Novel intranasal vaccine targeting SARS-CoV-2 receptor binding domain to mucosal microfold cells and adjuvanted with TLR3 agonist Riboxxim™ elicits strong antibody and T-cell responses in mice.

SARS-CoV-2 continues to circulate in the human population necessitating regular booster immunization for its long-term control. Ideally, vaccines should ideally not only protect against symptomatic disease, but also prevent transmission via asymptomatic shedding and cover existing and future variants of the virus. This may ultimately only be possible through induction of potent and long-lasting immune responses in the nasopharyngeal tract, the initial entry site of SARS-CoV-2. To this end, we have designed a vaccine based on recombinantly expressed receptor binding domain (RBD) of SARS-CoV-2, fused to the C-terminus of C. perfringens enterotoxin, which is known to target Claudin-4, a matrix molecule highly expressed on mucosal microfold (M) cells of the nasal and bronchial-associated lymphoid tissues. To further enhance immune responses, the vaccine was adjuvanted with a novel toll-like receptor 3/RIG-I agonist (Riboxxim™), consisting of synthetic short double stranded RNA. Intranasal prime-boost immunization of mice induced robust mucosal and systemic anti-SARS-CoV-2 neutralizing antibody responses against SARS-CoV-2 strains Wuhan-Hu-1, and several variants (B.1.351/beta, B.1.1.7/alpha, B.1.617.2/delta), as well as systemic T-cell responses. A combination vaccine with M-cell targeted recombinant HA1 from an H1N1 G4 influenza strain also induced mucosal and systemic antibodies against influenza. Taken together, the data show that development of an intranasal SARS-CoV-2 vaccine based on recombinant RBD adjuvanted with a TLR3 agonist is feasible, also as a combination vaccine against influenza.

PLGA-particle vaccine carrying TLR3/RIG-I ligand Riboxxim synergizes with immune checkpoint blockade for effective anti-cancer immunotherapy.

With emerging supremacy, cancer immunotherapy has evolved as a promising therapeutic modality compared to conventional antitumor therapies. Cancer immunotherapy composed of biodegradable poly(lactic-co-glycolic acid) (PLGA) particles containing antigens and toll-like receptor ligands induces vigorous antitumor immune responses in vivo. Here, we demonstrate the supreme adjuvant effect of the recently developed and pharmaceutically defined double-stranded (ds)RNA adjuvant Riboxxim especially when incorporated into PLGA particles. Encapsulation of Riboxxim together with antigens potently activates murine and human dendritic cells, and elevated tumor-specific CD8+ T cell responses are superior to those obtained using classical dsRNA analogues. This PLGA particle vaccine affords primary tumor growth retardation, prevention of metastases, and prolonged survival in preclinical tumor models. Its advantageous therapeutic potency was further enhanced by immune checkpoint blockade that resulted in reinvigoration of cytotoxic T lymphocyte responses and tumor ablation. Thus, combining immune checkpoint blockade with immunotherapy based on Riboxxim-bearing PLGA particles strongly increases its efficacy.

Structural bases of norovirus RNA dependent RNA polymerase inhibition by novel suramin-related compounds.

Noroviruses (NV) are +ssRNA viruses responsible for severe gastroenteritis; no effective vaccines/antivirals are currently available. We previously identified Suramin (9) as a potent inhibitor of NV-RNA dependent RNA polymerase (NV-RdRp). Despite significant in vitro activities versus several pharmacological targets, Suramin clinical use is hampered by pharmacokinetics/toxicity problems. To improve Suramin access to NV-RdRp in vivo, a Suramin-derivative, 8, devoid of two sulphonate groups, was synthesized, achieving significant anti-human-NV-RdRp activity (IC50 = 28 nM); the compound inhibits also murine NV (mNV) RdRp. The synthesis process led to the isolation/characterization of lower molecular weight intermediates (3-7) hosting only one sulphonate head. The crystal structures of both hNV/mNV-RdRps in complex with 6, were analyzed, providing new knowledge on the interactions that a small fragment can establish with NV-RdRps, and establishing a platform for structure-guided optimization of potency, selectivity and drugability.

