The substantial numbers of JUNV and LASV cases reported each year among humans, along with the high case-fatality rate associated with these pathogens, render them a substantial public health threat in many areas of the world. Of additional, significant concern is the potential intentional release of these agents in acts of bioterrorism. Safe and effective vaccines to combat these health and security threats are critically needed.
In this report, we describe the successful use of reverse genetics to generate a genetically homogeneous recombinant JUNV Candid1 vaccine and attenuated JUNV/LASV chimeric viruses that may serve as potential bivalent vaccine candidates for these two diverse arenaviruses. An important advantage of this strategy is that it relies on the preexisting JUNV Candid1 vaccine strain, which was proved to be safe and effective (4
). Since its approval for human use in Argentina in the 1980s, more than 200,000 persons throughout central Argentina have been vaccinated to date (18
). However, despite this record, concerns remain regarding the well-documented genetic heterogeneity of the current Candid1 vaccine, which likely resulted from the vaccine's extensive in vivo
passage history (13
) (Fig. ). The ability to efficiently generate pure stocks of recombinant JUNV Candid1 virus directly from cDNA clones provides the foundation for improvements in this vaccine and studies to precisely determine the contributions made by various mutations to the attenuated phenotype of the vaccine. In addition, this platform allowed us to investigate glycoprotein interactions involved in arenavirus maturation, propagation, and virulence by generation of JUNV Candid1 and LASV chimeric VRPs and infectious viruses.
The mechanisms and regulation of GPC maturation of arenaviruses have been the focus of much investigation over the past 20 years (9
). With the ultimate goal of developing vaccine candidates for JUNV and LASV in mind, we employed a general strategy of envelope exchange using precisely defined chimeric GPC domain constructs between these two significant New World and Old World arenaviruses. We generated a full complement of chimeric JUNV and LASV GPC cDNA plasmids containing various combinations of the SSP, G1/G2 ectodomains, and G2 TM-CT domains either together or individually. Using these constructs, we successfully demonstrated that efficient virus replication and propagation require the presence of homologous virus species-specific SSP and the G2 C terminus.
Envelope protein exchange among arenaviruses and other negative-sense RNA viruses to generate multivalent recombinant live attenuated arenavirus vaccines has been previously demonstrated (6
). In addition, several studies have shown that the SSP of the Arenaviridae
is required for GPC maturation and eventual production of infectious virions (16
). Furthermore, studies involving JUNV have demonstrated that interactions between the SSP and G2-cytoplasmic domains are critical to mask endogenous dibasic ER retention signals located within G2 (2
), thus allowing for the further processing and eventual incorporation of mature SSP, G1, and G2 into nascent virion particles.
Schlie et al. (44
) have recently described the relationship of LASV CT to GPC maturation. That report suggested that LASV CT was required for correct GPC oligomerization, which seemed to be a prerequisite for SKI-1/S1P processing. The correct interaction between SSP and CT apparently triggers a change in conformation of the GPC into a trimer that is selectively cleaved; thus, processing of SKI-1 appears to serve a quality control step where only trimers are incorporated in virions. Without proper oligomerization and interactions of the SSP and G2, an uncleaved form of GPC can be found on the cellular plasma membrane; however, this immature glycoprotein precursor is unable to form infectious virus particles (2
During the initial stages of our research, we anticipated some challenges for the successful generation of a recombinant JUNV Candid1 expressing LASV GPC, based on two relevant reports: first, the well-defined interaction of JUNV SSP with the G2 cytoplasmic tail (48
), and second, the suggested interaction of LCMV GPC with the Z protein (11
). In light of this information, we set out to define functionally relevant GPC domains that could be exchanged and still generate a virus that replicates efficiently while maintaining the avirulent phenotype of the parental Candid1 in animal models (and presumably in humans). Surprisingly, our studies demonstrated that recombinant VRPs containing the full-length LASV GPC [JCd1-Las(ABCD)] and nonhomologous G1/G2 ectodomains [JCd1-Las(BC)] can indeed be generated, and they were found to replicate at levels similar to those for wt JCd1 GPC VRPs in cell culture. Nonhomologous SSP and G2-cytoplamic domain exchanges led to significantly depressed production of infectious VRPs that was undetectable when measured with GFP fluorescence and only slightly above background when measured by luciferase activity.
Even more surprisingly, reverse genetics generated infectious chimeric viruses containing exchanges of the full-length LASV GPC [rJCd1V-LASV(ABCD)] or the extravirion ectodomains of G1/G2 [rJCd1V-LASV(BC)] were found to have near-equivalent cell culture growth characteristics compared with authentic JUNV. These results clearly demonstrated that the sequence mismatch of (i) the LASV G1/G2 glycoproteins with JUNV N and Z proteins or (ii) the JUNV SSP and G2 cytoplasmic domain with the G1/G2 ectodomains, did not result in significant defects in virus replication or production of infectious particles. Earlier studies with LCMV and LASV have provided evidence of interaction of the Z protein with the GP and suggested that this interaction is of importance for arenavirus maturation (11
). Our results would be consistent with the sequence divergence between Old World (LASV) and New World (JUNV) arenavirus glycoproteins and Z proteins being insufficient to prevent this interaction. In addition, the earlier study (11
) had demonstrated direct interaction of the SSP with Z. Interestingly, we found no difference in chimeric virus replication whether the Z protein was matched with the SSP protein [rJCd1-LASV(BC)] or not matched [rJCd1-LASV(ABCD)], suggesting a lack of a strict sequence requirement for such interactions to occur.
