A measles-vectored vaccine candidate expressing prefusion-stabilized SARS-CoV-2 spike protein brought to phase I/II clinical trials: protection of African green monkeys from COVID-19 disease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and is responsible for the largest human pandemic in 100 years. Thirty-four vaccines are currently approved for use worldwide, and approximately 67% of the world population has received a complete primary series of one, yet countries are dealing with new waves of infections, variant viruses continue to emerge, and breakthrough infections are frequent secondary to waning immunity. Here, we evaluate a measles virus (MV)-vectored vaccine expressing a stabilized prefusion SARS-CoV-2 spike (S) protein (MV-ATU3-S2PΔF2A; V591) with demonstrated immunogenicity in mouse models (see companion article [J. Brunet, Z. Choucha, M. Gransagne, H. Tabbal, M.-W. Ku et al., J Virol 98:e01693-23, 2024, https://doi.org/10.1128/jvi.01693-23]) in an established African green monkey model of disease. Animals were vaccinated with V591 or the control vaccine (an equivalent MV-vectored vaccine with an irrelevant antigen) intramuscularly using a prime/boost schedule, followed by challenge with an early pandemic isolate of SARS-CoV-2 at 56 days post-vaccination. Pre-challenge, only V591-vaccinated animals developed S-specific antibodies that had virus-neutralizing activity as well as S-specific T cells. Following the challenge, V591-vaccinated animals had lower infectious virus and viral (v) RNA loads in mucosal secretions and stopped shedding virus in these secretions earlier. vRNA loads were lower in these animals in respiratory and gastrointestinal tract tissues at necropsy. This correlated with a lower disease burden in the lungs as quantified by PET/CT at early and late time points post-challenge and by pathological analysis at necropsy.IMPORTANCESevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the largest human pandemic in 100 years. Even though vaccines are currently available, countries are dealing with new waves of infections, variant viruses continue to emerge, breakthrough infections are frequent, and vaccine hesitancy persists. This study uses a safe and effective measles vaccine as a platform for vaccination against SARS-CoV-2. The candidate vaccine was used to vaccinate African green monkeys (AGMs). All vaccinated AGMs developed robust antigen-specific immune responses. After challenge, these AGMs produced less virus in mucosal secretions, for a shorter period, and had a reduced disease burden in the lungs compared to control animals. At necropsy, lower levels of viral RNA were detected in tissue samples from vaccinated animals, and the lungs of these animals lacked the histologic hallmarks of SARS-CoV-2 disease observed exclusively in the control AGMs.

Immunogenicity, safety, and tolerability of a recombinant measles-vectored Lassa fever vaccine: a randomised, placebo-controlled, first-in-human trial.

BACKGROUND: Lassa fever is a substantial health burden in west Africa. We evaluated the safety, tolerability, and immunogenicity of a recombinant, live-attenuated, measles-vectored Lassa fever vaccine candidate (MV-LASV). METHODS: This first-in-human phase 1 trial-consisting of an open-label dose-escalation stage and an observer-blinded, randomised, placebo-controlled treatment stage-was conducted at a single site at the University of Antwerp, Antwerp, Belgium, and involved healthy adults aged 18-55 years. Participants in the dose-escalation stage were sequentially assigned to a low-dose group (two intramuscular doses of MV-LASV at 2 × 104 times the median tissue culture infectious dose) or a high-dose group (two doses at 1 × 105 times the median tissue culture infectious dose). Participants in the double-blinded treatment stage were randomly assigned in a 2:2:1 ratio to receive low dose, high dose, or placebo. The primary endpoint was the rate of solicited and unsolicited adverse events up to study day 56 and was assessed in all participants who received at least one dose of investigational product. The trial is registered with ClinicalTrials.gov, NCT04055454, and the European Union Drug Regulating Authorities Clinical Trials Database, 2018-003647-40, and is complete. FINDINGS: Between Sept 26, 2019, and Jan 20, 2020, 60 participants were enrolled and assigned to receive placebo (n=12) or MV-LASV (n=48). All 60 participants received at least one study treatment. Most adverse events occurred during the treatment phase, and frequencies of total solicited or unsolicited adverse events were similar between treatment groups, with 96% of participants in the low-dose group, 100% of those in the high-dose group, and 92% of those in the placebo group having any solicited adverse event (p=0·6751) and 76% of those in the low-dose group, 70% of those in the high-dose group, and 100% of those in the placebo group having any unsolicited adverse event (p=0·1047). The only significant difference related to local solicited adverse events, with higher frequencies observed in groups receiving MV-LASV (24 [96%] of 25 participants in the low-dose group; all 23 [100%] participants in the high-dose group) than in the placebo group (6 [50%] of 12 participants; p=0·0001, Fisher-Freeman-Halton test). Adverse events were mostly of mild or moderate severity, and no serious adverse events were observed. MV-LASV also induced substantial concentrations of LASV-specific IgG (geometric mean titre 62·9 EU/ml in the low-dose group and 145·9 EU/ml in the high-dose group on day 42). INTERPRETATION: MV-LASV showed an acceptable safety and tolerability profile, and immunogenicity seemed to be unaffected by pre-existing immunity against the vector. MV-LASV is therefore a promising candidate for further development. FUNDING: Coalition for Epidemic Preparedness Innovations.