PPNDS inhibits murine Norovirus RNA-dependent RNA-polymerase mimicking two RNA stacking bases.

Norovirus (NV) is a major cause of gastroenteritis worldwide. Antivirals against such important pathogens are on demand. Among the viral proteins that orchestrate viral replication, RNA-dependent-RNA-polymerase (RdRp) is a promising drug development target. From an in silico-docking search focused on the RdRp active site, we selected the compound PPNDS, which showed low micromolar IC50vs. murine NV-RdRp in vitro. We report the crystal structure of the murine NV-RdRp/PPNDS complex showing that two molecules of the inhibitor bind in antiparallel stacking interaction, properly oriented to block exit of the newly synthesized RNA. Such inhibitor-binding mode mimics two stacked nucleotide-bases of the RdRp/ssRNA complex.

Delivery of suramin as an antiviral agent through liposomal systems.

Norovirus RNA-dependent RNA polymerase (RdRp) is a promising target enzyme for the development of new antiviral drugs. Starting from the crystal structure of norovirus RdRp, we had previously performed an in silico docking search using a library of low-molecular-weight compounds that enabled us to select molecules with predicted enzyme inhibitory activity. Among these, the polysulfonated naphthylurea suramin proved to inhibit in vitro both murine and human norovirus polymerases, with IC50 values in the low micromolar range. The negatively charged inhibitor, however, displayed poor cell permeability in cell-based experiments. Therefore, we produced different suramin-loaded liposome formulations and evaluated their activities in cell-based assays using murine norovirus cultivated in RAW 264.7 macrophages, as a model for norovirus genus. The results obtained show that suramin, when delivered through liposomes, can effectively inhibit murine norovirus replication.

Naphthalene-sulfonate inhibitors of human norovirus RNA-dependent RNA-polymerase.

Noroviruses are members of the Caliciviridae family of positive sense RNA viruses. In humans Noroviruses cause rapid onset diarrhea and vomiting. Currently Norovirus infection is responsible for 21 million gastroenteritis yearly cases in the USA. Nevertheless, despite the obvious public health and socio-economic relevance, no effective vaccines/antivirals are yet available to treat Norovirus infection. Since the activity of RNA-dependent RNA polymerase (RdRp) plays a key role in genome replication and in the synthesis/amplification of subgenomic RNA, the enzyme is considered a promising target for antiviral drug development. In this context, following the identification of suramin and NF023 as Norovirus RdRp inhibitors, we analyzed the potential inhibitory role of naphthalene di-sulfonate (NAF2), a fragment derived from these two molecules. Although NAF2, tested in enzymatic polymerase inhibition assays, displayed low activity against RdRp (IC50=14µM), the crystal structure of human Norovirus RdRp revealed a thumb domain NAF2 binding site that differs from that characterized for NF023/suramin. To further map the new potential inhibitory site, we focused on the structurally related molecule pyridoxal-5'-phosphate-6-(2'-naphthylazo-6'-nitro-4',8'-disulfonate) tetrasodium salt (PPNDS). PPNDS displayed below-micromolar inhibitory activity versus human Norovirus RdRp (IC50=0.45µM), similarly to suramin and NF023. Inspection of the crystal structure of the RdRp/PPNDS complex showed that the inhibitor bound to the NAF2 thumb domain site, highlighting the relevance of such new binding site for exploiting Norovirus RdRp inhibitors.

TLR7/8 agonists trigger immunostimulatory properties of human 6-sulfo LacNAc dendritic cells.

Imiquimod and resiquimod represent Toll-like receptor (TLR) 7 and 8 agonists, which emerged as attractive candidates for tumor therapy. To elucidate immune cells, which mainly contribute to TLR7/8-mediated antitumoral activity, we investigated the impact of imiquimod and resiquimod on native human 6-sulfo LacNAc (slan) dendritic cells (DCs). We found that both TLR7/8 agonists significantly improve the release of various proinflammatory cytokines by slanDCs and promote their tumor-directed cytotoxic activity. Furthermore, resiquimod efficiently augmented the ability of slanDCs to stimulate T cells and natural killer cells. These results indicate that imiquimod and resiquimod trigger various immunostimulatory properties of slanDCs, which may contribute to their antitumor effects.

