Animal experiments show impact of vaccination on reduction of SARS-CoV-2 virus circulation: A model for vaccine development?

BACKGROUND: In the pre-clinical phase, SARS-CoV-2 vaccines were tested in animal models, including exposure trials, to investigate protection against SARS-CoV-2. These studies paved the way for clinical development. The objective of our review was to provide an overview of published animal exposure results, focussing on the capacity of vaccines to reduce/prevent viral shedding. METHOD: Using Medline, we retrieved eighteen papers on eight different vaccine platforms in four animal models. Data were extracted on presence/absence of viral RNA in nose, throat, or lungs, and neutralizing antibody levels in the blood. RESULTS: All vaccines showed a tendency of reduced viral load after exposure. Particularly nasal swab results are likely to give an indication about the impact on virus excretion in the environment. Similarly, the reduction or prevention of viral replication in the bronchoalveolar environment might be related with disease prevention, explaining the high efficacy in clinical trials. DISCUSSION: Although it remains difficult to compare the results directly, the potential for a strong reduction of transmission was shown, indicating that the animal models predicted what is observed in the field after large scale human vaccination. This merits further attention for standardization of exposure experiments, with the intention to speed up future vaccine development.

Monocyte-driven atypical cytokine storm and aberrant neutrophil activation as key mediators of COVID-19 disease severity.

Epidemiological and clinical reports indicate that SARS-CoV-2 virulence hinges upon the triggering of an aberrant host immune response, more so than on direct virus-induced cellular damage. To elucidate the immunopathology underlying COVID-19 severity, we perform cytokine and multiplex immune profiling in COVID-19 patients. We show that hypercytokinemia in COVID-19 differs from the interferon-gamma-driven cytokine storm in macrophage activation syndrome, and is more pronounced in critical versus mild-moderate COVID-19. Systems modelling of cytokine levels paired with deep-immune profiling shows that classical monocytes drive this hyper-inflammatory phenotype and that a reduction in T-lymphocytes correlates with disease severity, with CD8+ cells being disproportionately affected. Antigen presenting machinery expression is also reduced in critical disease. Furthermore, we report that neutrophils contribute to disease severity and local tissue damage by amplification of hypercytokinemia and the formation of neutrophil extracellular traps. Together our findings suggest a myeloid-driven immunopathology, in which hyperactivated neutrophils and an ineffective adaptive immune system act as mediators of COVID-19 disease severity.

ZikaPLAN: Zika Preparedness Latin American Network.

The ongoing Zika virus (ZIKV) outbreak in Latin America, the Caribbean, and the Pacific Islands has underlined the need for a coordinated research network across the whole region that can respond rapidly to address the current knowledge gaps in Zika and enhance research preparedness beyond Zika. The European Union under its Horizon 2020 Research and Innovation Programme awarded three research consortia to respond to this need. Here we present the ZikaPLAN (Zika Preparedness Latin American Network) consortium. ZikaPLAN combines the strengths of 25 partners in Latin America, North America, Africa, Asia, and various centers in Europe. We will conduct clinical studies to estimate the risk and further define the full spectrum and risk factors of congenital Zika virus syndrome (including neurodevelopmental milestones in the first 3 years of life), delineate neurological complications associated with ZIKV due to direct neuroinvasion and immune-mediated responses in older children and adults, and strengthen surveillance for birth defects and Guillain-Barré Syndrome. Laboratory-based research to unravel neurotropism and investigate the role of sexual transmission, determinants of severe disease, and viral fitness will underpin the clinical studies. Social messaging and engagement with affected communities, as well as development of wearable repellent technologies against Aedes mosquitoes will enhance the impact. Burden of disease studies, data-driven vector control, and vaccine modeling as well as risk assessments on geographic spread of ZIKV will form the foundation for evidence-informed policies. While addressing the research gaps around ZIKV, we will engage in capacity building in laboratory and clinical research, collaborate with existing and new networks to share knowledge, and work with international organizations to tackle regulatory and other bottlenecks and refine research priorities. In this way, we can leverage the ZIKV response toward building a long-term emerging infectious diseases response capacity in the region to address future challenges.

