Arboviruses represent major challenges to public health, particularly in tropical, and subtropical regions, and a substantial risk to other parts of the world as respective vectors extend their habitats. In recent years, two viruses transmitted by Aedes mosquitoes, Chikungunya and Zika virus, have gathered increased interest. After decades of regionally constrained outbreaks, both viruses have recently caused explosive outbreaks on an unprecedented scale, causing immense suffering and massive economic burdens in affected regions. Chikungunya virus causes an acute febrile illness that often transitions into a chronic manifestation characterized by debilitating arthralgia and/or arthritis in a substantial subset of infected individuals. Zika infection frequently presents as a mild influenza-like illness, often subclinical, but can cause severe complications such as congenital malformations in pregnancy and neurological disorders, including Guillain-Barré syndrome. With no specific treatments or vaccines available, vector control remains the most effective measure to manage spread of these diseases. Given that both viruses cause antibody responses that confer long-term, possibly lifelong protection and that such responses are cross-protective against the various circulating genetic lineages, the development of Zika and Chikungunya vaccines represents a promising route for disease control. In this review we provide a brief overview on Zika and Chikungunya viruses, the etiology and epidemiology of the illnesses they cause and the host immune response against them, before summarizing past and current efforts to develop vaccines to alleviate the burden caused by these emerging diseases. The development of the urgently needed vaccines is hampered by several factors including the unpredictable epidemiology, feasibility of rapid clinical trial implementation during outbreaks and regulatory pathways. We will give an overview of the current developments.

The translation of flaviviral RNA genomes yields a single polyprotein that is processed into the mature proteins by viral and host cell proteases. Mature capsid protein C is freed from the polyprotein by the viral NS2B/3 protease, cleaving in the C-terminal region of protein C in front of the signal sequence for prM. Protein C has been shown to be involved in viral assembly and RNA packaging. To examine further the role of protein C and its production by proteolysis, we replaced the NS2B/3 capsid cleavage site in tick-borne encephalitis virus (

Background Chikungunya virus (CHIKV) infection can result in chikungunya fever (CHIKF), a self-limited acute febrile illness that can progress to chronic arthralgic sequelae in a large percentage of patients. A new measles virus-vectored vaccine was developed to prevent CHIKF, and we tested it for immunogenicity and efficacy in a nonhuman primate model. Methods Nine cynomolgus macaques were immunized and boosted with the measles virus-vectored chikungunya vaccine or sham-vaccinated. Sera were taken at multiple times during the vaccination phase to assess antibody responses against CHIKV. Macaques were challenged with a dose of CHIKV previously shown to cause fever and viremia, and core body temperature, viremia, and blood cell and chemistry panels were monitored. Results The vaccine was well tolerated in all macaques, and all seroconverted (high neutralizing antibody [PRNT80 titers, 40–640] and enzyme-linked immunosorbent assay titers) after the boost. Furthermore, the vaccinated primates were protected against viremia, fever, elevated white blood cell counts, and CHIKF-associated cytokine changes after challenge with the virulent La Reunión CHIKV strain. Conclusions These results further document the immunogenicity and efficacy of a measles-vectored chikungunya vaccine that shows promise in Phase I–II clinical trials. These findings are critical to human health because no vaccine to combat CHIKF is yet licensed.

The mature capsid protein C of flaviviruses is generated through the proteolytic cleavage of the precursor polyprotein by the viral NS2B/3 protease. This cleavage is a prerequisite for the subsequent processing of the viral surface protein prM, and the concerted progression of these events plays a key role in the process of the assembly of infectious virions. Protein C of tick-borne encephalitis virus (TBEV) contains two amino acid sequence motifs within the carboxy-terminal region that match the canonical NS2B/3 recognition site. Sitespecific mutagenesis in the context of the full-length TBEV genome was used to investigate the in vivo cleavage specificity of the viral protease in this functionally important domain. The results indicate that the downstream site is necessary and sufficient for efficient cleavage and virion assembly; in contrast, the upstream site is dispensable and placed in a structural context that renders it largely inaccessible to the viral protease. Mutants with impaired C-prM cleavage generally exhibited a significantly increased cytotoxicity. In spite of the clear preference of the protease for only one of the two naturally occurring motifs, the enzyme was unexpectedly tolerant to both the presence of a noncanonical threonine residue at position P2 and the position of cleavage relative to the adjacent internal prM signal sequence. The insertion of three amino acid residues downstream of the cleavage site did not change the viral phenotype. Thus, this study further illuminates the specificity of the TBEV protease and reveals that the carboxy-terminal region of protein C has a remarkable functional flexibility in its role in the assembly of infectious virions.

The infectivity of flavivirus particles depends on a maturation process that is triggered by the proteolytic cleavage of the precursor of the M protein (prM). This activation cleavage is naturally performed by ubiquitous cellular proteases of the furin family, which typically recognize the multibasic sequence motif R-X-R/K-R. Previously, we demonstrated that a tick-borne encephalitis virus (TBEV) mutant with an altered cleavage motif, R-X-R, produced immature, noninfectious particles that could be activated by exogenous trypsin, which cleaves after single basic residues. Here, we report the adaptation of this mutant to chymotrypsin, a protease specific for large, hydrophobic amino acid residues. Using selection pressure in cell culture, two different mutations conferring a chymotrypsin-dependent phenotype were identified. Surprisingly, one of these mutations (Ser85Phe) occurred three positions upstream of the natural cleavage site. The other mutation (Arg89His) arose at the natural cleavage position but involved a His residue, which is not a typical chymotrypsin cleavage site. Efficient cleavage of protein prM and activation by the heterologous protease were confirmed using various recombinant TBEV mutants. Mutants with only the originally selected mutations exhibited unimpaired export kinetics and were genotypically stable during at least six cell culture passages. However, in contrast to the wild-type virus or trypsin-dependent mutants, chymotrypsin-dependent mutants were not neurovirulent in suckling mice. Our results demonstrate that flaviviruses with altered protease specificities can be generated and suggest that this approach can be used for the construction of viral mutants or vectors that can be activated on demand and have restricted tissue tropism and virulence.