Josiah (clade IV) and Sauerwald (also known as LASV-803213; clade II) [7], 2 LASV strains previously reported to be lethal in strain 13/N guinea pigs, were compared in cell culture experiments to 4 additional strains: N-322 (clade II), N-213 and N-383 (both clade III), and Togo (clade VI). We hypothesized that strains were more likely to cause disease in guinea pigs if relative (1) viral growth was high; (2) interferon induction was low; and (3) apoptosis or other types of cell death was absent. We found that, in general, higher titers (and higher infectivity) were observed in A549 cells than in GPC-16 cells (Figure 1A). Strain Josiah grew most robustly in both cell lines; strain Sauerwald reached lowest maximal titers in both cell lines (9- and 10-fold less than Josiah in A549 and GPC-16 cells, respectively). Growth kinetics of other strains were between those of Josiah and Sauerwald. No strain induced IFN-β transcription, apoptosis, or cell death by other pathways in A549 cells (Figure 1B and 1C).

Having found no major differences between LASV strains in vitro, we chose to determine disease course of the 4 additional strains in guinea pigs, along with Sauerwald. Groups of 5 guinea pigs were inoculated SC with 1 of 5 LASV strains representing 3 clades, and followed up to 42 dpi (Figure 1 and Supplementary Table 4). Of the 20 animals, 2 met euthanasia criteria (1 from Togo at 24 dpi and 1 from N-231 at 21 dpi). A period of weight loss was seen in either a subset (N-322) or all animals (remaining strains) within each experimental group. The most substantial mean weight loss was seen in animals infected with N-231 and N-383. With the exception of the N-322 group, all groups had characteristic elevated temperatures that peaked 9–15 dpi (11 of 15 [73.3%] at 12 dpi). IgG against Josiah nucleoprotein (NP) was detected by immunofluorescence in plasma of all animals except 1 N-231–infected animal euthanized due to disease at 21 dpi (Supplementary Table 4). Blood and tissues collected at euthanasia were evaluated by qRT-PCR. In the N-322 group, no viral RNA was detected at study end point (42 dpi). In all other experimental groups, viral RNA was detected in at least 1 tissue in all animals: in all spleens, and in a variety of other tissues depending on the individual (Figure 1 and Supplementary Table 4). As expected, highest viral loads and tissue RNA distribution were seen in animals that met euthanasia criteria; in these animals, RNA was found in all blood and 15 of 16 (93.8%) tissue samples.

While only a small number of animals exhibited fatal disease, clinical signs were pronounced enough to use this model for therapeutic screening. Two of the 5 strains characterized above, representing virus clades responsible for several recent Lassa fever outbreaks in Nigeria, were selected to evaluate VRP vaccine efficacy against nonhomologous virus infection. Animals were vaccinated SC with purified VRPs based on LASV Josiah (clade IV) in HBSS, or mock-vaccinated with HBSS vehicle control. Twenty-eight days later, VRP and HBSS control groups were challenged SC either with Sauerwald (clade II) or N-231 (clade III). Prechallenge, modest Josiah NP seroreactivity was detected in approximately half of the VRP-vaccinated animals, as previously described (Supplementary Table 5) [4, 5]. Vaccination provided complete protection against clinical signs following heterologous challenge, and no viral RNA was detected in any blood or tissue samples at study completion. In contrast, mock-vaccinated groups exhibited signs of moderate to severe disease (Figure 2). Timing and degree of weight loss and temperature elevation mirrored findings in model development studies, and 1 animal in the N-231 group again succumbed to fatal disease. In addition, viral RNA was detectable in at least 1 tissue from all mock-vaccinated survivors at study completion (42 dpi; Supplementary Table 5). At study end point, all animals were seroreactive to Josiah NP, and reactivity was increased in vaccinated animals with detectable responses prechallenge.

Heterologous protection is particularly important in LASV vaccine development due to substantial virus diversity; overall strain variation in partial NP gene sequences is up to 27% at the nucleotide level and 15% at the amino acid level [8]. Most therapeutic screening to date uses LASV strain Josiah, but inbred guinea pigs have been infected with other strains [9], including in our work characterizing LBR-USA-2015 (NJ2015; clade IV) [10]. In general, studies have focused on a few viral strains and reported binary outcome data (lethal/nonlethal) with limited descriptions of other clinical manifestations. We aimed to develop new infection models that recapitulate the spectrum of human disease, including mild to moderate clinical presentations, and address the need for models representing the genetic diversity of LASV.

All candidate LASV strains we examined were able to replicate in human cells without inducing apoptosis or expression of antiviral IFN-β. Interestingly, growth analysis showed that strain N-322, the least pathogenic in this study, grew as robustly as strain Josiah in A549 cells and GPC-16 cells. Conversely, strain Sauerwald resulted in reproducible clinical signs in the model despite reduced viral titers produced in cell culture. Mouse models have linked T cells to LASV pathology [11, 12], and strain-dependent disease in guinea pigs may be influenced by mechanisms not readily measured in cell culture.

Guinea pigs infected with the strains herein exhibited measurable alterations in clinical parameters useful for assessing cross-protective vaccine efficacy. Potential for cross-protective vaccine development is supported by reports of T-cell and antibody cross-reactivity induced by a ChAdOx1-Lassa-GPC vaccine [13], and cross-protection with a recombinant vesicular stomatitis virus expressing strain Josiah glycoproteins [14]. In addition, an ML29 reassortant virus that carries NP and glycoprotein (GP) dominant antigens of LASV and the L polymerase and Z matrix protein of the nonpathogenic genetically related Mopeia virus protected against Sauerwald challenge [7]. VRPs are promising vaccine candidates due to their safety and efficacy; they replicate in the first cells they encounter but do not spread, thus retaining a high safety profile, and show uniform protection against disease [3]. Previously, we reported LASV VRP safety, singledose prophylactic efficacy [3], and postexposure prevention of fatal outcomes [4]. Here we also report single-dose heterologous protection against geographically and genetically diverse LASV strains. Emerging strains do not differ greatly from previously detected strains within a region [15]; genetic variation correlates with geographic distance rather than time [8]. The viruses evaluated represent distinct endemic geographic regions and thus serve as proxy for current and future strains. These data further support the promise of cross-protective vaccine development for LASV and demonstrate utility of the VRP vaccine platform against both endemic and emerging virus strains.