The viral capping enzyme nsP1: a novel target for the inhibition of chikungunya virus infection.

The chikungunya virus (CHIKV) has become a substantial global health threat due to its massive re-emergence, the considerable disease burden and the lack of vaccines or therapeutics. We discovered a novel class of small molecules ([1,2,3]triazolo[4,5-d]pyrimidin-7(6H)-ones) with potent in vitro activity against CHIKV isolates from different geographical regions. Drug-resistant variants were selected and these carried a P34S substitution in non-structural protein 1 (nsP1), the main enzyme involved in alphavirus RNA capping. Biochemical assays using nsP1 of the related Venezuelan equine encephalitis virus revealed that the compounds specifically inhibit the guanylylation of nsP1. This is, to the best of our knowledge, the first report demonstrating that the alphavirus capping machinery is an excellent antiviral drug target. Considering the lack of options to treat CHIKV infections, this series of compounds with their unique (alphavirus-specific) target offers promise for the development of therapy for CHIKV infections.

Antiviral Activity of Favipiravir (T-705) against a Broad Range of Paramyxoviruses In Vitro and against Human Metapneumovirus in Hamsters.

The clinical impact of infections with respiratory viruses belonging to the family Paramyxoviridae argues for the development of antiviral therapies with broad-spectrum activity. Favipiravir (T-705) has demonstrated potent antiviral activity against multiple RNA virus families and is presently in clinical evaluation for the treatment of influenza. Here we demonstrate in vitro activity of T-705 against the paramyxoviruses human metapneumovirus (HMPV), respiratory syncytial virus, human parainfluenza virus, measles virus, Newcastle disease virus, and avian metapneumovirus. In addition, we demonstrate activity against HMPV in hamsters. T-705 treatment inhibited replication of all paramyxoviruses tested in vitro, with 90% effective concentration (EC90) values of 8 to 40 µM. Treatment of HMPV-challenged hamsters with T-705 at 200 mg/kg of body weight/day resulted in 100% protection from infection of the lungs. In all treated and challenged animals, viral RNA remained detectable in the respiratory tract. The observation that T-705 treatment had a significant effect on infectious viral titers, with a limited effect on viral genome titers, is in agreement with its proposed mode of action of viral mutagenesis. However, next-generation sequencing of viral genomes isolated from treated and challenged hamsters did not reveal (hyper)mutation. Polymerase activity assays revealed a specific effect of T-705 on the activity of the HMPV polymerase. With the reported antiviral activity of T-705 against a broad range of RNA virus families, this small molecule is a promising broad-range antiviral drug candidate for limiting the viral burden of paramyxoviruses and for evaluation for treatment of infections with (re)emerging viruses, such as the henipaviruses.

Extra-hepatic replication and infection of hepatitis E virus in neuronal-derived cells.

Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and a member of the genus Orthohepevirus in the family Hepeviridae. Infection usually leads to acute hepatitis that can become fulminant, particularly among pregnant women and in patients with preexisting liver disease, or may evolve to a chronic state, especially in immunosuppressed individuals. HEV has been shown to produce a range of extra-hepatic manifestations including aplastic anaemia, acute thyroiditis, glomerulonephritis as well as neurological disorders such as Guillain-Barré syndrome, neuralgic amyotrophy and encephalitis. The pathogenesis of these neurological injuries remains largely unknown, and it is also uncertain whether or not HEV can directly infect neuronal cells. In this study, we investigated whether HEV is capable of completing the viral life cycle in human neuronal-derived cell lines such as neuroepithelioma (SK-N-MC), desmoplastic cerebellar medulloblastoma (DAOY), glioblastoma multiforme (DBTRG), glioblastoma astrocytoma (U-373 MG) and oligodendrocytic (M03.13) cells. Following transfection of these cells with HEV Gaussia luciferase reporter virus, all tested cell lines supported HEV RNA replication. Furthermore, extra- and intracellular viral capsid was detected by an HEV antigen ELISA as a marker for virus assembly and release. Permissiveness for HEV cell entry could be demonstrated for the oligodendrocytic cell line M03.13. In conclusion, these results indicate that HEV tropism is not restricted to the liver and HEV can potentially complete the full viral life cycle in neuronal-derived tissues explaining neurologic disorders during HEV infection.

