Rational design of a safe recombinant Candid#1 vaccine

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Arenaviruses are endemic in rodent populations and can be transmitted to humans to cause severe life- threatening hemorrhagic fevers. Relevant US agencies (DHHS, DHS and DoD) and the WHO recognize these viruses as high priority pathogens that pose a serious threat to public health and national security. The live- attenuated Candid#1 strain of Junín virus (JUNV) is currently used in Argentina to protect against Argentine hemorrhagic fever, but this virus carries the distinct liability that attenuation is solely dependent on a single phenylalanine-to-isoleucine substitution at position 427 (F427I) in the GP2 fusion subunit of the JUNV envelope glycoprotein (GPC). Indeed, reversion at the attenuating position occurs readily in cell culture and in laboratory animals. The current proposal seeks to capitalize on our understanding of GPC structure and function to design recombinant Candid#1 (rCan) viruses that stably maintain attenuation without compromising protective efficacy. We have discovered an epistatic interaction between the attenuating F427I mutation in GP2 and a lysine-to-serine mutation at position 33 (K33S) in the stable signal peptide (SSP) subunit of GPC that provides an evolutionary barrier against reversion to the pathognomonic F427. Pilot studies indicate that K33S rCan is indeed attenuated in guinea pigs and capable of eliciting protective immunity against lethal challenge with JUNV. We hypothesize further that safety in a K33S rCan vaccine can be additionally enhanced by incorporating well-characterized and genetically stable GPC deletions. By characterizing rCan viruses that embody these strategies, we aim to enhance safety in a second-generation rCan vaccine. Towards this goal, we will pursue the following specific aims: Aim 1. Determine the degree of attenuation and genetic stability of K33S rCan in mice. We will utilize well-established mouse models to determine the degree of attenuation in rCan variants and confirm the genetic stability of the attenuating F427I mutation. Aim 2. Assess the balance of attenuation, immunogenicity and protective immunity of K33S rCan in the guinea pig model of lethal JUNV infection. Guinea pigs serve as the gold-standard model for assessing Candid#1 attenuation and protective efficacy. We will expand upon our pilot findings to optimize the balance between attenuation and protective efficacy. We will evaluate production of virus-neutralizing antibodies, an accepted surrogate of protection, and the ability of the vaccine to elicit virus-specific CD8+ T cells. Aim 3. Design and characterize rCan variants bearing redundant and genetically stable mutations that promote attenuation. We have identified two deletions in GPC that support rCan infectivity. We will characterize rCan variants bearing these deletions to integrate additional layers of attenuation. Taken together, our efforts will establish an optimal balance of attenuation, genetic stability and efficacy in a second-generation rCan vaccine, and elucidate the molecular basis for attenuation and the immunologic correlates of protection. These strategies may also be applicable towards the development of an urgently needed live-attenuated Lassa virus vaccine.