Modification of small interfering RNAs to prevent off-target effects by the sense strand.

OBJECTIVE: There is a broad therapeutic potential for the application of small interfering RNAs (siRNAs). However, one has to ensure that siRNAs act specifically, only targeting the expression of one gene. Off-target effects raised by the sense strand have to be eliminated. METHODS AND RESULTS: We examined a particular bidirectional siRNA molecule, able to knockdown intercellular adhesion molecule-1 (ICAM-1) and tumor necrosis factor receptor-1 (TNFR-1) by the sense or antisense strand, respectively. Transfection of human venous endothelial cells with an unmodified siRNA molecule led to equal silencing of ICAM-1 and TNFR-1. In contrast, modified siRNA was able to knockdown ICAM-1 and TNFR-1 separately, with only the antisense strand. DISCUSSION: We found the modified siRNAs to inhibit off-target effects originated by the sense strand. Our approach demonstrates one possibility to modify siRNAs before starting a clinical approach to eliminate off-target effects.

Activation of dendritic cells by the novel Toll-like receptor 3 agonist RGC100.

Toll-like receptor (TLR) 3 agonists emerged as attractive candidates for vaccination strategies against tumors and pathogens. An important mechanism of action of such agonists is based on the activation of TLR3-expressing dendritic cells (DCs), which display a unique capacity to induce and stimulate T-cell responses. In this context, it has been demonstrated that targeting of TLR3 by double-stranded RNA such as poly(I:C) results in potent activation of DCs. Major disadvantages of poly(I:C) comprise its undefined chemical structure and very poor homogeneity, with subsequent unpredictable pharmacokinetics and high toxicity. In the present study, we evaluated the physicochemical properties and biological activity of the novel TLR3 agonist RGC100. RGC100 has a defined chemical structure, with a defined length (100 bp) and molecular weight (64.9 KDa) and a good solubility. RGC100 is stable in serum and activates myeloid DCs through TLR3 targeting, as evidenced by gene silencing experiments. Activation of mouse and human myeloid CD1c(+) DCs by RGC100 leads to secretion of several proinflammatory cytokines. In addition, RGC100 improves the ability of CD1c(+) DCs to stimulate T-cell proliferation. Due to its physicochemical properties and its immunostimulatory properties, RGC100 may represent a promising adjuvant for prophylactic and therapeutic vaccination strategies.

Structure-based inhibition of Norovirus RNA-dependent RNA polymerases.

Caliciviridae are RNA viruses with a single-stranded, positively oriented polyadenylated genome, responsible for a broad spectrum of diseases such as acute gastroenteritis in humans. Recently, analyses on the structures and functionalities of the RNA-dependent RNA polymerase (RdRp) from several Caliciviruses have been reported. The RdRp is predicted to play a key role in genome replication, as well as in synthesis and amplification of additional subgenomic RNA. Starting from the crystal structures of human Norovirus (hNV) RdRp, we performed an in silico docking search to identify synthetic compounds with predicted high affinity for the enzyme active site. The best-ranked candidates were tested in vitro on murine Norovirus (MNV) and hNV RdRps to assay their inhibition of RNA polymerization. The results of such combined computational and experimental screening approach led to the identification of two high-potency inhibitors: Suramin and NF023, both symmetric divalent molecules hosting two naphthalene-trisulfonic acid heads. We report here the crystal structure of MNV RdRp alone and in the presence of the two identified inhibitors. Both inhibitory molecules occupy the same RdRp site, between the fingers and thumb domains, with one inhibitor head close to residue 42 and to the protein active site. To further validate the structural results, we mutated Trp42 to Ala in MNV RdRp and the corresponding residue (i.e., Tyr41 to Ala) in hNV RdRp. Both NF023 and Suramin displayed reduced inhibitory potency versus the mutated hNV RdRp, thus hinting at a conserved inhibitor binding mode in the two polymerases.

