Immunogenicity and efficacy of one and two doses of Ad26.COV2.S COVID vaccine in adult and aged NHP.

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 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.

Ad26 vector-based COVID-19 vaccine encoding a prefusion-stabilized SARS-CoV-2 Spike immunogen induces potent humoral and cellular immune responses.

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 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-γ. 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).

A unifying structural and functional model of the coronavirus replication organelle: Tracking down RNA synthesis.

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 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.

Adaptive Mutations in Replicase Transmembrane Subunits Can Counteract Inhibition of Equine Arteritis Virus RNA Synthesis by Cyclophilin Inhibitors.

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 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 ∼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 pan-nidovirus 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.

Cyclophilins and cyclophilin inhibitors in nidovirus replication.

Cyclophilins (Cyps) belong to the family of peptidyl-prolyl isomerases (PPIases). The PPIase activity of most Cyps is inhibited by the immunosuppressive drug cyclosporin A and several of its non-immunosuppressive analogs, which can also block the replication of nidoviruses (arteriviruses and coronaviruses). Cyclophilins have been reported to play an essential role in the replication of several other RNA viruses, including human immunodeficiency virus-1, hepatitis C virus, and influenza A virus. Likewise, the replication of various nidoviruses was reported to depend on Cyps or other PPIases. This review summarizes our current understanding of this class of nidovirus-host interactions, including the potential function of in particular CypA and the inhibitory effect of Cyp inhibitors. Also the involvement of the FK-506-binding proteins and parvulins is discussed. The nidovirus data are placed in a broader perspective by summarizing the most relevant data on Cyp interactions and Cyp inhibitors for other RNA viruses.

Coronaviruses and arteriviruses display striking differences in their cyclophilin A-dependence during replication in cell culture.

Cyclophilin A (CypA) is an important host factor in the replication of a variety of RNA viruses. Also the replication of several nidoviruses was reported to depend on CypA, although possibly not to the same extent. These prior studies are difficult to compare, since different nidoviruses, cell lines and experimental set-ups were used. Here, we investigated the CypA dependence of three distantly related nidoviruses that can all replicate in Huh7 cells: the arterivirus equine arteritis virus (EAV), the alphacoronavirus human coronavirus 229E (HCoV-229E), and the betacoronavirus Middle East respiratory syndrome coronavirus (MERS-CoV). The replication of these viruses was compared in the same parental Huh7 cells and in CypA-knockout Huh7 cells generated using CRISPR/Cas9-technology. CypA depletion reduced EAV yields by ~ 3-log, whereas MERS-CoV progeny titers were modestly reduced (3-fold) and HCoV-229E replication was unchanged. This study reveals that the replication of nidoviruses can differ strikingly in its dependence on cellular CypA.

Host Factors in Coronavirus Replication.

Coronaviruses are pathogens with a serious impact on human and animal health. They mostly cause enteric or respiratory disease, which can be severe and life threatening, e.g., in the case of the zoonotic coronaviruses causing severe acute respiratory syndrome (SARS) and Middle East Respiratory Syndrome (MERS) in humans. Despite the economic and societal impact of such coronavirus infections, and the likelihood of future outbreaks of additional pathogenic coronaviruses, our options to prevent or treat coronavirus infections remain very limited. This highlights the importance of advancing our knowledge on the replication of these viruses and their interactions with the host. Compared to other +RNA viruses, coronaviruses have an exceptionally large genome and employ a complex genome expression strategy. Next to a role in basic virus replication or virus assembly, many of the coronavirus proteins expressed in the infected cell contribute to the coronavirus-host interplay. For example, by interacting with the host cell to create an optimal environment for coronavirus replication, by altering host gene expression or by counteracting the host's antiviral defenses. These coronavirus-host interactions are key to viral pathogenesis and will ultimately determine the outcome of infection. Due to the complexity of the coronavirus proteome and replication cycle, our knowledge of host factors involved in coronavirus replication is still in an early stage compared to what is known for some other +RNA viruses. This review summarizes our current understanding of coronavirus-host interactions at the level of the infected cell, with special attention for the assembly and function of the viral RNA-synthesising machinery and the evasion of cellular innate immune responses.

