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Integration of Chikungunya research

Final Report Summary - ICRES (Integration of Chikungunya research)

Executive Summary:
The purpose of this project was to advance understanding and control of Chikungunya fever by integrating the expertise of EU laboratories with a long and strong track record of research on alphaviruses with EU laboratories that started work on this disease following the 2005 outbreak in La Réunion (France) and laboratories from SE Asia working on this virus in the current epidemic in this region. The aim was to coordinate research within, and to some extent beyond, the EU to build capacity and generate the outputs required to enhance surveillance, diagnosis and understanding of disease processes and to provide pre-clinically evaluated candidates for treatment and prevention.

All of these aims were met. Considerable progress was made in integrating chikungunya research across the EU and beyond. This has led to collaborations which will extend beyond the grant funding and has built capacity in the EU and elsewhere. The project greatly added to an understanding of the epidemiology and pathogenesis of Chikungunya. This included detailed characterisation of mouse and macaque models of this disease. New diagnostics were developed, lead antivirals were identified and a preclinical vaccine, ready for entry into human clinical trials, was designed, engineered and tested.

In October 2013, the consortium arranged the first International Congress on Chikungunya. This brought together consortium members with over a hundred other researchers from twenty four countries. This led to strong dissemination of the consortium’s results and establishment of additional collaborations. In December 2013, as predicted, Chikungunya spread to the Americas. To date around a million people have been infected there. Over the past 12-months consortium members have engaged with US colleagues to disseminate our expertise and reagents.

Project Context and Objectives:
The principle objectives of the ICRES project were:
1. Generate new molecular and cellular tools for research and applied studies including high-throughput screening and vaccines
2. Standardise, quality assure and distribute key diagnostic tests and develop new ones
3. Determine key virus genetic changes across time, geographical regions and species
4. Discover interactions between virus and human cells to inform the rational design of therapeutics
5. Determine pathogenesis of the acute and chronic disease in humans, including whether virus persists in joints, the cell types involved and the relationship to immune responses
6. Characterise rodent and non-human primate models of acute and chronic infection to further study the pathogenesis and to provide models for antiviral and vaccine screens
7. Screen libraries of characterized pharmaceutical and bioactive compounds for antiviral activity
8. Develop a vaccine which at the end of this project is ready to enter clinical trials

These objectives were all met.

Project Results:
A new molecular clone, derived from a virus isolate from Mauritius, ICRES1, was generated and its phenotype verified as identical to CHIKV LR2006-OPY1 in macaques. A virus replicon particle (VRP) system was established. ICRES-1 derived molecular constructs expressing full length virus sequence, virus replicase or virus structural proteins, various mutations and vaccine candidates and additions to these, including several biochemical and fluorescent markers were engineered and tested in vitro, and in some cases in vivo. ZsGreen, eGFP, Tomato, mCherry, Cherrypicker, FPTurbo650 and two variants of IFP were used as fluorescent markers and firefly, firefly luciferase 2, nano-luciferase, red-shift luciferease, Renilla and Gaussia luciferases as biochemical markers. All markers were inserted either inside the nsP3 region or as a separate expression unit between the non-structural and structural regions of ICRES-1. Genetic stability of a number of constructs was determined and found to be strong. A system to study the virus replicase outside the context of infectious virus was also developed. In total, over 100 different molecular constructs were generated.

Cells lines inducible for the expression of individual virus proteins and cells persistently expressing the CHIKV replicase driving a marker gene were generated. The latter was used to establish a high throughput in vitro screening system for antivirals.

Two different inducible cell line systems showed that expression of nsP2 leads to cell death. In contrast cell lines with truncated or inactivated nsP2, or expressing polyproteins where nsP2 is not released, survive. Two combinations of nsP2 mutations which make this protein non-cytotoxic for rodent and human cells were characterised. The non-cytotoxic phenotype does not seem to result from changes to subcellular localisation but may be associated with severely reduced enzymatic activities.

Monoclonal or polyclonal mono-specific antibodies to all the individual virus proteins were generated

Methods for diagnostics and key laboratory assays including, plaque assays, ELISPOT, FACS, ELISA and RT-PCR were reviewed and standardised across the consortium. Cell lines used for assays were exchanged between partners to ensure standardisation. Human sera were screened for reactivity to recombinant E1, E2 and capsid proteins. Many antibodies, from as early as 4 days post-infection, reacted to domain B of E2. Capsid was found to be unsuitable as an ELISA antigen for acute diagnosis, as anti-capsid antibodies only appeared in later convalescence (68.6% of 35 samples collected <6 months post-infection). A novel high throughput, safe, neutralisation test using virus replicon particles expressing Gaussia luciferase was developed and optimised. Unlike conventional virus neutralisation tests, this can be used at low level containment (CL2) and the readout is a rapid and sensitive biochemical assay which does not require the long incubation period of plaque assays.

