YF17D-based vaccines - standing on the shoulders of a giant.

Live-attenuated yellow fever vaccine (YF17D) was developed in the 1930s as the first ever empirically derived human vaccine. Ninety years later, it is still a benchmark for vaccines made today. YF17D triggers a particularly broad and polyfunctional response engaging multiple arms of innate, humoral and cellular immunity. This unique immunogenicity translates into an extraordinary vaccine efficacy and outstanding longevity of protection, possibly by single-dose immunization. More recently, progress in molecular virology and synthetic biology allowed engineering of YF17D as a powerful vector and promising platform for the development of novel recombinant live vaccines, including two licensed vaccines against Japanese encephalitis and dengue, even in paediatric use. Likewise, numerous chimeric and transgenic preclinical candidates have been described. These include prophylactic vaccines against emerging viral infections (e.g. Lassa, Zika and SARS-CoV-2) and parasitic diseases (e.g. malaria), as well as therapeutic applications targeting persistent infections (e.g. HIV and chronic hepatitis), and cancer. Efforts to overcome historical safety concerns and manufacturing challenges are ongoing and pave the way for wider use of YF17D-based vaccines. In this review, we summarize recent insights regarding YF17D as vaccine platform, and how YF17D-based vaccines may complement as well as differentiate from other emerging modalities in response to unmet medical needs and for pandemic preparedness.

SeParate: multiway fluorescence-activated droplet sorting based on integration of serial and parallel triaging concepts.

Fluorescence-activated droplet sorting (FADS) has emerged as a versatile high-throughput sorting tool that is, unlike most fluorescence-activated cell sorting (FACS) platforms, capable of sorting droplet-compartmentalized cells, cell secretions, entire enzymatic reactions and more. Recently, multiplex FADS platforms have been developed for the sorting of multi-fluorophore populations towards different outlets in addition to the standard, more commonly used, 2-way FADS platform. These multiplex FADS platforms consist of either multiple 2-way junctions one after the other (i.e. serial sorters) or of one junction sorting droplets in more than 2 outlets (i.e. parallel sorters). In this work, we present SeParate, a novel platform based on integrating s̲e̲rial and p̲a̲r̲allel sorting principles for accura̲t̲e̲ multiplex droplet sorting that is able to mitigate limitations of current multiplex sorters. We show the SeParate platform and its capability in highly accurate 4-way sorting of a multi-fluorophore population into four subpopulations with the potential to expand to more. More specifically, the SeParate platform was thoroughly validated using mixed populations of fluorescent beads and picoinjected droplets, yielding sorting accuracies up to 100% and 99.9%, respectively. Finally, transfected HEK-293T cells were sorted employing two different optical setups, resulting in an accuracy up to 99.5%. SeParate's high accuracy for a diverse set of samples, including highly variable biological specimens, together with its scalability beyond the demonstrated 4-way sorting, warrants a broad applicability for multi-fluorophore studies in life sciences, environmental sciences and others.

Optimized vaccine candidate MVA-S(3P) fully protects against SARS-CoV-2 infection in hamsters.

The development of novel optimized vaccines against coronavirus disease 2019 (COVID-19) that are capable of controlling the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic and the appearance of different variants of concern (VoC) is needed to fully prevent the transmission of the virus. In the present study, we describe the enhanced immunogenicity and efficacy elicited in hamsters by a modified vaccinia virus Ankara (MVA) vector expressing a full-length prefusion-stabilized SARS-CoV-2 spike (S) protein [termed MVA-S(3P)]. Hamsters vaccinated with one or two doses of MVA-S(3P) developed high titers of S-binding IgG antibodies and neutralizing antibodies against the ancestral Wuhan SARS-CoV-2 virus and VoC beta, gamma, and delta, as well as against omicron, although with a somewhat lower neutralization activity. After SARS-CoV-2 challenge, vaccinated hamsters did not lose body weight as compared to matched placebo (MVA-WT) controls. Consistently, vaccinated hamsters exhibited significantly reduced viral RNA in the lungs and nasal washes, and no infectious virus was detected in the lungs in comparison to controls. Furthermore, almost no lung histopathology was detected in MVA-S(3P)-vaccinated hamsters, which also showed significantly reduced levels of proinflammatory cytokines in the lungs compared to unvaccinated hamsters. These results reinforce the use of MVA-S(3P) as a vaccine candidate against COVID-19 in clinical trials.

Establishment of a robust rat hepatitis E virus fecal-oral infection model and validation for antiviral studies.