The main constraint we found for the generation of chimeric JUNV Candid1 infectious viruses where the JUNV glycoprotein has been replaced with the LASV glycoprotein was the requirement to match the virus species origin of the SSP and G2 cytoplasmic tail domains. Interestingly, our Western blot analysis and VRP experiments suggested different mechanisms to explain why these chimeras did not sustain production of infectious viruses. The defect where the mismatch was the G2 cytoplasmic tail [pC-JCd1-Las(ABC)] appeared to be more severe, in that in addition to lack of generation of infectious virus, the GPC was not processed efficiently by the S1P/SKI-I protease, and it presented an abnormal distribution at the cell surface (Fig. ). In contrast, the Las(ABC) chimeric protein processing was almost completely abrogated, while the Las(BCD) protein did not show any defect in cleavage or in cell surface expression.
In a very recent report, Schlie et al. (44
) have shown that the exchange of the LASV GPC CT with that of the closely related LCMV also blocked SKI-1/S1P cleavage. The exact reason for the block remains unclear, but current data suggest that the chimeric GPC was not in the correct ternary or quaternary conformation to allow SKI-1/S1P cleavage. Strikingly, introduction of matching JUNV SSP and JUNV TM/CT into the LASV GPC promoted its cleavage and the production of infectious virus, suggesting that interaction between the matched pair of SSP and TM/CT is solely responsible for the rescue of the defect in their original unproductive GPC chimeras (the equivalent of ABC and BCD proteins discussed here).
In contrast to the defect where the mismatch was the G2 cytoplasmic tail [pC-JCd1-Las(ABC)], the defect with the mismatch in the SSP [JCd1-Las(BCD)] produced a GPC that was efficiently cleaved by S1P/SKI-I and presented a normal cell surface distribution but, surprisingly, failed to support robust generation of VRPs or infectious chimeric virus. These results further suggest that chimeric GPC folds and traffics to the cellular compartment where S1P/SKI-1 cleavage occurs but is then defective in a subsequent step prior to maturation of infectious virus particles. Further studies on the relationship of GPC maturation and budding of virus particles will be necessary to elucidate the precise basis of this additional defect. Taken together, these results indicate that homologous SSP and G2-cytoplasmic tail domains, regardless of their origin (JUNV or LASV), are essential for the efficient generation of an infectious virus.
Using a lethal model of arenavirus infection in suckling/weanling mice, we established a clear age-dependent phenotype between JUNV and LASV. In this model, suckling mice aged 2 days old at the time of infection were highly susceptible to lethal infection by JUNV. However, LASV was found to be phenotypically avirulent in these mice, even at dosages exceeding 1,000 times the JUNV LD50 (Fig. ). This virulence phenotype was reversed when older mice were used. In 14-day-old suckling mice, JUNV Candid1 was found to be completely attenuated, whereas LASV was shown to have near 100% lethality (Fig. ). Strikingly, a Candid1-like pattern of age-dependent lethality was also observed with both envelope exchange JUNV-LASV chimeras, i.e., JCd1-LASV(ABCD) and rJCd1-LASV(BC) (Fig. ). Even though these chimeras did not cause lethality in 14-day-old suckling mice, all ani- mals infected with either rJCd1-LASV(ABCD) or rJCd1- LASV(BC) developed robust anti-NP antibody responses by 28 days postinfection, equivalent to those observed in rJUNV-Candid1 survivor animals.
The underlying pathophysiological mechanisms of this age-related phenomenon remain unknown. However, this finding may be related to the well-described observation of LASV dependence on host immune dysregulation for lethality in humans and animal models, rather than direct virus-induced cell toxicity (20
). Neonatal mice are known to preferentially develop type II immune responses for several days postbirth without the maturation and robust action of type I responses (reviewed in references 1
, and 24
) that are thought to be responsible for the observed immunopathology of fatal Lassa fever. Further study of these JUNV-LASV chimeras may shed light on the complex interplay of the neonatal murine immune system and the molecular mechanisms by which arenaviruses establish persistent infection in rodent hosts. Regardless of the physiological mechanism, it is clear that the LASV GPC alone is not sufficient to alter the attenuated phenotype of the JUNV-Candid1 vaccine strain in 14-day-old weanling mice.
Given how divergent JUNV and LASV are, it was surprising that the infectious JUNV-Candid1-LASV chimeric viruses generated here retained infectivity in tissue culture and in vivo
, comparable to that of authentic JUNV-Candid1 virus. This is particularly so in light of the well-demonstrated major impact that minor sequence changes can have on viral virulence. For example, the virulent JUNV-XJ13 and the vaccine JUNV-Candid1 strains differ in only nine and six amino acids (nine in the L polymerase, five in the glycoproteins, and one in NP), but clearly exhibit very different virulence phenotypes in a variety of animal models (e.g., mice, guinea pigs, and rhesus macaques) (5
The attenuated phenotype of these JUNV-LASV chimeras may allow for their use in the rational development of bivalent JUNV and LASV live attenuated vaccine platforms that employ an envelope protein exchange strategy. While i.c. inoculation of mice has proved to be a convenient and useful model for study of arenaviruses, the precise relevance of such studies to viral pathogenesis and virus attenuation in humans is questionable. Further characterization of these constructs in a vaccination-challenge animal model (such as guinea pigs or nonhuman primates) will be essential and required before more definitive statements on the immunogenicity and efficacy of these JUNV-LASV chimeras as vaccine candidates can be made. The results presented here represent a first step toward the production of rationally designed, safe, and effective live attenuated bivalent arenavirus vaccines that can be used to combat these significant health threats.