A Refined Guinea Pig Model of Foot-and-Mouth Disease Virus Infection for Assessing the Efficacy of Antiviral Compounds.

An antiviral containment strategy for foot-and-mouth disease (FMD) outbreaks could support or replace current contingency plans in case of an outbreak in Europe and could spare many healthy animals from being pre-emptively culled. Recently, substantial progress has been made towards the development of small molecule drugs that inhibit FMD virus (FMDV) replication in vitro. For the initial in vivo evaluation of antiviral lead molecules, a refined FMDV-infection model in guinea pigs (GP) is herewith described. This GP model was validated by demonstrating the antiviral effect of T-1105 (an influenza virus inhibitor with reported activity against FMDV). Sixteen animals were orally administered with T-1105 twice daily (400 mg/kg/day) for five consecutive days and inoculated intraplantarly with 100 GPID50 of the GP-adapted FMDV strain O1 Manisa 1 h after the first administration. The efficacy of T-1105 was compared with that of prophylactic vaccination with a highly potent double-oil emulsion-inactivated O1 Manisa vaccine. Ten animals received a single, full (2 ml) cattle vaccine dose and were inoculated 3 weeks later. Fourteen T-1105-treated and all vaccinated GP were completely protected from generalization of vesicular lesions. At 2 dpi, viral RNA was detected in serum of 9/16 T-1105-treated and of 6/10 vaccinated animals. At 4 dpi, viral RNA was detected in serum, organs and oral swabs of half of the T-1105-treated animals and only in the serum of 1/10 of the vaccinated animals. Mean viral RNA levels in serum and organs of T-1105-treated and vaccinated animals were reduced compared to untreated controls (P < 0.01). T-1105 conferred a substantial clinical and virological protection against infection with O1 Manisa, similar to the protection afforded by vaccination. These results validate the suitability of the enhanced GP model for the purpose of initial evaluation of inhibitors of FMDV replication and illustrate the potential of selective inhibitors of viral replication to control FMD outbreaks.

Prophylactic treatment with the nucleoside analogue 2'-C-methylcytidine completely prevents transmission of norovirus.

OBJECTIVES: Norovirus outbreaks of acute gastroenteritis are highly prevalent, extensive and can disturb the functioning of health institutions, leading to the closure of hospital wards and causing life-threatening infections in long-term care facilities. There is no vaccine available; hence there is a pressing need for antivirals for the treatment (in immunodeficient patients) and prophylaxis of norovirus infections. We explored in a mouse model whether an inhibitor of norovirus replication can prevent/reduce transmission of the virus. METHODS: We reported recently that the viral polymerase inhibitor 2'-C-methylcytidine (2CMC) efficiently protects against murine norovirus (MNV)-induced diarrhoea and mortality in mice. Here, we established an MNV-transmission model, determined the 50% infectious dose and assessed the ability of an antiviral molecule to prevent or reduce transmission of (murine) norovirus when given either to the infected (seeder) mice or to the uninfected (sentinel) mice. RESULTS: A robust norovirus transmission model was established using genogroup V (murine) norovirus in AG129 mice. The 50% infectious dose was determined to be ∼ 270 CCID50 (50% cell culture infectious dose). Treatment of infected mice with 2CMC reduced viral shedding and markedly reduced transmission to uninfected sentinels. Also, prophylactic treatment of sentinels with 2CMC resulted in protection against infection with MNV. CONCLUSIONS: These findings constitute an important first step towards developing an efficient prophylaxis for the control of norovirus outbreaks.

