A randomized, double-blinded Phase 3 study to demonstrate lot-to-lot consistency and to confirm immunogenicity and safety of the live-attenuated chikungunya virus vaccine candidate VLA1553 in healthy adults†.

BACKGROUND: The global spread of the chikungunya virus (CHIKV) increases the exposure risk for individuals travelling to or living in endemic areas. This Phase 3 study was designed to demonstrate manufacturing consistency between three lots of the single shot live-attenuated CHIKV vaccine VLA1553, and to confirm the promising immunogenicity and safety data obtained in previous trials. METHODS: This randomized, double-blinded, lot-to-lot consistency, Phase 3 study, assessed immunogenicity and safety of VLA1553 in 408 healthy adults (18-45 years) in 12 sites across the USA. The primary endpoint was a comparison of the geometric mean titre (GMT) ratios of CHIKV-specific neutralizing antibodies between three VLA1553 lots at 28 days post-vaccination. Secondary endpoints included immunogenicity and safety over 6 months post-vaccination. RESULTS: GMTs were comparable between the lots meeting the acceptance criteria for equivalence. The average GMT (measured by 50% CHIKV micro plaque neutralization test; µPRNT50) peaked with 2643 at 28 days post-vaccination and decreased to 709 at 6 months post-vaccination. An excellent seroresponse rate (defined as µPRNT50 titre ≥ 150 considered protective) was achieved in 97.8% of participants at 28 days post-vaccination and still persisted in 96% at 6 months after vaccination. Upon VLA1553 immunization, 72.5% of participants experienced adverse events (AEs), without significant differences between lots (related solicited systemic AE: 53.9% of participants; related solicited local AE: 19.4%). Overall, AEs were mostly mild or moderate and resolved without sequela, usually within 3 days. With 3.9% of participants experiencing severe AEs, 2.7% were classified as related, whereas none of the six reported serious adverse events was related to the administration of VLA1553. CONCLUSIONS: All three lots of VLA1553 recapitulated the safety and immunogenicity profiles of a preceding Phase 3 study, fulfilling pre-defined consistency requirements. These results highlight the manufacturability of VLA1553, a promising vaccine for the prevention of CHIKV disease for those living in or travelling to endemic areas.

Safety and immunogenicity of a single-shot live-attenuated chikungunya vaccine: a double-blind, multicentre, randomised, placebo-controlled, phase 3 trial.

BACKGROUND: VLA1553 is a live-attenuated vaccine candidate for active immunisation and prevention of disease caused by chikungunya virus. We report safety and immunogenicity data up to day 180 after vaccination with VLA1553. METHODS: This double-blind, multicentre, randomised, phase 3 trial was done in 43 professional vaccine trial sites in the USA. Eligible participants were healthy volunteers aged 18 years and older. Patients were excluded if they had history of chikungunya virus infection or immune-mediated or chronic arthritis or arthralgia, known or suspected defect of the immune system, any inactivated vaccine received within 2 weeks before vaccination with VLA1553, or any live vaccine received within 4 weeks before vaccination with VLA1553. Participants were randomised (3:1) to receive VLA1553 or placebo. The primary endpoint was the proportion of baseline negative participants with a seroprotective chikungunya virus antibody level defined as 50% plaque reduction in a micro plaque reduction neutralisation test (µPRNT) with a µPRNT50 titre of at least 150, 28 days after vaccination. The safety analysis included all individuals who received vaccination. Immunogenicity analyses were done in a subset of participants at 12 pre-selected study sites. These participants were required to have no major protocol deviations to be included in the per-protocol population for immunogenicity analyses. This trial is registered at ClinicalTrials.gov, NCT04546724. FINDINGS: Between Sept 17, 2020 and April 10, 2021, 6100 people were screened for eligibility. 1972 people were excluded and 4128 participants were enrolled and randomised (3093 to VLA1553 and 1035 to placebo). 358 participants in the VLA1553 group and 133 participants in the placebo group discontinued before trial end. The per-protocol population for immunogenicity analysis comprised 362 participants (266 in the VLA1553 group and 96 in the placebo group). After a single vaccination, VLA1553 induced seroprotective chikungunya virus neutralising antibody levels in 263 (98·9%) of 266 participants in the VLA1553 group (95% CI 96·7-99·8; p<0·0001) 28 days post-vaccination, independent of age. VLA1553 was generally safe with an adverse event profile similar to other licensed vaccines and equally well tolerated in younger and older adults. Serious adverse events were reported in 46 (1·5%) of 3082 participants exposed to VLA1553 and eight (0·8%) of 1033 participants in the placebo arm. Only two serious adverse events were considered related to VLA1553 treatment (one mild myalgia and one syndrome of inappropriate antidiuretic hormone secretion). Both participants recovered fully. INTERPRETATION: The strong immune response and the generation of seroprotective titres in almost all vaccinated participants suggests that VLA1553 is an excellent candidate for the prevention of disease caused by chikungunya virus. FUNDING: Valneva, Coalition for Epidemic Preparedness Innovation, and EU Horizon 2020.