Over 40 samples of CHIKV from serum, CSF, skin blisters from acute cases were isolated from La Reunion and 25 new complete length virus sequences, predominantly from SE Asia, were determined.

Phylogenetic analysis of the isolates from Southeast Asia indicated that virus from Kerala, India spread to Malaysia in 2007, and from there, seeded large outbreaks in Malaysia, Thailand and China. A unique mutation was introduced into the Malaysian virus population early during the epidemic. This has a minor effect on infection of Ae. albopictus mosquitoes. In Malaysia both Asian and Central/East African (CEA) genotypes co-circulate. Both disseminate more efficiently in Ae. albopictus than Ae. aegypti mosquitoes while CEA replicates and disseminates to salivary glands more efficiently than Asian in Ae. albopictus. This may explain why CEA genotype spread across Malaysia while the Asian genotype outbreaks were far more limited.

Sixteen full CHIKV genomes from the Research Institute for Tropical Medicine at the Department of Health, Philippines (2012) have been sequenced and analysed. All but one were confirmed to cluster in the Asian genotype.

In November 2013 Chikungunya, Asian genotype, spread to the Americas. To investigate whether our vaccine candidates (see below), developed from the LR2006OPY1 (ICRES-1) virus provided protection against the Asian genotype, molecular constructs and VRP systems based on the Asian genotype were constructed. These were used to produce VRPs for neutralization assays and will be used to determine whether antibodies resulting from vaccination of macaques with our advanced vaccine candidates are also capable of neutralizing the Asian genotype virus responsible for the epidemic in the Americas.

Analysis of the interactions between virus components, RNA and proteins, and cellular components was undertaken by yeast-two-hybrid analysis of virus and cell protein interactions, analysis of cellular proteins which interact with virus replicase complexes by tagged immunoprecipitation and SILAC analysis, an siRNA knock down screen to identify important cellular determinates of virus replication and anti-viral screening against a library of 3,086 known bioactive compounds. Six antiviral compounds were studied in detail. These assays identified a series of cellular molecules that interact with, are required for, or affect, virus replication. These are now under further investigation.

We used magnetic isolation of alphavirus replicase complex containing endosomes and the SILAC method to detect over- or under-representation of host proteins in virus replication structures. The purified complexes were enzymatically active indicating that they were functional assemblies. Using reciprocal experiments >10 cellular proteins were found to be four or more fold over-represented in endosomes from infected cells. This included well known interactors with alphavirus replicase including hnRNPC, hnRNPK, G3BP1 and G3BP2 (their interaction G3BPs with alphavirus NsP3 was analysed in detail) as well as proteins never before found associated with alphavirus replicase complexes. These included PCBP1 and several hnRNPs including hnRNPM. All these proteins were found to co-localise with the replicase complexes of alphaviruses in immunofluorescent microscopy.

The genome-wide RNAi screen was based on human HEK-293 cells transfected with a whole genome siRNA library, consisting of approximately 62,000 siRNAs targeting about 17,000 annotated genes and 6,000 predicted genes and including at least 2 siRNAs per gene, challenged with CHIKV-eGFP. The primary screen led to the identification of 279 proviral cellular factors (inhibition of virus replication upon gene knockdown) and 269 antiviral factors (enhanced virus replication upon gene knockdown). To avoid false-positives due to off-target effects all these genes were then validated in a second-round screening resulting in a highly reproducible list containing 156 confirmed genes required for viral replication and 41 genes controlling the infection. The genes under further study include those encoding surface proteins that may act as receptors, histone modification complexes, lipid metabolism and mRNA degradation pathway proteins. Ingenuity pathway analyses indicate a strong enrichment in the pathways ‘Post-Translational Modification’, ‘RNA Post-Transcriptional Modification’ and ‘Lipid Metabolism’. Based on the 156 confirmed genes required for viral replication, we obtained small molecule inhibitors that interfere with viral replication. Several of these efficiently reduced virus replication without significant disturbance of cellular metabolism. Our screen identified fatty acid synthase and ATP citrate lyase as critical cellular factors required for CHIKV replication. Immunofluorescence analysis showed that FASN co-localizes with CHIKV replication complexes upon infection.

We have compared CHIKV infection in human and macaque fibroblasts, melanocytes, monocytes and macrophages. Interestingly, infection of macrophages occurs most efficiently via phagocytic uptake of apoptotic debris from productively infected cells such as fibroblasts. Virus material can persist for several weeks in infected macrophages; using nsP3-ZsGreen to infect macrophages we were able to detect fluorescent proteins up to 35 days post-infection. Finally, we are able to show that macrophage infection induces a burst of released inflammation cytokines (including, IL-4, IL-6, IL-10, IL-13, TNF-α and G-MCSF).

