Safe and effective coronavirus disease-19 (COVID-19) vaccines are urgently needed to control the ongoing pandemic. While single-dose vaccine regimens would provide multiple advantages, two doses... Show moreSafe and effective coronavirus disease-19 (COVID-19) vaccines are urgently needed to control the ongoing pandemic. While single-dose vaccine regimens would provide multiple advantages, two doses may improve the magnitude and durability of immunity and protective efficacy. We assessed one-and two-dose regimens of the Ad26.COV2.S vaccine candidate in adult and aged nonhuman primates (NHPs). A two-dose Ad26.COV2.S regimen induced higher peak binding and neutralizing antibody responses compared with a single dose. In one-dose regimens, neutralizing antibody responses were stable for at least 14 wk, providing an early indication of durability. Ad26.COV2.S induced humoral immunity and T helper cell (Th cell) 1-skewed cellular responses in aged NHPs that were comparable to those in adult animals. Aged Ad26.COV2.S-vaccinated animals challenged 3 mo after dose 1 with a SARS-CoV-2 spike G614 variant showed near complete lower and substantial upper respiratory tract protection for both regimens. Neutralization of variants of concern by NHP sera was reduced for B.1.351 lineages while maintained for the B.1.1.7 lineage independent of Ad26.COV2.S vaccine regimen. Show less
Bos, R.; Rutten, L.; Lubbe, J.E.M. van der; Bakkers, M.J.G.; Hardenberg, G.; Wegmann, F.; ... ; Schuitemaker, H. 2020
Development of effective preventative interventions against SARS-CoV-2, the etiologic agent of COVID-19 is urgently needed. The viral surface spike (S) protein of SARS-CoV-2 is a key target for... Show moreDevelopment of effective preventative interventions against SARS-CoV-2, the etiologic agent of COVID-19 is urgently needed. The viral surface spike (S) protein of SARS-CoV-2 is a key target for prophylactic measures as it is critical for the viral replication cycle and the primary target of neutralizing antibodies. We evaluated design elements previously shown for other coronavirus S protein-based vaccines to be successful, e.g., prefusion-stabilizing substitutions and heterologous signal peptides, for selection of a S-based SARS-CoV-2 vaccine candidate. In vitro characterization demonstrated that the introduction of stabilizing substitutions (i.e., furin cleavage site mutations and two consecutive prolines in the hinge region of S2) increased the ratio of neutralizing versus non-neutralizing antibody binding, suggestive for a prefusion conformation of the S protein. Furthermore, the wild-type signal peptide was best suited for the correct cleavage needed for a natively folded protein. These observations translated into superior immunogenicity in mice where the Ad26 vector encoding for a membrane-bound stabilized S protein with a wild-type signal peptide elicited potent neutralizing humoral immunity and cellular immunity that was polarized towards Th1 IFN-gamma. This optimized Ad26 vector-based vaccine for SARS-CoV-2, termed Ad26.COV2.S, is currently being evaluated in a phase I clinical trial (ClinicalTrials.gov Identifier: NCT04436276). Show less
Zoonotic coronavirus (CoV) infections, such as those responsible for the current severe acute respiratory syndrome-CoV 2 (SARS-CoV-2) pandemic, cause grave international public health concern. In... Show moreZoonotic coronavirus (CoV) infections, such as those responsible for the current severe acute respiratory syndrome-CoV 2 (SARS-CoV-2) pandemic, cause grave international public health concern. In infected cells, the CoV RNA-synthesizing machinery associates with modified endoplasmic reticulum membranes that are transformed into the viral replication organelle (RO). Although double-membrane vesicles (DMVs) appear to be a pan-CoV RO element, studies to date describe an assortment of additional CoV-induced membrane structures. Despite much speculation, it remains unclear which RO element(s) accommodate viral RNA synthesis. Here we provide detailed 2D and 3D analyses of CoV ROs and show that diverse CoVs essentially induce the same membrane modifications, including the small open double-membrane spherules (DMSs) previously thought to be restricted to gamma- and delta-CoV infections and proposed as sites of replication. Metabolic labeling of newly synthesized viral RNA followed by quantitative electron microscopy (EM) autoradiography revealed abundant viral RNA synthesis associated with DMVs in cells infected with the beta-CoVs Middle East respiratory syndrome-CoV (MERS-CoV) and SARS-CoV and the gamma-CoV infectious bronchitis virus. RNA synthesis could not be linked to DMSs or any other cellular or virus-induced structure. Our results provide a unifying model of the CoV RO and clearly establish DMVs as the central hub for viral RNA synthesis and a potential drug target in CoV infection. Show less