Effectiveness of CHIKV vaccine VLA1553 demonstrated by passive transfer of human sera.

Chikungunya virus (CHIKV) is a reemerging mosquito-borne alphavirus responsible for numerous outbreaks. Chikungunya can cause debilitating acute and chronic disease. Thus, the development of a safe and effective CHIKV vaccine is an urgent global health priority. This study evaluated the effectiveness of the live-attenuated CHIKV vaccine VLA1553 against WT CHIKV infection by using passive transfer of sera from vaccinated volunteers to nonhuman primates (NHP) subsequently exposed to WT CHIKV and established a serological surrogate of protection. We demonstrated that human VLA1553 sera transferred to NHPs conferred complete protection from CHIKV viremia and fever after challenge with homologous WT CHIKV. In addition, serum transfer protected animals from other CHIKV-associated clinical symptoms and from CHIKV persistence in tissue. Based on this passive transfer study, a 50% micro-plaque reduction neutralization test titer of ≥ 150 was determined as a surrogate of protection, which was supported by analysis of samples from a seroepidemiological study. In conclusion, considering the unfeasibility of an efficacy trial due to the unpredictability and explosive, rapidly moving nature of chikungunya outbreaks, the definition of a surrogate of protection for VLA1553 is an important step toward vaccine licensure to reduce the medical burden caused by chikungunya.

Immunoproteasome subunit deficiency has no influence on the canonical pathway of NF-κB activation.

Activation of the pro-inflammatory transcription factor NF-κB requires signal-induced proteasomal degradation of the inhibitor of NF-κB (IκB) in order to allow nuclear translocation. Most cell types are capable of expressing two types of 20S proteasome core particles, the constitutive proteasome and immunoproteasome. Inducible under inflammatory conditions, the immunoproteasome is mainly characterized through an altered cleavage specificity compared to the constitutive proteasome. However, the question whether immunoproteasome subunits affect NF-κB signal transduction differently from constitutive subunits is still up for debate. To study the effect of immunoproteasomes on LPS- or TNF-α-induced NF-κB activation, we used IFN-γ stimulated peritoneal macrophages and mouse embryonic fibroblasts derived from mice deficient for the immunoproteasome subunits low molecular mass polypeptide (LMP) 2, or LMP7 and multicatalytic endopeptidase complex-like 1 (MECL-1). Along the canonical signaling pathway of NF-κB activation no differences in the extent and kinetic of IκB degradation were observed. Neither the nuclear translocation and DNA binding of NF-κB nor the production of the NF-κB dependent cytokines TNF-α, IL-6, and IL-10 differed between immunoproteasome deficient and proficient cells. Hence, we conclude that immunoproteasome subunits have no specialized function for canonical NF-κB activation.

Newly translated proteins are substrates for ubiquitin, ISG15, and FAT10.