Antiviral strategies to control calicivirus infections.

Caliciviridae are human or non-human pathogenic viruses with a high diversity. Some members of the Caliciviridae, i.e. human pathogenic norovirus or rabbit hemorrhagic disease virus (RHDV), are worldwide emerging pathogens. The norovirus is the major cause of viral gastroenteritis worldwide, accounting for about 85% of the outbreaks in Europe between 1995 and 2000. In the United States, 25 million cases of infection are reported each year. Since its emergence in 1984 as an agent of fatal hemorrhagic diseases in rabbits, RHDV has killed millions of rabbits and has been dispersed to all of the inhabitable continents. In view of their successful and apparently increasing emergence, the development of antiviral strategies to control infections due to these viral pathogens has now become an important issue in medicine and veterinary medicine. Antiviral strategies have to be based on an understanding of the epidemiology, transmission, clinical symptoms, viral replication and immunity to infection resulting from infection by these viruses. Here, we provide an overview of the mechanisms underlying calicivirus infection, focusing on the molecular aspects of replication in the host cell. Recent experimental data generated through an international collaboration on structural biology, virology and drug design within the European consortium VIZIER is also presented. Based on this analysis, we propose antiviral strategies that may significantly impact on the epidemiological characteristics of these highly successful viral pathogens.

Structural and biochemical analysis of human pathogenic astrovirus serine protease at 2.0 A resolution.

Astroviruses are single-stranded RNA viruses with a replication strategy based on the proteolytic processing of a polyprotein precursor and subsequent release of the viral enzymes of replication. So far, the catalytic properties of the astrovirus protease as well as its structure have remained uncharacterized. In this study, the three-dimensional crystal structure of the predicted protease of human pathogenic astrovirus has been solved to 2.0 A resolution. The protein displays the typical properties of trypsin-like enzymes but also several characteristic features: (i) a catalytic Asp-His-Ser triad in which the aspartate side chain is oriented away from the histidine, being replaced by a water molecule; (ii) a non-common conformation and composition of the S1 pocket; and (iii) the lack of the typical surface beta-ribbons together with a "featureless" shape of the substrate-binding site. Hydrolytic activity assays indicate that the S1 pocket recognises Glu and Asp side chains specifically, which, therefore, are predicted to occupy the P1 position on the substrate cleavage site. The positive electrostatic potential featured by the S1 region underlies this specificity. The comparative structural analysis highlights the peculiarity of the astrovirus protease, and differentiates it from the human and viral serine proteases.

The active form of the norovirus RNA-dependent RNA polymerase is a homodimer with cooperative activity.

Norovirus (NV) is a leading cause of gastroenteritis worldwide and a major public health concern. So far, the replication strategy of NV remains poorly understood, mainly because of the lack of a cell system to cultivate the virus. In this study, the function and the structure of a key viral enzyme of replication, the RNA-dependent RNA polymerase (RdRp, NS7), was examined. The overall structure of the NV NS7 RdRp was determined by X-ray crystallography to a 2.3 A (0.23 nm) resolution (PDB ID 2B43), displaying a right-hand fold typical of the template-dependent polynucleotide polymerases. Biochemical analysis evidenced that NV NS7 RdRp is active as a homodimer, with an apparent K(d) of 0.649 microM and a positive cooperativity (Hill coefficient n(H)=1.86). Crystals of the NV NS7 homodimer displayed lattices containing dimeric arrangements with high shape complementarity statistics. This experimental data on the structure and function of the NV RdRp may set the cornerstone for the development of polymerase inhibitors to control the infection with NV, a medically relevant pathogen.

Detection of herpesvirus and adenovirus co-infections with diagnostic DNA-microarrays.