Chikungunya infection can result, particularly in neonates, in neurological disease. In culture we showed that the virus infects astrocytes (primary human and mouse cells) but not microglia and rarely neurons. Astrocytes rapidly control the infection through apoptosis and mount a robust innate immune response.

We confirmed the nsP2 protein is involved in activation of interferon (IFN) response and at the same time it also acts as an antagonist of this response. Mutation of Pro 718 to Gly greatly increased IFN production and the nsP2 mutations which made the corresponding replicons non-cytotoxic, induced efficient IFN production in primary infected human cells.

A mouse model of infection was established in three of our laboratories. In vivo imaging of the infection using luciferase marker virus, screening of fourteen vaccine candidates and assessment of virus neurotropism were all carried out. The roles of viperin, CD74, antibodies and CD4+ T-cells were studied.

For purpose of vaccine evaluation, various mouse models were set up and assessed. Viral persistence and inflammation were assessed within the ankle of female C57BL/6 mice inoculated with CHIKV. This infection recapitulates the self-limiting arthritis, tenosynovitis, and myositis seen in humans. Rheumatic disease was associated with a prolific infiltrate of monocytes, macrophages, and NK cells and the production of monocyte chemoattractant protein 1 (MCP-1), tumour necrosis factor alpha, and gamma interferon. We established in vivo imaging to study spread of the infection in mice. The roles of viperin, antibodies, TLRs and CD4+ T-cells were also studied in the mouse.

The Chikungunya macaque model was refined by showing that it is possible to target CHIKV infection to the joints and establish a predictable site at which the course of infection and inflammation can be reproducibly studied. A longitudinal study of immune parameters of infection was carried out in comparison with another alphavirus, Ross River virus.

In a study of the chronic phase of the infection in macaques we showed that virus RNA could be detected in the serum, CSF and spleens 35 days post-infection, though no virus could be isolated from the synovial fluid. One of three virus sequences obtained during chronic infection exhibited a large in frame deletion in the nsP2/nsP3 region (nucleotides 3185 to 4270) raising the possibility that a defective interfering RNA might have arisen. The macaque model was used to evaluate our vaccine candidates.

To test the hypothesis that acute virus infection of monocytes and late virus persistence in macrophages are important contributors to the pathogenesis of acute and chronic Chikungunya disease, a virus engineered not to replicate in cells of the mononuclear phagocyte lineage was constructed by incorporation into the virus genome of recognition sites for a monocyte/macrophage specific miRNA (mir-142-3P). This virus was shown not to replicate in monocytes and macrophages in vitro. In mice, unlike wt virus, the miRNA recognition element containing virus did not replicate in spleen marginal zone macrophages. This virus has a low level viraemia in macaques. Virus persistence in joints is currently being evaluated.

In La Reunion, 120 patients 16-18 months post-CHIKV infection; with polyarthralgia and referred to a rheumatologist were studied. Almost 21% of had inflammatory polyarthritis. The others had arthralgia not associated with inflammatory arthropathy. Sixty months post-CHIKV infection peripheral blood mononuclear cells and serum were analysed for cytokines, cell counts, transaminases, anti-cyclic citrullinated peptides, rheumatoid factor, anti-nuclear antigens and other clinical biomarkers of inflammation. A second cohort of patients (220 in total, including 19% with inflammatory polyarthritis post-CHIKV) was established in 2011 (60 months PI). Overall, findings in the 360 patients presenting painful manifestations following CHIKV infection showed that the risk of developing inflammatory polyarthritis was higher if the initial acute phase lasted longer than 3 weeks. Although some patients suffered from clinically classical RA or PA rheumatisms, they had no detectable levels of rheumatoid factor, no autoantibodies against nucleic acids and few had anti-CCP antibodies

Interestingly, IFN type I expression persisted in PBMCs of chronic patients at 12 months post-infection and was also observed in chronic patients at 3 years. Analysis of the T cell response against three viral antigens (E2, NSP1, Capsid) found no significant difference between recovered and chronic patients suggesting that chronic arthritis post-chikungunya is not due to a failure to mount an adaptive immune response. Unfortunately, we were not able to isolate CHIKV from frozen (preserved in isopentane) synovial tissues from chronic patients. Studies are ongoing to try to detect virus sequences by ISH and RTPCR on formalin fixed tissues. A limited set of samples (mostly fixed in formalin) were collected during the acute epidemic and from chronic patients. Immunohistopathological (IHP) and in situ hybridisation studies carried out on these samples show a strong level of angiogenesis in the synovial tissue while immune cells (CD56 < CD3 < CD68) are relatively sparse.