The hepatitis E virus (HEV) is a major cause of hepatitis, with an estimated 3.3 million symptomatic cases annually. There is no HEV-specific treatment besides the off-label use of ribavirin and a vaccine is only available in China and Pakistan. To aid the development of therapeutic and preventive strategies, there is a need for convenient HEV infection models in small laboratory animals. To this end, we make use of the rat hepatitis E virus. Human infections with this virus have been reported in recent years, making it a relevant pathogen for the establishment of a small animal infection model. We here report that oral gavage of a feces suspension, containing a pre-defined viral RNA load, results in a reproducible synchronized infection in athymic nude rats. This route of administration mimics fecal-oral transmission in a standardized fashion. The suitability of the model to study the effect of antiviral drugs was assessed by using ribavirin, which significantly reduced viral loads in the feces, liver, and other tissues.

A High-Throughput Yellow Fever Neutralization Assay.

Quick and accurate detection of neutralizing antibodies (nAbs) against yellow fever is essential in serodiagnosis during outbreaks for surveillance and to evaluate vaccine efficacy in population-wide studies. All of this requires serological assays that can process a large number of samples in a highly standardized format. Albeit being laborious, time-consuming, and limited in throughput, the classical plaque reduction neutralization test (PRNT) is still considered the gold standard for the detection and quantification of nAbs due to its sensitivity and specificity. Here, we report the development of an alternative fluorescence-based serological assay (SNTFLUO) with an equally high sensitivity and specificity that is fit for high-throughput testing with the potential for automation. Finally, our novel SNTFLUO was cross-validated in several reference laboratories and against international WHO standards, showing its potential to be implemented in clinical use. SNTFLUO assays with similar performance are available for the Japanese encephalitis, Zika, and dengue viruses amenable to differential diagnostics. IMPORTANCE Fast and accurate detection of neutralizing antibodies (nAbs) against yellow fever virus (YFV) is key in yellow fever serodiagnosis, outbreak surveillance, and monitoring of vaccine efficacy. Although classical PRNT remains the gold standard for measuring YFV nAbs, this methodology suffers from inherent limitations such as low throughput and overall high labor intensity. We present a novel fluorescence-based serum neutralization test (SNTFLUO) with equally high sensitivity and specificity that is fit for processing a large number of samples in a highly standardized manner and has the potential to be implemented for clinical use. In addition, we present SNTFLUO assays with similar performance for Japanese encephalitis, Zika, and dengue viruses, opening new avenues for differential diagnostics.

MVA-CoV2-S Vaccine Candidate Neutralizes Distinct Variants of Concern and Protects Against SARS-CoV-2 Infection in Hamsters.

To control the coronavirus disease 2019 (COVID-19) pandemic and the emergence of different variants of concern (VoCs), novel vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are needed. In this study, we report the potent immunogenicity and efficacy induced in hamsters by a vaccine candidate based on a modified vaccinia virus Ankara (MVA) vector expressing a human codon optimized full-length SARS-CoV-2 spike (S) protein (MVA-S). Immunization with one or two doses of MVA-S elicited high titers of S- and receptor-binding domain (RBD)-binding IgG antibodies and neutralizing antibodies against parental SARS-CoV-2 and VoC alpha, beta, gamma, delta, and omicron. After SARS-CoV-2 challenge, MVA-S-vaccinated hamsters showed a significantly strong reduction of viral RNA and infectious virus in the lungs compared to the MVA-WT control group. Moreover, a marked reduction in lung histopathology was also observed in MVA-S-vaccinated hamsters. These results favor the use of MVA-S as a potential vaccine candidate for SARS-CoV-2 in clinical trials.

HIV protease inhibitors Nelfinavir and Lopinavir/Ritonavir markedly improve lung pathology in SARS-CoV-2-infected Syrian hamsters despite lack of an antiviral effect.

Nelfinavir is an HIV protease inhibitor that has been widely prescribed as a component of highly active antiretroviral therapy, and has been reported to exert in vitro antiviral activity against SARS-CoV-2. We here assessed the effect of Nelfinavir in a SARS-CoV-2 infection model in hamsters. Despite the fact that Nelfinavir, [50 mg/kg twice daily (BID) for four consecutive days], did not reduce viral RNA load and infectious virus titres in the lung of infected animals, treatment resulted in a substantial improvement of SARS-CoV-2-induced lung pathology. This was accompanied by a dense infiltration of neutrophils in the lung interstitium which was similarly observed in non-infected hamsters. Nelfinavir resulted also in a marked increase in activated neutrophils in the blood, as observed in non-infected animals. Although Nelfinavir treatment did not alter the expression of chemoattractant receptors or adhesion molecules on human neutrophils, in vitro migration of human neutrophils to the major human neutrophil attractant CXCL8 was augmented by this protease inhibitor. Nelfinavir appears to induce an immunomodulatory effect associated with increasing neutrophil number and functionality, which may be linked to the marked improvement in SARS-CoV-2 lung pathology independent of its lack of antiviral activity. Since Nelfinavir is no longer used for the treatment of HIV, we studied the effect of two other HIV protease inhibitors, namely the combination Lopinavir/Ritonavir (Kaletra™) in this model. This combination resulted in a similar protective effect as Nelfinavir against SARS-CoV2 induced lung pathology in hamsters.