In immunocompromised patients, the diagnosis of infections with herpesviruses and adenoviruses relies mainly on PCR amplification of viral genomic DNA from clinical samples. In the case of co-infections with two or more viruses, single amplification of viral DNA from clinical samples has proven to be time-consuming and expensive, hampering the efficient diagnosis and therapy of viral co-infections. In this study, a diagnostic DNA-microarray allowing simultaneous detection of herpes simplex virus types 1 and 2 (HSV 1/2), varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus-6 types A and B (HHV-6 A/B), and adenovirus in clinical samples was developed and validated. The assay displays a high analytical sensitivity (10genomeequivalents(GE)/reaction) and specificity, being cost-effective and time-saving. Because the DNA-microarray uses the same analytical conditions as real-time quantitative PCR, it can be used as a screening device for multiple viral infections, followed by selective viral load measurement depending on the clinical context. Those features make the DNA-microarray an attractive device for the management of viral infections in immunocompromised patients.

Functional characterization of the cleavage specificity of the sapovirus chymotrypsin-like protease.

Sapovirus is a positive-stranded RNA virus with a translational strategy based on processing of a polyprotein precursor by a chymotrypsin-like protease. So far, the molecular mechanisms regulating cleavage specificity of the viral protease are poorly understood. In this study, the catalytic activities and substrate specificities of the predicted forms of the viral protease, the 3C-like protease (NS6) and the 3CD-like protease-polymerase (NS6-7), were examined in vitro. The purified NS6 and NS6-7 were able to cleave synthetic peptides (15 to 17 residues) displaying the cleavage sites of the sapovirus polyprotein, both NS6 and NS6-7 proteins being active forms of the viral protease. High-performance liquid chromatography and subsequent mass spectrometry analysis of digested products showed a specific trans cleavage of peptides bearing Gln-Gly, Gln-Ala, Glu-Gly, Glu-Pro, or Glu-Lys at the scissile bond. In contrast, peptides bearing Glu-Ala or Gln-Asp at the scissile bond (NS4-NS5 and NS5-NS6, or NS6-NS7 junctions, respectively) were resistant to trans cleavage by NS6 or NS6-7 proteins, whereas cis cleavage of the Glu-Ala scissile bond of the NS5-NS6 junction was evidenced. Interestingly, the presence of a Phe at position P4 overruled the resistance to trans cleavage of the Glu-Ala junction (NS5-NS6), whereas substitutions at the P1 and P2' positions altered the cleavage efficiency. The differential cleavage observed is supported by a model of the substrate-binding site of the sapovirus protease, indicating that the P4, P1, and P2' positions in the substrate modulate the cleavage specificity and efficiency of the sapovirus chymotrypsin-like protease.

The thiazolobenzimidazole TBZE-029 inhibits enterovirus replication by targeting a short region immediately downstream from motif C in the nonstructural protein 2C.

TBZE-029 {1-(2,6-difluorophenyl)-6-trifluoromethyl-1H,3H-thiazolo[3,4-a]benzimidazole} is a novel selective inhibitor of the replication of several enteroviruses. We show that TBZE-029 exerts its antiviral activity through inhibition of viral RNA replication, without affecting polyprotein processing. To identify the viral target of TBZE-029, drug-resistant coxsackievirus B3 (CVB3) was selected. Genotyping of resistant clones led to the identification of three amino acid mutations in nonstructural protein 2C, clustered at amino acid positions 224, 227, and 229, immediately downstream of NTPase/helicase motif C. The mutations were reintroduced, either alone or combined, into an infectious full-length CVB3 clone. In particular the mutations at positions 227 and 229 proved essential for the altered sensitivity of CVB3 to TBZE-029. Resistant virus exhibited cross-resistance to the earlier-reported antienterovirus agents targeting 2C, namely, guanidine hydrochloride, HBB [2-(alpha-hydroxybenzyl)-benzimidazole], and MRL-1237 {1-(4-fluorophenyl)-2-[(4-imino-1,4-dihydropyridin-1-yl)methyl]benzimidazole hydrochloride}. The ATPase activity of 2C, however, remained unaltered in the presence of TBZE-029.

Differential cleavage of the norovirus polyprotein precursor by two active forms of the viral protease.

