified a variant of MACV strain Carvallo termed Car⁹¹ that was attenuated in guinea pigs. Infection of guinea pigs with an earlier passage of Carvallo, termed Car⁶⁸, resulted in a lethal disease with a 63% mortality rate. Sequencing analysis revealed that compared to Car⁶⁸, Car⁹¹ had a 35 nucleotide (nt) deletion and a point mutation within the L-segment intergenic region (IGR), and three silent changes in the polymerase gene that did not impact amino acid coding. No changes were found on the S-segment. Because it was apathogenic, we determined if Car⁹¹ could protect guinea pigs against Guanarito virus (GTOV), a distantly related NW arenavirus. While naïve animals succumbed to GTOV infection, 88% of the Car⁹¹-exposed guinea pigs were protected. These findings indicate that attenuated MACV vaccines can provide heterologous protection against NW arenaviruses. The disruption in the L-segment IGR, including a single point mutant and 35 nt partial deletion, were the only major variance detected between virulent and avirulent isolates, implicating its role in attenuation. Overall, our data support the development of live-attenuated arenaviruses as broadly protective pan-arenavirus vaccines.

Figure 1

Infection of Hartley guinea pigs with MACV strain Car⁶⁸, Car⁹¹ and Chicava. (A) Guinea pigs were challenged with 1,000 pfu of Car⁹¹, Car⁶⁸ and Chic by the i.p. route. Survival was monitored for 30 days post-infection. Asterisks denote statistical significance. (B) Percent weight loss for individual guinea pigs in the indicated groups was plotted based on day 0 starting weight. Animals succumbing to infection are shown in red. (C) The mean group temperatures were plotted. The normal temperature range is shaded in grey. To aid visualization, data for Car⁹¹-infected animals is depicted in orange.

Figure 2

Evaluation of serum from MACV infected guinea pigs. (A) Viremia was determined on Vero cell monolayers using guinea pig serum taken from survivors on day 30 (black) or when animals were euthanized (red). The solid line represents the GMT values. (B) Antibody binding ELISA titers were determined by incubating sera from Car⁹¹-infected animals with PsVs pseudotyped with GPC from Strain Carvallo. Antiserum samples were serially diluted prior to incubation. The dashed line denotes the limit of detection. The solid line represents the GMT value. (C) Titer of neutralizing antibody in Car⁹¹-infected animals was determined and PRNT50 and PRNT80 titers were plotted. The dashed line denotes the limit of detection. The solid line represents the GMT values.

Figure 3

Sequence of MACV strain Car⁹¹ and Car⁶⁸. (A) Predicted L-segment amino acid changes between Car⁹¹ and Car⁶⁸. (B) Alignment of the Car⁹¹ and Car⁶⁸ IGR. The underlined region denotes the region deleted in Car⁹¹. Also underlined is the single nucleotide change at position 399. (C) Predicted hairpin tertiary structure of the Car⁹¹ and Car⁶⁸ L-segment IGR.

Figure 4

In vitro characterization of Car⁹¹, Car⁶⁸ and Chic. (A) Particle-to-pfu ratios for each of the indicated MACV strains were determined using a ViroCyt system. Four virus preparations for each virus were tested in triplicate. The geometric means are indicated by the solid line. (B) The indicated cell types were infected with Car⁹¹ (circles), Car⁶⁸ (squares) or Chic (triangles) at an MOI of 0.1 and replication was quantitated at 24, 48 and 72 hpi by plaque assay. All samples were titered in duplicate and the mean + /− SD were graphed.

Figure 5

Protective efficacy of MACV strain Car⁹¹ against lethal GTOV challenge in guinea pigs. (A) Survival plot of naïve (red squares) and Car⁹¹-vaccinated (black circles) guinea pigs infected by the i.p. route with 2,000 pfu GTOV. Survival was plotted for 30 day post-infection. (B) Percent loss from starting weight was plotted for each group as described above. (C) Temperature was monitored as in Fig. 3.

Figure 6

Binding and neutralizing antibody responses in guinea pigs infected with GTOV. (A) Antibody binding titers were determined by coating 96-well plates with the indicated PsVs and incubating them with serially diluted antiserum samples from before (circles/PRE) or after (squares/POST) challenge with GTOV. The dashed line denotes the limit of detection. The red circle denotes the single animal (Animal #4) that succumbed to infection. The blue symbols denote the same animal before and after GTOV challenge. Note that titers against MACV prior to GTOV challenge are also depicted in Fig. 4B. (B) PRNT80 titers against MACV (Car⁶⁸) prior to and after challenge with GTOV were determined as above. Note that PRNT80 titers prior to GTOV challenge are also depicted in Fig. 4C. (C) PRNT50 titers against JUNV, MACV and GTOV were determined as in Fig. 2. Titers were determined as described above. The dashed line indicates the limit of detection. For all panels, asterisks denote statistical significance.