Alisporivir inhibits MERS- and SARS-coronavirus replication in cell culture, but not SARS-coronavirus infection in a mouse model.

Currently, there is no registered treatment for infections with emerging zoonotic coronaviruses like SARS- and MERS-coronavirus. We here report that in cultured cells low-micromolar concentrations of alisporivir, a non-immunosuppressive cyclosporin A-analog, inhibit the replication of four different coronaviruses, including MERS- and SARS-coronavirus. Ribavirin was found to further potentiate the antiviral effect of alisporivir in these cell culture-based infection models, but this combination treatment was unable to improve the outcome of SARS-CoV infection in a mouse model. Nevertheless, our data provide a basis to further explore the potential of Cyp inhibitors as host-directed, broad-spectrum inhibitors of coronavirus replication.

A Kinome-Wide Small Interfering RNA Screen Identifies Proviral and Antiviral Host Factors in Severe Acute Respiratory Syndrome Coronavirus Replication, Including Double-Stranded RNA-Activated Protein Kinase and Early Secretory Pathway Proteins.

UNLABELLED: To identify host factors relevant for severe acute respiratory syndrome-coronavirus (SARS-CoV) replication, we performed a small interfering RNA (siRNA) library screen targeting the human kinome. Protein kinases are key regulators of many cellular functions, and the systematic knockdown of their expression should provide a broad perspective on factors and pathways promoting or antagonizing coronavirus replication. In addition to 40 proteins that promote SARS-CoV replication, our study identified 90 factors exhibiting an antiviral effect. Pathway analysis grouped subsets of these factors in specific cellular processes, including the innate immune response and the metabolism of complex lipids, which appear to play a role in SARS-CoV infection. Several factors were selected for in-depth validation in follow-up experiments. In cells depleted for the ß2 subunit of the coatomer protein complex (COPB2), the strongest proviral hit, we observed reduced SARS-CoV protein expression and a >2-log reduction in virus yield. Knockdown of the COPB2-related proteins COPB1 and Golgi-specific brefeldin A-resistant guanine nucleotide exchange factor 1 (GBF1) also suggested that COPI-coated vesicles and/or the early secretory pathway are important for SARS-CoV replication. Depletion of the antiviral double-stranded RNA-activated protein kinase (PKR) enhanced virus replication in the primary screen, and validation experiments confirmed increased SARS-CoV protein expression and virus production upon PKR depletion. In addition, cyclin-dependent kinase 6 (CDK6) was identified as a novel antiviral host factor in SARS-CoV replication. The inventory of pro- and antiviral host factors and pathways described here substantiates and expands our understanding of SARS-CoV replication and may contribute to the identification of novel targets for antiviral therapy. IMPORTANCE: Replication of all viruses, including SARS-CoV, depends on and is influenced by cellular pathways. Although substantial progress has been made in dissecting the coronavirus replicative cycle, our understanding of the host factors that stimulate (proviral factors) or restrict (antiviral factors) infection remains far from complete. To study the role of host proteins in SARS-CoV infection, we set out to systematically identify kinase-regulated processes that influence virus replication. Protein kinases are key regulators in signal transduction, controlling a wide variety of cellular processes, and many of them are targets of approved drugs and other compounds. Our screen identified a variety of hits and will form the basis for more detailed follow-up studies that should contribute to a better understanding of SARS-CoV replication and coronavirus-host interactions in general. The identified factors could be interesting targets for the development of host-directed antiviral therapy to treat infections with SARS-CoV or other pathogenic coronaviruses.

Design, synthesis and evaluation of a series of acyclic fleximer nucleoside analogues with anti-coronavirus activity.