The enterovirus protease inhibitor rupintrivir exerts cross-genotypic anti-norovirus activity and clears cells from the norovirus replicon.

Potent and safe inhibitors of norovirus replication are needed for the treatment and prophylaxis of norovirus infections. We here report that the in vitro anti-norovirus activity of the protease inhibitor rupintrivir is extended to murine noroviruses and that rupintrivir clears human cells from their Norwalk replicon after only two passages of antiviral pressure. In addition, we demonstrate that rupintrivir inhibits the human norovirus (genogroup II [GII]) protease and further explain the inhibitory effect of the molecule by means of molecular modeling on the basis of the crystal structure of the Norwalk virus protease. The combination of rupintrivir with the RNA-dependent RNA polymerase inhibitors 2'-C-methylcytidine and favipiravir (T-705) resulted in a merely additive antiviral effect. The fact that rupintrivir is active against noroviruses belonging to genogroup I (Norwalk virus), genogroup V (murine norovirus), and the recombinant 3C-like protease of a GII norovirus suggests that the drug exerts cross-genotypic anti-norovirus activity and will thus most likely be effective against the clinically relevant human norovirus strains. The design of antiviral molecules targeting the norovirus protease could be a valuable approach for the treatment and/or prophylaxis of norovirus infections.

Proof of concept for the inhibition of foot-and-mouth disease virus replication by the anti-viral drug 2'-C-methylcytidine in severe combined immunodeficient mice.

Recent European contingency plans envisage emergency vaccination as an animal-friendly control strategy for foot-and-mouth disease (FMD). Anti-viral drugs may be used as an alternative or complementary measure. We here demonstrate that the nucleoside analogue 2'-C-methylcytidine (2'CMC) protects severe combined immunodeficient (SCID) mice against lethal FMD virus infection. In brief, SCID mice were inoculated with serotype A FMD virus and treated for five consecutive days with 2'CMC. All 15 treated mice remained healthy until the end of the study at 14 days post-infection (dpi). At that time, viral RNA was no longer detected in 13 of 15 treated mice. All eight untreated mice suffered from an acute generalized disease and were euthanized for ethical reasons on average at 4 dpi. These results illustrate the potential of small molecules to control FMD.

Laboratory validation of a lateral flow device for the detection of CyHV-3 antigens in gill swabs.

Cyprinid herpesvirus-3 (CyHV-3) induces the highly contagious koi herpesvirus disease (KHVD) and may result in significant economic losses to the ornamental and food-producing carp industry. Suspicion of KHVD is triggered by clinical signs and confirmed using laboratory techniques. The latter are labour- and time-consuming, require specialised equipment and trained personnel. For rapid, on-site detection of CyHV-3, a lateral flow device (LFD) was developed using two monoclonal antibodies directed towards the viral glycoprotein ORF65. The LFD was highly specific with analytical and diagnostic specificities of 100%. Analytical sensitivity ranged between 1.25×10(2) and 2.40×10(4) plaque forming units per ml for isolates originating from geographically distinct regions. In experimentally infected carp, CyHV-3 was detected as early as 4-5 days post infection. Diagnostic sensitivities of 52.6% and 72.2% relative to PCR were recorded, depending on the viral isolate used. When onset of mortality was taken as reference, diagnostic sensitivities increased to 67.0% and 93.3%. The diagnostic sensitivity for freshly found-dead animals was 100%, irrespective of the virus isolate used. Given the high specificity and ease-of-use for on-site detection of CyHV-3, the LFD was regarded fit for purpose as a first-line diagnostic tool for the identification of acute CyHV-3 infections in KHVD affected (koi) carp.

Inhibition of norovirus replication by the nucleoside analogue 2'-C-methylcytidine.