Biodistribution and toxicology evaluation of a recombinant measles Schwarz-based Lassa vaccine in cynomolgus macaques.

MV-LASV is an investigational measles Schwarz-based vaccine for the prevention of Lassa fever. A repeated-dose toxicity study in cynomolgus macaques was performed to assess the biodistribution and local and systemic toxicological effects. Monkeys received three immunizations of MV-LASV or saline intramuscularly with a 2-week interval. An increase in anti-measles antibodies confirmed the reaction of the immune system to the vaccine backbone. Clinical observations, body weight, body temperature, local tolerance, electrocardiogram parameters, various clinical pathology parameters (hematology, coagulation urinalysis, serum chemistry, and C-reactive protein) were monitored. Gross pathology and histopathology of various tissues were evaluated. MV-LASV induced a mild increase in fibrinogen and C-reactive protein concentrations. This coincided with microscopic inflammation at the injection sites which partially or fully resolved following a 3-week recovery period. Viral RNA was found in secondary lymphoid organs and injection sites and gall bladder. No viral shedding to the environment was observed. Overall, the vaccine was locally and systemically well tolerated, supporting a first-in-human study.

Immunogenicity and Efficacy of a Measles Virus-Vectored Chikungunya Vaccine in Nonhuman Primates.

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.

Generation and genetic stability of tick-borne encephalitis virus mutants dependent on processing by the foot-and-mouth disease virus 3C protease.

Mature protein C of tick-borne encephalitis virus (TBEV) is cleaved from the polyprotein precursor by the viral NS2B/3 protease (NS2B/3(pro)). We showed previously that replacement of the NS2B/3(pro) cleavage site at the C terminus of protein C by the foot-and-mouth disease virus (FMDV) 2A StopGo sequence leads to the production of infectious virions. Here, we show that infectious virions can also be produced from a TBEV mutant bearing an inactivated 2A sequence through the expression of the FMDV 3C protease (3C(pro)) either in cis or in trans (from a TBEV replicon). Cleavage at the C terminus of protein C depended on the catalytic activity of 3C(pro) as well as on the presence of an optimized 3C(pro) cleavage site. Passage of the TBEV mutants bearing a 3C(pro) cleavage site either in the absence of 3C(pro) or in the presence of a catalytically inactive 3C(pro) led to the appearance of revertants in which protein C cleavage by NS2B/3(pro) had been regained. In three different revertants, a cleavage site for NS2B/3(pro), namely RR*C, was now present, leading to an elongated protein C. Furthermore, two revertants acquired additional mutations in the C terminus of protein C, eliminating two basic residues. Although these latter mutants showed wild-type levels of early RNA synthesis, their foci were smaller and an accumulation of protein C in the cytoplasm was observed. These findings suggest a role of the positive charge of the C terminus of protein C for budding of the nucleocapsid and further support the notion that TBEV protein C is a multifunctional protein.

Extension of flavivirus protein C differentially affects early RNA synthesis and growth in mammalian and arthropod host cells.

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 (TBEV) and West Nile virus (WNV) by the 2A protein of foot-and-mouth disease virus (TBEV-2A and WNV-2A). This obviated the need for NS2B/3 processing at the C terminus of mature protein C while simultaneously producing a 19-amino-acid extension on protein C. Infectious virions were generated with both viruses; the phenotype depended on the host cell. TBEV-2A replicated well in BHK-21 cells but was essentially incapable of replication in tick cells. In contrast, WNV-2A replicated well in mosquito cells but showed a small-plaque phenotype in Vero cells, with frequent production of larger plaques. Sequencing of viral RNA from the larger plaques showed substitutions in the signal sequence for prM, presumably improving coordinated protein processing at the C-prM junction. Furthermore, both TBEV-2A and WNV-2A were also defective in unpackaging and/or early RNA synthesis. Together, these results indicate a role for flavivirus protein C in both viral assembly and RNA replication, possibly by interacting with host cell factors required to set up the cell for RNA replication.

Current Efforts in the Development of Vaccines for the Prevention of Zika and Chikungunya Virus Infections.

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.

Functional analysis of potential carboxy-terminal cleavage sites of tick-borne encephalitis virus capsid protein.

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. Site-specific 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.

Changing the protease specificity for activation of a flavivirus, tick-borne encephalitis virus.

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.