The combined treatment of Molnupiravir and Favipiravir results in a potentiation of antiviral efficacy in a SARS-CoV-2 hamster infection model.

BACKGROUND: Favipiravir and Molnupiravir, orally available antivirals, have been reported to exert antiviral activity against SARS-CoV-2. First efficacy data have been recently reported in COVID-19 patients. METHODS: We here report on the combined antiviral effect of both drugs in a SARS-CoV-2 Syrian hamster infection model. The infected hamsters were treated twice daily with the vehicle (the control group) or a suboptimal dose of each compound or a combination of both compounds. FINDINGS: When animals were treated with a combination of suboptimal doses of Molnupiravir and Favipiravir at the time of infection, a marked combined potency at endpoint is observed. Infectious virus titers in the lungs of animals treated with the combination are reduced by ∼5 log10 and infectious virus are no longer detected in the lungs of >60% of treated animals. When start of treatment was delayed with one day a reduction of titers in the lungs of 2.4 log10 was achieved. Moreover, treatment of infected animals nearly completely prevented transmission to co-housed untreated sentinels. Both drugs result in an increased mutation frequency of the remaining viral RNA recovered from the lungs of treated animals. In the combo-treated hamsters, an increased frequency of C-to-T mutations in the viral RNA is observed as compared to the single treatment groups which may explain the pronounced antiviral potency of the combination. INTERPRETATION: Our findings may lay the basis for the design of clinical studies to test the efficacy of the combination of Molnupiravir/Favipiravir in the treatment of COVID-19. FUNDING: stated in the acknowledgment.

Broad betacoronavirus neutralization by a stem helix-specific human antibody.

The spillovers of betacoronaviruses in humans and the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants highlight the need for broad coronavirus countermeasures. We describe five monoclonal antibodies (mAbs) cross-reacting with the stem helix of multiple betacoronavirus spike glycoproteins isolated from COVID-19 convalescent individuals. Using structural and functional studies, we show that the mAb with the greatest breadth (S2P6) neutralizes pseudotyped viruses from three different subgenera through the inhibition of membrane fusion, and we delineate the molecular basis for its cross-reactivity. S2P6 reduces viral burden in hamsters challenged with SARS-CoV-2 through viral neutralization and Fc-mediated effector functions. Stem helix antibodies are rare, oftentimes of narrow specificity, and can acquire neutralization breadth through somatic mutations. These data provide a framework for structure-guided design of pan-betacoronavirus vaccines eliciting broad protection.

A single-dose live-attenuated YF17D-vectored SARS-CoV-2 vaccine candidate.

The expanding pandemic of coronavirus disease 2019 (COVID-19) requires the development of safe, efficacious and fast-acting vaccines. Several vaccine platforms are being leveraged for a rapid emergency response1. Here we describe the development of a candidate vaccine (YF-S0) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that uses live-attenuated yellow fever 17D (YF17D) vaccine as a vector to express a noncleavable prefusion form of the SARS-CoV-2 spike antigen. We assess vaccine safety, immunogenicity and efficacy in several animal models. YF-S0 has an excellent safety profile and induces high levels of SARS-CoV-2 neutralizing antibodies in hamsters (Mesocricetus auratus), mice (Mus musculus) and cynomolgus macaques (Macaca fascicularis), and-concomitantly-protective immunity against yellow fever virus. Humoral immunity is complemented by a cellular immune response with favourable T helper 1 polarization, as profiled in mice. In a hamster model2 and in macaques, YF-S0 prevents infection with SARS-CoV-2. Moreover, a single dose conferred protection from lung disease in most of the vaccinated hamsters within as little as 10 days. Taken together, the quality of the immune responses triggered and the rapid kinetics by which protective immunity can be attained after a single dose warrant further development of this potent SARS-CoV-2 vaccine candidate.