We here report on the activity of 2'-C-methylcytidine (2CMC) [a nucleoside polymerase inhibitor of the hepatitis C virus (HCV)] on the in vitro replication of (murine) norovirus (MNV). 2CMC inhibits (i) virus-induced CPE formation, (ii) viral RNA synthesis and (iii) infectious progeny formation with EC(50) values of ∼2µM. 2CMC acts at a time-point that coincides with the onset of viral RNA synthesis. Even following 30 passages of selective pressure no MNV-resistant virus was selected, which is in line with the high barrier to resistance of the nucleoside analogue for HCV. When combined with the broad-spectrum RNA virus inhibitor ribavirin, a marked antagonistic activity was observed indicating that these molecules should not be combined for the treatment of norovirus infections. Our results suggest that 2'-C-methyl nucleoside analogues should be further explored for the treatment and prophylaxis of norovirus infections.

Favipiravir (T-705) inhibits in vitro norovirus replication.

Human noroviruses are the primary cause of foodborne gastroenteritis. Potent and safe inhibitors are needed for the treatment/prophylaxis of norovirus infections. We demonstrate that Favipiravir [T-705, a drug in advanced clinical development for the treatment of infections with the influenza virus] inhibits in vitro murine norovirus replication. Time-of-drug addition studies reveal that T-705 exerts its activity at a time-point that coincides with onset of viral RNA synthesis, which is in line with the viral polymerase as the presumed target.

Classical swine fever outbreak containment using antiviral supplementation: a potential alternative to emergency vaccination and stamping-out.

Classical swine fever (CSF) outbreaks may result in huge economic losses to countries with densely populated pig areas (DPLAs). The EU minimum control measures require depopulation of infected farms, movement restrictions, zoning and surveillance (EU Minimum strategy). Emergency vaccination is authorised for DPLAs although the EU Minimum strategy plus culling in a 1-km ring around infected premises is preferred. Nonetheless, vaccination in a 2-km ring has been found equally effective as 1-km ring culling using stochastic modelling. Alternatives control measures (e.g. antiviral agents, in particular small molecule inhibitors of the CSFV replication) are being explored. Hence, the present study was set up to simulate inter-herd CSFV spread when antiviral molecules are supplemented to pig feed in a 1-km ring around infected farms. The effectiveness of the antiviral strategy for containing CSF outbreaks was compared to six other control scenarios including the EU Minimum strategy, the EU preferred policy for DPLAs and the use of 2-km ring vaccination. The InterSpread Plus model was adapted to the 2006 Belgian pig population and outbreak simulations were performed with a fast spreading CSFV strain entering a DPLA in Belgium. Four out of the seven control strategies resulted in outbreaks that were controlled by the end of the simulation period (i.e. 365 days). The distributions of the number of infected herds and the duration of the predicted outbreaks for these four control strategies were not different. This is the first report investigating CSF outbreak containment using antiviral molecules. Although antiviral supplementation was not found to perform any better than some other conventional strategies, such as pre-emptive culling and emergency vaccination, it might be worthwhile considering it further as additional tool in a response to CSF outbreaks.

Adefovir serum levels do not differ between responders and nonresponders.

Primary or secondary failure of adefovir dipivoxil (ADV) therapy of chronic hepatitis B is not infrequent. The reasons for suboptimal responses are not well defined. In HIV and hepatitis C virus infection, failure of antiviral drug therapy has been linked with low blood drug levels. We have studied 20 well-defined patients with chronic hepatitis B who were treated with ADV for drug and virus kinetics. Importantly, neither Cmax levels (mean 26 ng/mL, range 14-59 ng/mL) nor the time to maximal drug levels (mean 4 h, range 2-8 h) differed between patients showing a complete virological response to adefovir (n = 10), patients with secondary treatment failure (n = 7) and patients with suboptimal primary response (hepatitis B virus-DNA >10,000 IU/mL after 6 months of treatment; n = 3). Thus, adefovir treatment failure is unlikely to be due to an inability to mount sufficient drug levels in the blood.

Development of a foot-and-mouth disease infection model in severe combined immunodeficient mice for the preliminary evaluation of antiviral drugs.