Wilde, A.H. de; Boomaars-van Der Zanden, A.L.; Jong, A.W.M. de; Barcena, M.; Snijder, E.J.; Posthuma, C.C. 2019
Previously, the cyclophilin inhibitors cyclosporine (CsA) and alisporivir (ALV) were shown to inhibit the replication of diverse RNA viruses, including arteriviruses and coronaviruses, which both... Show morePreviously, the cyclophilin inhibitors cyclosporine (CsA) and alisporivir (ALV) were shown to inhibit the replication of diverse RNA viruses, including arteriviruses and coronaviruses, which both belong to the order Nidovirales. In this study, we aimed to identify arterivirus proteins involved in the mode of action of cyclophilin inhibitors and to investigate how these compounds inhibit arterivirus RNA synthesis in the infected cell. Repeated passaging of the arterivirus prototype equine arteritis virus (EAV) in the presence of CsA revealed that reduced drug sensitivity is associated with the emergence of adaptive mutations in nonstructural protein 5 (nsp5), one of the transmembrane subunits of the arterivirus replicase polyprotein. Introduction of singular nsp5 mutations (nsp5 Q21R, Y113H, or A134V) led to an similar to 2-fold decrease in sensitivity to CsA treatment, whereas combinations of mutations further increased EAV's CsA resistance. The detailed experimental characterization of engineered EAV mutants harboring CsA resistance mutations implicated nsp5 in arterivirus RNA synthesis. Particularly, in an in vitro assay, EAV RNA synthesis was far less sensitive to CsA treatment when nsp5 contained the adaptive mutations mentioned above. Interestingly, for increased sensitivity to the closely related drug ALV, CsA-resistant nsp5 mutants required the incorporation of an additional adaptive mutation, which resided in nsp2 (H114R), another transmembrane subunit of the arterivirus replicase. Our study provides the first evidence for the involvement of nsp2 and nsp5 in the mechanism underlying the inhibition of arterivirus replication by cyclophilin inhibitors.IMPORTANCE Currently, no approved treatments are available to combat infections with nidoviruses, a group of positive-stranded RNA viruses, including important zoonotic and veterinary pathogens. Previously, the cyclophilin inhibitors cyclosporine (CsA) and alisporivir (ALV) were shown to inhibit the replication of diverse nidoviruses (both arteriviruses and coronaviruses), and they may thus represent a class of pannidovirus inhibitors. In this study, using the arterivirus prototype equine arteritis virus, we have established that resistance to CsA and ALV treatment is associated with adaptive mutations in two transmembrane subunits of the viral replication machinery, nonstructural proteins 2 and 5. This is the first evidence for the involvement of specific replicase subunits of arteriviruses in the mechanism underlying the inhibition of their replication by cyclophilin inhibitors. Understanding this mechanism of action is of major importance to guide future drug design, both for nidoviruses and for other RNA viruses inhibited by these compounds. Show less
The interplay between nidoviruses and the infected host cell was investigated. Arterivirus RNA-synthesising activity was shown to depend on intact membranes and on a cytosolic host protein which... Show moreThe interplay between nidoviruses and the infected host cell was investigated. Arterivirus RNA-synthesising activity was shown to depend on intact membranes and on a cytosolic host protein which does not cosediment with the RTC. Furthermore, the immunosuppressant drug cyclosporin A (CsA) blocks replication of EAV and the swine arterivirus PRRSV in cell culture. Cyclophilin A appears to be an important host factor for EAV replication. CsA may be a nidovirus-wide inhibitor of replication since this compound also blocked replication of the coronaviruses SARS-CoV, HCoV-229E, and MHV. We further described a kinase siRNA library screen that identified ninety antiviral and forty proviral hits and signalling pathways involved in the SARS-CoV replicative cycle. PKR (antiviral) and COPB2 (proviral) were validated in follow-up experiments. We also investigated MERS-CoV replication characteristics and we described an assay t o screen for compounds that block MERS-CoV infection. CsA and pegylated IFN-_ (PEG-IFN) significantly inhibited infection, and MERS-CoV was shown to be much more sensitive to PEG-IFN treatment than SARS-CoV, an observation that may have implications for the treatment of MERS-CoV infection. The data presented in this thesis might contribute to better understand virus replication and hopefully provide additional starting points for the development of antiviral strategies. Show less
Wilde, A.H. de; Raj, V.S.; Oudshoorn, D.; Bestebroer, T.M.; Nieuwkoop, S. van; Limpens, R.W.A.L.; ... ; Hoogen, B.G. van den 2013