Evidence brief: Stirred tank bioreactor process for chikungunya vaccine candidate VEEV-ΔC-CHIKV

Abstract

Chikungunya virus (CHIKV), a mosquito-borne alphavirus with significant public health impact, has caused recurrent epidemics across Africa, Asia, and the Americas. Despite the recent approval of a live-attenuated vaccine (Chix), scalable manufacturing processes for CHIKV vaccines remain critical to meet global demand. In this context, we previously constructed a chimeric virus, VEEV-ΔC-CHIKV, by replacing the backbone of ΔC-CHIKV with that of the VEEV replicon while retaining the CHIKV antigenic glycoprotein (E3-E2-6K-E1). A series of experiments have demonstrated that the chimeric virus exhibits safety, stability, and immunogenicity, making it a promising candidate for a new attenuated live vaccine strain. Therefore, to further validate its viral properties and explore the potential for large-scale production, we developed an optimized bioreactor-based production process for VEEV-ΔC-CHIKV. Utilizing adherent Vero cells in mechanically stirred bioreactors, we systematically optimized key parameters, including carrier type selection, cell seeding density, human albumin supplementation, and nutrient modulation, to maximize viral titers while minimizing the accumulation of metabolic byproducts. Results indicated that Cytodex™ 1 microcarriers outperformed Fibra-Cel carriers, enabling 10 consecutive harvests with viral titers ≥ 105 PFU/mL (compared to 4 harvests for Fibra-Cel). When the initial cell density was 6.5 × 105 cells/mL, the total virus yield was 77% higher than at a cell density of 2.5 × 105 cells/mL. Adding 1.0% human albumin doubled total virus production compared to the addition of 0.5%. Nutrient modulation through the maintenance of glucose (>3 g/L) and glutamine (>0.5 g/L) in fed-batch systems enhanced peak titers to 106.4 PFU/mL, representing a 2.2-fold increase compared to non-fed controls. This study establishes a scalable and cost-effective bioreactor platform for the production of the CHIKV vaccine, highlighting the critical interplay between nutrient optimization and viral yield enhancement, while emphasizing the advantages of automated process control in bioreactors to facilitate large-scale, high-yield viral production.

1. Introduction

Chikungunya virus (CHIKV), a member of the Alphavirus genus within the Togaviridae family [1], is an RNA enveloped virus with a diameter of approximately 70 nm. It consists of three structural proteins (capsid protein C, envelope proteins E1 and E2) and four non-structural proteins (nsP1, nsP2, nsP3, and nsP4). The viral genome is a single-stranded, non-segmented, positive-sense RNA that spans approximately 11–12 kb. CHIKV is primarily transmitted by Aedes mosquitoes, particularly in tropical and subtropical regions [2,3].

Humans are generally susceptible to CHIKV infection, which can manifest as either symptomatic or asymptomatic. The disease caused by CHIKV typically manifests as an acute febrile illness accompanied by joint pain, which can persist for weeks to years in many cases [1,4,5]. In newly emerged or epidemic areas, individuals of all age groups are susceptible; however, in endemic regions such as Africa and Southeast Asia, some symptoms in children infected with CHIKV are more severe [6,7]. CHIKV exhibits a global distribution but is predominantly endemic in Africa, South America, and Southeast Asia [8,9]. Furthermore, it has caused large-scale outbreaks in the Indian Ocean region in recent years. Since 2004, CHIKV has infected millions of people across Africa and Asia [5]. Between 2005 and 2007, massive outbreaks were reported in parts of Southeast Asia and neighboring islands, and cases emerged in Europe in 2007 [10,11]. It is estimated that half of the global population is at risk of infection [12]. According to the latest outbreak data on CHIKV released by the World Health Organization (WHO) and the European Centre for Disease Prevention and Control (ECDC), as of December 2024, 119 countries and territories have reported local transmission. However, high attack rates and ongoing susceptibility increase the risk of large-scale human infections. Additionally, due to limitations in surveillance and diagnostics, the potential for re-emergence remains high in previously affected areas [13]. From January to July 2025, approximately 240,000 CHIKV cases and 90 CHIKV-related deaths have been reported in 16 countries/ territories. Cases have been reported in the Americas, Africa, Asia, and Europe [14].