The single-nucleotide changes that mutated the amino acid were selected since they arose experimentally during mutagen treatment26 and also to avoid stochastic effects of that could arise from changing RNA secondary structures by mutating more nucleotides. is responsible for a global disease burden of millions of cases each year with autochthonous transmission in over 100 countries and territories worldwide. There is currently no approved treatment or vaccine for CHIKV. One live-attenuated vaccine (LAV) developed by the United States Army progressed to Phase II human clinical trials but was withdrawn when 8% of volunteers developed joint pain associated with vaccination. Attenuation of the Armys CHIKV LAV strain 181 clone 25 (CHIKV-181/25) relies on two mutations in the envelope S3I-201 (NSC 74859) 2 (E2) glycoprotein responsible for cell binding and entry, making it particularly prone to reversion, a common concern for replication-competent vaccines. High error rates associated with RNA computer virus replication have posed a challenge for LAV development where stable incorporation of attenuating elements is necessary for establishing safety in pre-clinical models. Herein, we incorporate two replicase mutations into CHIKV-181/25 which modulate CHIKV replication fidelity combined with additional attenuating features that cannot be eliminated by point mutation. The mutations were stably incorporated in the LAV and did not increase virulence in mice. Two fidelity-variant CHIKV LAVs generated neutralizing antibodies and were protective from CHIKV disease in adult mice. Unexpectedly, our fidelity-variant candidates were more mutable than CHIKV-181/25 and exhibited restricted replication in mice and mosquitoes, a possible consequence of hypermutation. Our data demonstrate safety and efficacy but highlight a further need to evaluate fidelity-altering phenotypes before use as a LAV given the potential for virulent reversion. larval (f) cells at starting MOI?=?1. mosquito (C6/36) cells. An approximately threefold boost in infection efficiency was observed in BHK cells 12?h post inoculation in CHIKV-181/25-P2.P4, but titers were no different 24 or 48?h post-infection (Fig. ?(Fig.1e).1e). As expected, both CHIKV-181/25-P2.P4.IRES and CHIKV-181/25-P2.P4.E3/E1 attenuated derivative strains infected cells at slower rates and achieved lower titers at 48?h (determined to be the peak in preliminary experiments) than their parent strain in BHK-21 cells (mosquito infectivity One complication to the development of a replication-competent vaccine for a vector-borne disease is the potential for vaccine transmission via vectors. To ensure that fidelity-variant CHIKV LAVs are not more transmissible than WT CHIKV or CHIKV-181/25 in the S3I-201 (NSC 74859) primary mosquito vector, mosquitoes colonized from Los Angeles, California, USA, were presented bloodmeals made up of LAVs at titers reported. Saliva was collected to assess transmission rates and whole bodies were assayed for contamination rate assessments 10 days post blood feeding. Infectious CHIKV and RNA were determined by plaque assay and qRT-PCR, respectively. Table values are percentage (positive/total). mosquitoes, the primary vector. It must be stressed, however, that vector competence of the LAVs explored in this study comes with the caveat that CHIKV-181/25-P2.P4.E3/E1 was presented at nearly 2-logs lower concentration due to limitations on LAV recovery yield in vitro. While this challenge prevents us from definitively saying that CHIKV-181/25-P2.P4-E3/E1 is non-transmissible in mosquitoes, the lack of a significant increase in CHIKV-181/25-P2.P4 vector competence is promising nonetheless. Furthermore, vaccine-induced viremia is required for mosquito vectors to become infected with a LAV, and sufficiently high titers must be present to facilitate transmission. While extremely rare, LAV VEEV used in equines around the Texas Gulf Coast in 1971 was detected in local mosquito populations in Louisiana, USA where the LAV was not administered56, underscoring the need to restrict vector competence. Unexpectedly, one derivative vaccine based on the CHIKV-181/25-P2.P4 candidate failed to elicit adequate protection in our studies. CHIKV-181/25-P2.P4.IRES, which is based on attenuation of an Indian Ocean Lineage CHIKV strain, proved challenging to produce in sufficient quantities for immunization and was non-protective at the available dosage. This candidate produced lower peak titers in vertebrate cells and failed to infect mosquito cells and was S3I-201 (NSC 74859) well tolerated in neonatal mice. However, immunization with CHIKV-181/25-P2.P4.IRES failed to produce a measurable neutralizing antibody response and pre-exposure was associated with a reproducible increase in disease severity following challenge with WT CHIKV. We propose that poor vaccine replication in mice resulting from reduced structural protein production, which was four- to six occasions lower than the WT computer virus for the related alphavirus, VEEV41, combined with the attenuation of CHIKV-181/25 was Rabbit Polyclonal to MuSK (phospho-Tyr755) insufficient to trigger the production of adequate immunity including neutralizing antibodies. This is in stark contrast to the CHIKV-IRES vaccine raised in the WT background, which was both protective and yielded sufficient viral titers for vaccination studies35C37. It is possible that poor immunological memory associated with CHIKV-181/25-P2.P4.IRES vaccination is directly responsible for this increase in disease severity. In contrast,.