Genetic Variation In Vitro and In Vivo of an Attenuated Lassa Vaccine Candidate

Abstract

The attenuated Lassa vaccine candidate ML29 is a laboratory-produced reassortant between Lassa and Mopeia viruses, two Old World arenaviruses that differ by 40% in nucleic acid sequence. In our previous studies, ML29 elicited sterilizing immunity against Lassa virus challenge in guinea pigs and marmosets and virus-specific cell-mediated immunity in both simian immunodeficiency virus (SIV)-infected and uninfected rhesus macaques. Here, we show that ML29 is stable after 12 passages in vitro without losing its plaque morphology or its attenuated phenotype in suckling mice. Additionally, we used deep sequencing to characterize the viral population comprising the original stock of ML29, the stock of ML29 after 12 passages in Vero cells, and the ML29 isolates obtained from vaccinated animals. Twenty-seven isolates bore approximately 77 mutations that exceeded 20% of the single-nucleotide polymorphism (SNP) changes at any single locus. Of these 77 mutations, 5 appeared to be host specific, for example, appearing in mice but not in primates. None of these mutations were reversions of ML29 to the sequences of the parental Lassa and Mopeia viruses. The host-specific mutations indicate viral adaptations to virus-host interactions, and such interactions make reasonable targets for antiviral approaches. Variants capable of chronic infection did not emerge from any of the primate infections, even in immune-deficient animals, indicating that the ML29 reassortant is reasonably stable in vivo. In conclusion, the preclinical studies of ML29 as a Lassa virus vaccine candidate have been advanced, showing high levels of protection in nonhuman primates and acceptable stability both in vitro and in vivo.

Arenavirus Variations Due to Host-Specific Adaptation

“…The small plaque phenotype and the ability to protect mice from lethal intracerebral inoculation was retained through all 12 passages, as was the consensus sequence of the open reading frames. However, deep sequence analysis revealed the gradual increase in single nucleotide polymorphisms (SNPs) with in vitro passage, though none of the SNPs exceeded 25% frequency within the viral population [107]. This agrees with the idea that one viral preparation can maintain a swarm of quasi-species with a variety of sequences; yet retain its phenotypic characteristics [102].…”

“…ML29 virus isolated during these first few weeks was subjected to pyro-sequencing and proved to have a lower number of SNPs (less variation) than virus obtained from in vitro passage, and surprisingly, some of the variations were host-specific (Table 2). The virus-host adaptations also seemed to accumulate with time in the primate host [107]. Protein prediction analysis showed that those mutations, at the amino acid level, induced structural changes in GPC and NP (Figure 4 and Figure 5).…”

Vaccine platforms to control Lassa fever

“…There is a compelling link between mutation rate, viral population dynamics and pathogenesis [119]. To address this issue, stability of ML29 was assessed after 12 passages (P1-P12) in FDA-approved Vero cells required for vaccine manufacturing [120]. There were no significant differences among the estimated mutation frequencies for parental ML29 P1, for ML29 P12, and for ML29 isolates from vaccinated animals supporting the high genetic stability of ML29 during multiplication in cultured cells and in vivo.…”

Arenavirus Genome Rearrangement for the Development of Live Attenuated Vaccines

“…In this regard, the Mopeia virus (MOPV)/ LASV ML29 reassortant is a LASV live attenuated candidate vaccine that has been shown to provide post-exposure protection and to be safe and immunogenic in guinea pigs and small nonhuman primates (33)(34)(35). However, the mechanisms of ML29 attenuation remain poorly understood and additional mutations in ML29 or reassortments between ML29 and circulating virulent strains of LASV could result in viruses with enhanced virulence (36). Here we have explored the distinctive organization of the arenavirus genome as an unique opportunity for the rational design of live attenuated vaccines based on rearrangement of the viral genes to disrupt normal viral gene expression and, therefore, viral fitness.…”