The ubiquitin-like modifier, FAT10, is involved in proteasomal degradation and antigen processing. As ubiquitin and the ubiquitin-like modifier, ISG15, cotranslationally modify proteins, we investigated whether FAT10 could also be conjugated to newly synthesized proteins. Indeed, we found that nascent proteins are modified with FAT10, but not with the same preference for newly synthesized proteins as observed for ISG15. Our data show that puromycin-labeled polypeptides are strongly modified by ISG15 and less intensely by ubiquitin and FAT10. Nevertheless, conjugates of all three modifiers copurify with ribosomes. Taken together, we show that unlike ISG15, ubiquitin and FAT10 are conjugated to a similar degree to newly translated and pre-existing proteins.

Chaperone BAG6 is dispensable for MHC class I antigen processing and presentation.

Antigen processing for direct presentation on MHC class I molecules is a multistep process requiring the concerted activity of several cellular complexes. The essential steps at the beginning of this pathway, namely protein synthesis at the ribosome and degradation via the proteasome, have been known for years. Nevertheless, there is a considerable lack of factors identified to function between protein synthesis and degradation during antigen processing. Here, we analyzed the impact of the chaperone BAG6 on MHC class I cell surface expression and presentation of virus-derived peptides. Although an essential role of BAG6 in antigen processing has been proposed previously, we found BAG6 to be dispensable in this pathway. Still, interaction of BAG6 and the model antigen tyrosinase was enhanced during proteasome inhibition pointing towards a role of BAG6 in antigen degradation. Redundant chaperone pathways potentially mask the contribution of BAG6 to antigen processing and presentation.

The immunoproteasome: a novel drug target for autoimmune diseases.

The immunoproteasome, a special class of the proteasome, is mainly expressed in cells of haematopoietic origin. Additionally, during inflammation, the immunoproteasome is induced by IFN-γ or TNF-α. In recent years it became apparent that the immunoproteasome has important functions other than processing proteins for MHC class I restricted presentation. The immunoproteasome plays a critical role in T cell expansion, cytokine production, and T helper cell differentiation. Inhibition of the immunoproteasome ameliorated disease symptoms in different animal models for autoimmune diseases. Hence, the unique role for LMP7 in controlling pathogenic immune responses provides a therapeutic rationale for targeting LMP7 in autoimmune disorders. In this review we summarise the effect of immunoproteasome inhibition in animal models for rheumatoid arthritis, inflammatory bowel disease, Hashimoto's thyroiditis, systemic lupus erythematosus, and multiple sclerosis.

Stable antigen is most effective for eliciting CD8+ T-cell responses after DNA vaccination and infection with recombinant vaccinia virus in vivo.

The induction of strong CD8(+) T-cell responses against infectious diseases and cancer has remained a major challenge. Depending on the source of antigen and the infectious agent, priming of CD8(+) T cells requires direct and/or cross-presentation of antigenic peptides on major histocompatibility complex (MHC) class I molecules by professional antigen-presenting cells (APCs). However, both pathways show distinct preferences concerning antigen stability. Whereas direct presentation was shown to efficiently present peptides derived from rapidly degraded proteins, cross-presentation is dependent on long-lived antigen species. In this report, we analyzed the role of antigen stability on DNA vaccination and recombinant vaccinia virus (VV) infection using altered versions of the same antigen. The long-lived nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) can be targeted for degradation by N-terminal fusion to ubiquitin or, as we show here, to the ubiquitin-like modifier FAT10. Direct presentation by cells either transfected with NP-encoding plasmids or infected with recombinant VV in vitro was enhanced in the presence of short-lived antigens. In vivo, however, the highest induction of NP-specific CD8(+) T-cell responses was achieved in the presence of long-lived NP. Our experiments provide evidence that targeting antigens for proteasomal degradation does not improve the immunogenicity of DNA vaccines and recombinant VVs. Rather, it is the long-lived antigen that is superior for the efficient activation of MHC class I-restricted immune responses in vivo. Hence, our results suggest a dominant role for antigen cross-priming in DNA vaccination and recombinant VV infection.