A series of doubly flexible nucleoside analogues were designed based on the acyclic sugar scaffold of acyclovir and the flex-base moiety found in the fleximers. The target compounds were evaluated for their antiviral potential and found to inhibit several coronaviruses. Significantly, compound 2 displayed selective antiviral activity (CC50 >3× EC50) towards human coronavirus (HCoV)-NL63 and Middle East respiratory syndrome-coronavirus, but not severe acute respiratory syndrome-coronavirus. In the case of HCoV-NL63 the activity was highly promising with an EC50 <10 µM and a CC50 >100 µM. As such, these doubly flexible nucleoside analogues are viewed as a novel new class of drug candidates with potential for potent inhibition of coronaviruses.

Screening of an FDA-approved compound library identifies four small-molecule inhibitors of Middle East respiratory syndrome coronavirus replication in cell culture.

Coronaviruses can cause respiratory and enteric disease in a wide variety of human and animal hosts. The 2003 outbreak of severe acute respiratory syndrome (SARS) first demonstrated the potentially lethal consequences of zoonotic coronavirus infections in humans. In 2012, a similar previously unknown coronavirus emerged, Middle East respiratory syndrome coronavirus (MERS-CoV), thus far causing over 650 laboratory-confirmed infections, with an unexplained steep rise in the number of cases being recorded over recent months. The human MERS fatality rate of ∼ 30% is alarmingly high, even though many deaths were associated with underlying medical conditions. Registered therapeutics for the treatment of coronavirus infections are not available. Moreover, the pace of drug development and registration for human use is generally incompatible with strategies to combat emerging infectious diseases. Therefore, we have screened a library of 348 FDA-approved drugs for anti-MERS-CoV activity in cell culture. If such compounds proved sufficiently potent, their efficacy might be directly assessed in MERS patients. We identified four compounds (chloroquine, chlorpromazine, loperamide, and lopinavir) inhibiting MERS-CoV replication in the low-micromolar range (50% effective concentrations [EC(50)s], 3 to 8 µM). Moreover, these compounds also inhibit the replication of SARS coronavirus and human coronavirus 229E. Although their protective activity (alone or in combination) remains to be assessed in animal models, our findings may offer a starting point for treatment of patients infected with zoonotic coronaviruses like MERS-CoV. Although they may not necessarily reduce viral replication to very low levels, a moderate viral load reduction may create a window during which to mount a protective immune response.

MERS-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin A or interferon-α treatment.

Coronavirus (CoV) infections are commonly associated with respiratory and enteric disease in humans and animals. The 2003 outbreak of severe acute respiratory syndrome (SARS) highlighted the potentially lethal consequences of CoV-induced disease in humans. In 2012, a novel CoV (Middle East Respiratory Syndrome coronavirus; MERS-CoV) emerged, causing 49 human cases thus far, of which 23 had a fatal outcome. In this study, we characterized MERS-CoV replication and cytotoxicity in human and monkey cell lines. Electron microscopy of infected Vero cells revealed extensive membrane rearrangements, including the formation of double-membrane vesicles and convoluted membranes, which have been implicated previously in the RNA synthesis of SARS-CoV and other CoVs. Following infection, we observed rapidly increasing viral RNA synthesis and release of high titres of infectious progeny, followed by a pronounced cytopathology. These characteristics were used to develop an assay for antiviral compound screening in 96-well format, which was used to identify cyclosporin A as an inhibitor of MERS-CoV replication in cell culture. Furthermore, MERS-CoV was found to be 50-100 times more sensitive to alpha interferon (IFN-α) treatment than SARS-CoV, an observation that may have important implications for the treatment of MERS-CoV-infected patients. MERS-CoV infection did not prevent the IFN-induced nuclear translocation of phosphorylated STAT1, in contrast to infection with SARS-CoV where this block inhibits the expression of antiviral genes. These findings highlight relevant differences between these distantly related zoonotic CoVs in terms of their interaction with and evasion of the cellular innate immune response.

