Researchers developed an engineered chikungunya vaccine that can complete only a single round of infection while still generating strong immune protection in mice. The experimental approach may offer a safer alternative to traditional live-attenuated vaccines for vulnerable populations.
The researchers showed that chikungunya virus engineered to depend on an artificial โTEVโ protease site for maturation can be produced as immature, replicationโlimited particles that, after a controlled inโvitro activation step, protect mice from lethal infection, viremia, and joint swelling.
Chikungunya remains a significant public health problem, with large recent outbreaks in Brazil and other regions and no universally safe vaccine for all age and risk groups. A licensed liveโattenuated vaccine (IXCHIQ) has raised safety concerns in older adults, leading regulators in several countries to restrict or suspend its use in people over 60 or 65 years old. The authors therefore wanted to explore a platform that preserves strong immune stimulation but minimizes the risk that a vaccine virus could replicate uncontrollably, especially in vulnerable populations.
Previous work had established that chikungunya virus and related alphaviruses need cleavage of a precursor glycoprotein (p62, or E3โE2) by the host enzyme furin to become fully infectious. Studies in other viruses, particularly dengue, had shown that immature or partially mature particles can influence disease and immune responses, and that furinโmediated maturation in acidic compartments is a key step for infectivity. Two chikungunya vaccines one liveโattenuated (IXCHIQ) and one virusโlike particle (VIMKUNYA)โwere already available, but each has limitations in either safety profile or target population.
First, the team produced immature chikungunya particles by infecting LoVo cells, a human cell line lacking functional furin, and compared them with mature particles made in furinโcompetent cells such as Vero cells using electron microscopy and infectivity assays. They then transiently restored furin expression in LoVo cells with a plasmid to confirm that this protease is required for efficient maturation and production of infectious virus.
Next, they engineered a recombinant chikungunya virus in which the natural furin cleavage site between E3 and E2 was replaced with a recognition site for Tobacco Etch virus (TEV) protease, generating โCHIKVโTEVโ constructs, including a version expressing a fluorescent mCherry reporter. RNA transcripts from these cDNA clones were electroporated into BHKโ21 cells, and the researchers monitored particle production via plaque assays, Western blotting for capsid protein, and fluorescence or immunofluorescence markers of infection.
To test controllable infectivity, CHIKVโTEV particles were incubated with or without recombinant TEV protease in vitro and then used to infect BHKโ21 cells, followed by a second infection using supernatant from the first experiment; they also performed five sequential passages in cell culture to probe whether infectivity could reโemerge over time. Finally, they evaluated vaccine performance in two mouse models: interferon receptorโdeficient IFNARโป/โป mice for survival after lethal challenge, and immunocompetent C57BL/6 mice for neutralizing antibody responses, viremia, footpad swelling, and crossโneutralization against Mayaro virus.
Electron microscopy revealed that immature particles produced in furinโdeficient LoVo cells were significantly larger (about 120 nm in diameter) than mature particles from Vero cells (about 76 nm), consistent with an immature structural state. In infection experiments, immature particles caused delayed viral progeny production in furinโcompetent Vero, HeLa, and C6/36 cells, but did not generate detectable progeny in LoVo cells unless furin expression was restored, demonstrating the central role of this host protease.
When the furin cleavage site in the viral genome was replaced by a TEV protease site, the resulting CHIKVโTEV particles were still produced and detectable by capsid protein in supernatant but failed to form plaques or cause cytopathic effects in Vero cells, indicating they were nonโinfectious in standard conditions. After inโvitro treatment with TEV protease, however, CHIKVโTEV particles regained the ability to infect BHKโ21 cells and produce progeny, while untreated particles remained nonโinfectious in both first and secondโround infections, and neither treated nor untreated viruses regained replication competence after five serial passages.
In IFNARโป/โป mice, a single immunization with TEVโtreated immature particles provided strong protection against lethal challenge with wildโtype chikungunya virus: all animals receiving 10ยณ or 10โด particles survived, and only a minority in the lowestโdose group (10ยฒ particles) succumbed, whereas most animals immunized with uncleaved particles and all mockโimmunized controls died rapidly. In C57BL/6 mice, TEVโtreated particles induced about 9โfold higher neutralizing antibody titers (PRNTโ โ > 686) than untreated particles (PRNTโ โ = 75), and mice vaccinated with treated particles showed no detectable viremia after challenge and minimal footpad swelling, while mock animals developed high viral loads and pronounced edema.
Sera from vaccinated C57BL/6 mice also showed modest crossโneutralization of Mayaro virus at low dilutions, with higher activity in animals immunized with TEVโtreated particles. Across these experiments, both TEVโtreated and untreated formulations reduced disease signs compared with mock controls, but TEVโtreated particles consistently produced stronger antibody responses and better control of inflammation.
The authors conclude that immature chikungunya particles are not entirely nonโinfectious in furinโcompetent cells, because furinโmediated cleavage in endosomes can still render them infectious, but that replacing the furin site with a TEV site creates particles that are effectively nonโinfectious in natural host environments. They argue that inโvitro TEV maturation produces a vaccine candidate that can undergo a single round of replication in the host sufficient to express all structural and nonโstructural proteins and amplify immune responses while producing progeny that remain immature and unable to propagate.
They describe this as a promising platform for developing โa safe, replication limited immunogenic particleโ for chikungunya and potentially for other furinโdependent viruses, including other alphaviruses, flaviviruses, and even SARSโCoVโ2. As they write, โour immature particle platform is intrinsically nonโreplicating unless deliberately activated, offering a safer alternative for highโrisk groups such as the elderly, immunocompromised individuals, and pregnant women,โ said Beate Mareike Kรผmmerer, a virologist at the Institute of Virology of the University of Bonn and senior author of the study
The authors note that they did not measure Tโcell responses, so any advantages related to CD8โบ Tโcell priming by limited replication remain hypothetical and need to be examined in future work. They also acknowledge that they did not sequence viral progeny under nonโpermissive conditions, because no infectious virus was recovered without TEV; longerโterm studies will be required to fully characterize the genetic stability and evolutionary robustness of the TEVโdependent construct.
In addition, the protective data come from mouse models, including IFNARโdeficient mice with impaired interferon signaling, which are more susceptible than humans and do not fully capture human immune complexity. The challenge doses, routes, and timing are experimental conditions, so the results cannot be directly extrapolated to human vaccine performance or to all demographic groups.
Reference:
โMaturationโdeficient chikungunya virus elicits protective immune responses in a murine challenge model,โ by Danillo Lucas Alves Esposito, Jhefferson Barbosa Guimarรฃes, Benedito Antonio Lopes da Fonseca, and Beate Mareike Kรผmmerer, published in npj Vaccines, volume 11, article number 98, 2026.