To identify antivirals we screened 3086 compounds derived from three commercially available libraries against the CHIKV replicon cell line. The compounds represent known bioactive substances, drugs used in the clinics, or compounds in advanced clinical trials for other indications. Secondary validation was carried out with infectious CHIKV containing the luciferase marker. Based on all the data obtained during the process, 7 best compounds were selected for further study; of these 6 inhibited the replication of CHIKV in both hamster and human cells with low micromolar EC50 values. Out of the 6 compounds, 3 were highly effective in reducing the levels of CHIKV RNA and CHIKV proteins in infected cells. These compounds were similarly effective against two other alphaviruses, Sindbis virus and Semliki Forest virus, in inhibiting virus production. Interestingly, two of the compounds also showed efficacy against a completely unrelated virus, the flavivirus yellow fever virus. These 3 compounds, together with another highly efficacious molecule that emerged from an independent line of investigation, are under further study. Independent virus clones showing variable levels of resistance against each of these four compounds were isolated by plaque purification from virus stock passaged in the presence of compounds for 20-30 passages. These are being sequenced. This data may indicate likely mechanisms of action. Testing of the three most promising antivirals in mouse models is ongoing.

A number of vaccine constructs were made. These included deletion of the capsid or 6K genes, deletions in nsP3 and mutations in nsP2. Different delivery systems were developed, RNA derived by vitro transcription; DNA launched RNA, a layered vector system and virus replicon particles. A vaccinia virus (MVA) expressing CHIKV structural proteins was also constructed. The constructs were tested in mice either alone or in homologous or heterologous prime-boost combinations. Foot swelling, viraemia, the capacity to induce strong humoral and cellular responses and protective efficacy were determined. The MVA-CHIKV was stable in cell culture, expressed the CHIKV structural proteins, elicited robust innate immune responses in human macrophages and monocyte derived dendritic cells, induced high titers of neutralising antibodies and strong, broad, polyfunctional and durable CHIKV-specific CD8+ T cell immune responses in mice as well as effector memory CHIKV-specific CD8+ T cells. A single dose protected all mice from a high-dose challenge with CHIKV.

On the basis of the results in mice four systems were tested in non-human primate trials: (1) an attenuated infection clone of CHIKV, (2) a DNA-launched replicon of the same attenuated mutant, (3) recombinant MVA virus, (4) envelope protein antigen delivered with the state-of-the art Matrix-M adjuvant. Pathogenesis including fever, foot swelling, viraemia and capacity to induce strong humoral and cellular responses and protective efficacy were tested. All vaccine candidates protected from infection.

The consortium met every 6-months. A highly successful 3-day international meeting, Chikungunya-2013, was organised in October 2013. This attracted sponsorship and 127 delegates from 24 countries.

Potential Impact:
This project will advance fundamental understanding on Chikungunya virus and indeed alphaviruses in general, and provide tools and systems for future studies, diagnostics and vaccines. The project will advance fundamental understanding on how the virus replicates; its interactions with mammalian cells, in particular macrophages; evolution of the virus and the nature of changes which adapt the virus for optimal replication in different species or cell types; the nature of innate and adaptive immune responses to the virus and virus antagonism or avoidance of these; the pathogenesis of the infection in rodents and primates and the nature and mechanism of virus persistence. Many tools of value for future fundamental and more applied research will be generated. These will include virus constructs, antibodies, cell lines and well-characterised animal models. The project will generate new and improved diagnostics, candidate antivirals and a pre-clinically verified vaccine.

It is estimated that CHIKV has infected millions, perhaps 5 million people, since its re-emergence in 2005. On La Réunion there was a significant mortality, >250,000 people were infected and there were around 250 deaths. The Disability Adjusted Life Years (DALY) burden during the 2006 epidemic in India has been estimated at over 25,000. In late 2013 CHIKV was reported in the Caribbean; we anticipate it will spread there. Prevention of CHIKV outbreaks with a vaccine or therapeutic to ensure rapid recovery or prevent or alleviate chronic disease would have a major impact. Furthermore, other alphaviruses, including Sindbis virus and Ross River virus also cause polyarthralgia and the vaccine platform chosen for CHIKV could be modified to provide other alphavirus vaccines or indeed vaccines to other viruses.

The project is building capacity in the EU on alphaviruses. This project involves several EU countries and key institutes from countries outside the EU. It brings the best expertise to bear on the problem. It is not only building capacity on alphaviruses but also on RNA viruses and arboviruses in general. It is furthering the careers of junior scientists with potential to become the research leaders of the future. Expertise in arbovirology needs to be rebuilt in Europe to meet the challenges of the future which are likely to include continual spread of new and existing vectors including mosquitoes (eg Ae. albopictus), midges and ticks in Europe. The high technology platforms used in the project, transcriptomics, next generation sequencing, yeast-two-hybrid, antiviral and RNAi screens also build EU capacity in these technologies which have many ‘users’. The training provided to the junior scientists increases the human capital in these areas adding to the competitiveness of the EU economy in the field of biomedicine.

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