Cyclophilin inhibitors block arterivirus replication by interfering with viral RNA synthesis.

Virus replication strongly depends on cellular factors, in particular, on host proteins. Here we report that the replication of the arteriviruses equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV) is strongly affected by low-micromolar concentrations of cyclosporine A (CsA), an inhibitor of members of the cyclophilin (Cyp) family. In infected cells, the expression of a green fluorescent protein (GFP) reporter gene inserted into the PRRSV genome was inhibited with a half-maximal inhibitory concentration (IC(50)) of 5.2 µM, whereas the GFP expression of an EAV-GFP reporter virus was inhibited with an IC(50) of 0.95 µM. Debio-064, a CsA analog that lacks its undesirable immunosuppressive properties, inhibited EAV replication with an IC(50) that was 3-fold lower than that of CsA, whereas PRRSV-GFP replication was inhibited with an IC(50) similar to that of CsA. The addition of 4 µM CsA after infection prevented viral RNA and protein synthesis in EAV-infected cells, and CsA treatment resulted in a 2.5- to 4-log-unit reduction of PRRSV or EAV infectious progeny. A complete block of EAV RNA synthesis was also observed in an in vitro assay using isolated viral replication structures. The small interfering RNA-mediated knockdown of Cyp family members revealed that EAV replication strongly depends on the expression of CypA but not CypB. Furthermore, upon fractionation of intracellular membranes in density gradients, CypA was found to cosediment with membranous EAV replication structures, which could be prevented by CsA treatment. This suggests that CypA is an essential component of the viral RNA-synthesizing machinery.

Cyclosporin A inhibits the replication of diverse coronaviruses.

Low micromolar, non-cytotoxic concentrations of cyclosporin A (CsA) strongly affected the replication of severe acute respiratory syndrome coronavirus (SARS-CoV), human coronavirus 229E and mouse hepatitis virus in cell culture, as was evident from the strong inhibition of GFP reporter gene expression and a reduction of up to 4 logs in progeny titres. Upon high-multiplicity infection, CsA treatment rendered SARS-CoV RNA and protein synthesis almost undetectable, suggesting an early block in replication. siRNA-mediated knockdown of the expression of the prominent CsA targets cyclophilin A and B did not affect SARS-CoV replication, suggesting either that these specific cyclophilin family members are dispensable or that the reduced expression levels suffice to support replication.

The in vitro RNA synthesizing activity of the isolated arterivirus replication/transcription complex is dependent on a host factor.

The cytoplasmic replication of positive-stranded RNA viruses is associated with characteristic, virus-induced membrane structures that are derived from host cell organelles. We used the prototype arterivirus, equine arteritis virus (EAV), to gain insight into the structure and function of the replication/transcription complex (RTC) of nidoviruses. RTCs were isolated from EAV-infected cells, and their activity was studied using a newly developed in vitro assay for viral RNA synthesis, which reproduced the synthesis of both viral genome and subgenomic mRNAs. A detailed characterization of this system and its reaction products is described. RTCs isolated from cytoplasmic extracts by differential centrifugation were inactive unless supplemented with a cytosolic host protein factor, which, according to subsequent size fractionation analysis, has a molecular mass in the range of 59-70 kDa. This host factor was found to be present in a wide variety of eukaryotes. Several EAV replicase subunits cosedimented with newly made viral RNA in a heavy membrane fraction that contained all RNA-dependent RNA polymerase activity. This fraction contained the characteristic double membrane vesicles (DMVs) that were previously implicated in EAV RNA synthesis and could be immunolabeled for EAV nonstructural proteins (nsps). Replicase subunits directly involved in viral RNA synthesis (nsp9 and nsp10) or DMV formation (nsp2 and nsp3) exclusively cosedimented with the active RTC. Subgenomic mRNAs appeared to be released from the complex, whereas newly made genomic RNA remained more tightly associated. Taken together, our data strongly support a link between DMVs and the RNA-synthesizing machinery of arteriviruses.