Protein translation in noroviruses requires translational processing of a polyprotein precursor by the viral protease. So far, the molecular mechanisms of catalytic cleavage by the viral protease are poorly understood. In this study, the catalytic activities and substrate specificities of the viral protease were examined in vitro by using synthetic peptides (11-15 residues) corresponding to the cleavage sites of the norovirus polyprotein. Both predicted forms of the viral protease, the 3C-like protease (3C(pro)) and the 3CD-like protease polymerase protein (3CD(propol)), displayed a specific trans cleavage activity of peptides bearing Gln-Gly at the scissile bond. In contrast, peptides bearing Glu-Gly at the scissile bond (p20/VPg and 3C(pro)/3D(pol) junctions) were resistant to trans-cleavage by 3C(pro) and 3CD(propol). Interestingly, the VPg/3C(pro) scissile bond (Glu-Ala) was cleaved only by 3CD(propol), and examination of relative cleavage efficiencies revealed significant differences in processing of peptides, indicating differential cleavage patterns for 3C(pro) and 3CD(propol).

Cidofovir and foscarnet for treatment of human herpesvirus 6 encephalitis in a neutropenic stem cell transplant recipient.

We report a stem cell transplant recipient who developed human herpesvirus 6 encephalitis. Human herpesvirus 6 load indicated massive intrathecal viral replication. Administration of cidofovir followed by foscarnet was associated with total clearance of human herpesvirus 6 infection. Cidofovir and foscarnet combination therapy may be beneficial for reducing mortality of (neutropenic) stem cell transplant recipients with human herpesvirus 6 encephalitis.

Structural and functional characterization of sapovirus RNA-dependent RNA polymerase.

Sapoviruses are one of the major agents of acute gastroenteritis in childhood. They form a tight genetic cluster (genus) in the Caliciviridae family that regroups both animal and human pathogenic strains. No permissive tissue culture has been developed for human sapovirus, limiting its characterization to surrogate systems. We report here on the first extensive characterization of the key enzyme of replication, the RNA-dependent RNA polymerase (RdRp) associated with the 3D(pol)-like protein. Enzymatically active sapovirus 3D(pol) and its defective mutant were expressed in Escherichia coli and purified. The overall structure of the sapovirus 3D(pol) was determined by X-ray crystallography to 2.32-A resolution. It revealed a right hand fold typical for template-dependent polynucleotide polymerases. The carboxyl terminus is located within the active site cleft, as observed in the RdRp of some (norovirus) but not other (lagovirus) caliciviruses. Sapovirus 3D(pol) prefers Mn(2+) over Mg(2+) but may utilize either as a cofactor in vitro. In a synthetic RNA template-dependent reaction, sapovirus 3D(pol) synthesizes a double-stranded RNA or labels the template 3' terminus by terminal transferase activity. Initiation of RNA synthesis occurs de novo on heteropolymeric templates or in a primer-dependent manner on polyadenylated templates. Strikingly, this mode of initiation of RNA synthesis was also described for norovirus, but not for lagovirus, suggesting structural and functional homologies in the RNA-dependent RNA polymerase of human pathogenic caliciviruses. This first experimental evidence makes sapovirus 3D(pol) an attractive target for developing drugs to control calicivirus infection in humans.

Chlorpromazine combined with cidofovir for treatment of a patient suffering from progressive multifocal leukoencephalopathy.

We report on a stem cell-transplanted patient with B cell chronic lymphatic leukemia who presented with a subacute onset of focal neurological deficits, gait abnormalities, emotional lability and dementia. Progressive multifocal leukoencephalopathy was diagnosed by magnetic resonance imaging (MRI) of the brain and detection of JC virus genome in the cerebrospinal fluid. Cidofovir and the 5HT2A receptor antagonist chlorpromazine were subsequently administered. A follow-up MRI of the brain 2 weeks after initiation of the antiviral therapy displayed progress of the demyelination, and the patient died 3 months after onset of the neurological symptoms. This report highlights the need for the development of novel and potent strategies for treatment of progressive multifocal leukoencephalopathy.