Deliberate Attenuation of Chikungunya Virus by Adaptation to Heparan Sulfate-Dependent Infectivity: A Model for Rational Arboviral Vaccine Design

Abstract

Mosquito-borne chikungunya virus (CHIKV) is a positive-sense, single-stranded RNA virus from the genus Alphavirus, family Togaviridae, which causes fever, rash and severe persistent polyarthralgia in humans. Since there are currently no FDA licensed vaccines or antiviral therapies for CHIKV, the development of vaccine candidates is of critical importance. Historically, live-attenuated vaccines (LAVs) for protection against arthropod-borne viruses have been created by blind cell culture passage leading to attenuation of disease, while maintaining immunogenicity. Attenuation may occur via multiple mechanisms. However, all examined arbovirus LAVs have in common the acquisition of positively charged amino acid substitutions in cell-surface attachment proteins that render virus infection partially dependent upon heparan sulfate (HS), a ubiquitously expressed sulfated polysaccharide, and appear to attenuate by retarding dissemination of virus particles in vivo. We previously reported that, like other wild-type Old World alphaviruses, CHIKV strain, La Réunion, (CHIKV-LR), does not depend upon HS for infectivity. To deliberately identify CHIKV attachment protein mutations that could be combined with other attenuating processes in a LAV candidate, we passaged CHIKV-LR on evolutionarily divergent cell-types. A panel of single amino acid substitutions was identified in the E2 glycoprotein of passaged virus populations that were predicted to increase electrostatic potential. Each of these substitutions was made in the CHIKV-LR cDNA clone and comparisons of the mutant viruses revealed surface exposure of the mutated residue on the spike and sensitivity to competition with the HS analog, heparin, to be primary correlates of attenuation in vivo. Furthermore, we have identified a mutation at E2 position 79 as a promising candidate for inclusion in a CHIKV LAV.

Prime-Boost Immunization Strategies against Chikungunya Virus

“…Region 1 consists of peptides 380 to 382 (amino acids 3033 to 3066) and sits in the acid-sensitive region (ASR) of the E2 glycoprotein. The ASR is thought to play a critical role in regulating CHIKV virulence and regeneration (54,55). Interestingly, these data conform to earlier reports, from investigations of CHIKV-infected humans, nonhuman primates, and mice (30,49,52), that neutralizing antibodies tend to target this region.…”

“…Here we examined the immunogenicity and efficacy of p62-E1 protein in the C57BL/6 mouse infection model (12,55) and showed that two immunizations with p62-E1 mixed with the AS03 or Matrix-M adjuvant were required to induce immunity against CHIKV challenge. The Matrix-M adjuvant elicited the highest magnitudes of anti-CHIKV IgG titers, and as in a recent preclinical study with an influenza virus antigen, formulation with Matrix-M induced a balanced IgG1/IgG2c ratio (52).…”

Chikungunya virus: epidemiology, replication, disease mechanisms, and prospective intervention strategies

“…179). Strain 181/25 is attenuated but immunogenic in both mice and NHPs (138,179,180), and vaccination with 181/25 protects NHPs against CHIKV challenge (179). Strain 181/25 showed no adverse effects in a phase I clinical trial (181), and although it was highly immunogenic in phase II clinical trials, approximately 8% of subjects developed mild, transient arthralgia (182).…”

Dissecting the Role of E2 Protein Domains in Alphavirus Pathogenicity

“…Assays to assess the effect of heparin on virus infection were performed as previously described (39). Virus was diluted to an MOI of 2.5 in Opti-MEM (Invitrogen) and then mixed with either heparin or bovine serum albumin (BSA; final concentration, 200 g/ml).…”