Recent European guidelines facilitate the use of emergency vaccines during outbreaks of foot-and-mouth disease. Antiviral drugs could be used as a complementary measure. This study aimed at developing a small animal model to assess the in vivo activity of early antiviral lead molecules with anti-foot-and-mouth disease virus (FMDV) activity in vitro. In a first attempt, several FMDV strains were titrated in Balb/c mice. Inoculations with O1 Manisa or C1 Noville did not induce clinical disease, whereas Asia1 Shamir induced death too rapidly [i.e. within 4 days post-inoculation (dpi)]. Therefore, we switched to severe combined immunodeficient mice which are frequently used as a model for viral infections and experimental therapeutics. Strain O1 Manisa did not induce clinical disease, but titrations with A22 Iraq, C1 Noville or Asia1 Shamir resulted in virus-induced morbidity (including respiratory problems and weight loss) with subsequent mortality. Inoculations with strain A22 Iraq resulted in a reproducible mean time of death of 6 dpi (this was shorter for the other strains). In this newly developed rodent model, strain A22 Iraq seems the most suited to assess the in vivo anti-FMDV activity of selective inhibitors of FMDV.

Genomics and structure/function studies of Rhabdoviridae proteins involved in replication and transcription.

Some mammalian rhabdoviruses may infect humans, and also infect invertebrates, dogs, and bats, which may act as vectors transmitting viruses among different host species. The VIZIER programme, an EU-funded FP6 program, has characterized viruses that belong to the Vesiculovirus, Ephemerovirus and Lyssavirus genera of the Rhabdoviridae family to perform ground-breaking research on the identification of potential new drug targets against these RNA viruses through comprehensive structural characterization of the replicative machinery. The contribution of VIZIER programme was of several orders. First, it contributed substantially to research aimed at understanding the origin, evolution and diversity of rhabdoviruses. This diversity was then used to obtain further structural information on the proteins involved in replication. Two strategies were used to produce recombinant proteins by expression of both full length or domain constructs in either E. coli or insect cells, using the baculovirus system. In both cases, parallel cloning and expression screening at small-scale of multiple constructs based on different viruses including the addition of fusion tags, was key to the rapid generation of expression data. As a result, some progress has been made in the VIZIER programme towards dissecting the multi-functional L protein into components suitable for structural and functional studies. However, the phosphoprotein polymerase co-factor and the structural matrix protein, which play a number of roles during viral replication and drives viral assembly, have both proved much more amenable to structural biology. Applying the multi-construct/multi-virus approach central to protein production processes in VIZIER has yielded new structural information which may ultimately be exploitable in the derivation of novel ways of intervening in viral replication.

The reduction of CSFV transmission to untreated pigs by the pestivirus inhibitor BPIP: a proof of concept.

5-[(4-Bromophenyl)methyl]-2-phenyl-5H-imidazo[4,5-c]pyridine (BPIP) is a representative molecule of a novel class of highly active in vitro inhibitors of the replication of Classical swine fever virus (CSFV). We recently demonstrated in a proof of concept study that the molecule has a marked effect on viral replication in CSFV-infected pigs. Here, the effect of antiviral treatment on virus transmission to untreated sentinel pigs was studied. Therefore, BPIP-treated pigs (n=4), intra-muscularly infected with CSFV, were placed into contact with untreated sentinel pigs (n=4). Efficient transmission of CSFV from four untreated seeder pigs to four untreated sentinels was observed. In contrast, only two out of four sentinel animals in contact with BPIP-treated seeder animals developed a short transient infection, of which one was likely the result of sentinel to sentinel transmission. A significant lower viral genome load was measured in tonsils of sentinels in contact with BPIP-treated seeder animals compared to the positive control group (p=0.015). Although no significant difference (p=0.126) in the time of onset of viraemia could be detected between the groups of contact animals, a tendency towards the reduction of virus transmission was observed. Since sentinel animals were left untreated in this exploratory trial, the study can be regarded as a worst case scenario and gives therefore an underestimation of the potential efficacy of the activity of BPIP on virus transmission.