STAT2 signaling restricts viral dissemination but drives severe pneumonia in SARS-CoV-2 infected hamsters.

Emergence of SARS-CoV-2 causing COVID-19 has resulted in hundreds of thousands of deaths. In search for key targets of effective therapeutics, robust animal models mimicking COVID-19 in humans are urgently needed. Here, we show that Syrian hamsters, in contrast to mice, are highly permissive to SARS-CoV-2 and develop bronchopneumonia and strong inflammatory responses in the lungs with neutrophil infiltration and edema, further confirmed as consolidations visualized by micro-CT alike in clinical practice. Moreover, we identify an exuberant innate immune response as key player in pathogenesis, in which STAT2 signaling plays a dual role, driving severe lung injury on the one hand, yet restricting systemic virus dissemination on the other. Our results reveal the importance of STAT2-dependent interferon responses in the pathogenesis and virus control during SARS-CoV-2 infection and may help rationalizing new strategies for the treatment of COVID-19 patients.

Small-molecule inhibitors of TBK1 serve as an adjuvant for a plasmid-launched live-attenuated yellow fever vaccine.

Plasmid-launched live-attenuated vaccines (PLLAV), also called infectious DNA (iDNA) vaccines, combine the assets of genetic immunization with the potency of replication-competent live viral vaccines. However, due to their origin as bacterial plasmid DNA, efficient delivery of PLLAV may be hampered by innate signaling pathways such as the cGAS-STING-mediated sensing of cytosolic DNA, resulting in an unfavorable proinflammatory and antiviral response locally at the site of immunization. Employing several complementary cell-based systems and using the yellow fever vaccine (YF17D) and the respective PLLAV-YF17D, we screened a panel of small molecules known to interfere with antiviral signaling for their proviral activity and identified two potent inhibitors of the TANK-binding kinase 1 (TBK1), BX795 and CYT387, to enhance YF17D replication and hence efficacy of PLLAV-YF17D transfection. In tissue culture, BX795 could fully revert the block that plasmid transfection poses on YF17D infection in a type I interferon dependent manner, as confirmed by (i) a marked change in gene expression signatures, (ii) a rescue of full YF17D replication, and (iii) a massively increased virus yield. Inhibitors of TBK1 may hence be considered an adjuvant to potentiate novel PLLAV vaccines, which might boost PLLAV delivery toward their use in vivo.

DEB025 (Alisporivir) inhibits hepatitis C virus replication by preventing a cyclophilin A induced cis-trans isomerisation in domain II of NS5A.

DEB025/Debio 025 (Alisporivir) is a cyclophilin (Cyp)-binding molecule with potent anti-hepatitis C virus (HCV) activity both in vitro and in vivo. It is currently being evaluated in phase II clinical trials. DEB025 binds to CypA, a peptidyl-prolyl cis-trans isomerase which is a crucial cofactor for HCV replication. Here we report that it was very difficult to select resistant replicons (genotype 1b) to DEB025, requiring an average of 20 weeks (four independent experiments), compared to the typically <2 weeks with protease or polymerase inhibitors. This indicates a high genetic barrier to resistance for DEB025. Mutation D320E in NS5A was the only mutation consistently selected in the replicon genome. This mutation alone conferred a low-level (3.9-fold) resistance. Replacing the NS5A gene (but not the NS5B gene) from the wild type (WT) genome with the corresponding sequence from the DEB025(res) replicon resulted in transfer of resistance. Cross-resistance with cyclosporine A (CsA) was observed, whereas NS3 protease and NS5B polymerase inhibitors retained WT-activity against DEB025(res) replicons. Unlike WT, DEB025(res) replicon replicated efficiently in CypA knock down cells. However, DEB025 disrupted the interaction between CypA and NS5A regardless of whether the NS5A protein was derived from WT or DEB025(res) replicon. NMR titration experiments with peptides derived from the WT or the DEB025(res) domain II of NS5A corroborated this observation in a quantitative manner. Interestingly, comparative NMR studies on two 20-mer NS5A peptides that contain D320 or E320 revealed a shift in population between the major and minor conformers. These data suggest that D320E conferred low-level resistance to DEB025 probably by reducing the need for CypA-dependent isomerisation of NS5A. Prolonged DEB025 treatment and multiple genotypic changes may be necessary to generate significant resistance to DEB025, underlying the high barrier to resistance.

Antiviral therapy for hepatitis C virus: beyond the standard of care.

Hepatitis C virus (HCV) represents a major health burden, with an estimated 180 million chronically infected individuals worldwide. These patients are at increased risk of developing liver cirrhosis and hepatocellular carcinoma. Infection with HCV is the leading cause of liver transplantation in the Western world. Currently, the standard of care (SoC) consists of pegylated interferon alpha (pegIFN-α) and ribavirin (RBV). However this therapy has a limited efficacy and is associated with serious side effects. Therefore more tolerable, highly potent inhibitors of HCV replication are urgently needed. Both Specifically Targeted Antiviral Therapy for HCV (STAT-C) and inhibitors that are believed to interfere with the host-viral interaction are discussed.

Debio 025, a cyclophilin binding molecule, is highly efficient in clearing hepatitis C virus (HCV) replicon-containing cells when used alone or in combination with specifically targeted antiviral therapy for HCV (STAT-C) inhibitors.

Debio 025 is a potent inhibitor of hepatitis C virus (HCV) replication (J. Paeshuyse et al., Hepatology 43:761-770, 2006). In phase I clinical studies, monotherapy (a Debio 025 dose of 1,200 mg twice a day) resulted in a mean maximal decrease in the viral load of 3.6 log(10) units (R. Flisiak et al., Hepatology 47:817-826, 2008), whereas a reduction of 4.6 log(10) units was obtained in phase II studies when Debio 025 was combined with interferon (R. Flisiak et al., J. Hepatol., 48:S62, 2008). We here report on the particular characteristics of the in vitro anti-HCV activities of Debio 025. The combination of Debio 025 with either ribavirin or specifically targeted antiviral therapy for HCV (STAT-C) inhibitors (NS3 protease or NS5B [nucleoside and nonnucleoside] polymerase inhibitors) resulted in additive antiviral activity in short-term antiviral assays. Debio 025 has the unique ability to clear hepatoma cells from their HCV replicon when it is used alone or in combination with interferon and STAT-C inhibitors. Debio 025, when it was used at concentrations that have been observed in human plasma (0.1 or 0.5 muM), was able to delay or prevent the development of resistance to HCV protease inhibitors as well as to nucleoside and nonnucleoside polymerase inhibitors. Debio 025 forms an attractive drug candidate for the treatment of HCV infections in combination with standard interferon-based treatment and treatments that directly target the HCV polymerase and/or protease.

Comparative in vitro anti-hepatitis C virus activities of a selected series of polymerase, protease, and helicase inhibitors.

We report here a comparative study of the anti-hepatitis C virus (HCV) activities of selected (i) nucleoside polymerase, (ii) nonnucleoside polymerase, (iii) alpha,gamma-diketo acid polymerase, (iv) NS3 protease, and (v) helicase inhibitors, as well as (vi) cyclophilin binding molecules and (vii) alpha 2b interferon in four different HCV genotype 1b replicon systems.

Ribavirin antagonizes the in vitro anti-hepatitis C virus activity of 2'-C-methylcytidine, the active component of valopicitabine.

Ribavirin antagonizes the in vitro anti-hepatitis C virus (HCV) activity of the pyrimidine nucleoside analogue 2'-C-methylcytidine, the active component of the experimental anti-HCV drug valopicitabine. In contrast, the combination of ribavirin with either the purine nucleoside analogue 2'-C-methyladenosine or the HCV protease inhibitor VX-950 resulted in an additive antiviral activity. These findings may have implications when planning clinical studies with valopicitabine.

Hemin potentiates the anti-hepatitis C virus activity of the antimalarial drug artemisinin.

We report that the antimalarial drug artemisinin inhibits hepatitis C virus (HCV) replicon replication in a dose-dependent manner in two replicon constructs at concentrations that have no effect on the proliferation of the exponentially growing host cells. The 50% effective concentration (EC(50)) for inhibition of HCV subgenomic replicon replication in Huh 5-2 cells (luciferase assay) by artemisinin was 78+/-21 microM. Hemin, an iron donor, was recently reported to inhibit HCV replicon replication [mediated by inhibition of the viral polymerase (C. Fillebeen, A.M. Rivas-Estilla, M. Bisaillon, P. Ponka, M. Muckenthaler, M.W. Hentze, A.E. Koromilas, K. Pantopoulos, Iron inactivates the RNA polymerase NS5B and suppresses subgenomic replication of hepatitis C virus, J. Biol. Chem. 280 (2005) 9049-9057.)] at a concentration that had no adverse effect on the host cells. When combined, artemisinin and hemin resulted, over a broad concentration range, in a pronounced synergistic antiviral activity. Also at a concentration (2 microM) that alone had no effect on HCV replication, hemin still potentiated the anti-HCV activity of artemisinin.