Vaccines and molecular tools for the control of the emerging bunyavirus, severe fever with thrombocytopenia syndrome phlebovirus (SFTSV)

Viruses in the newly formed order Bunyavirales are frequently arthropod-transmitted with many members capable of causing substantial human, animal and disease. The WHO has identified several bunyaviruses as key emerging pathogens, and this call entitled "One health approaches to accelerate vaccine development" also emphasizes interest in this group of viruses. This project focuses on severe fever with thrombocytopenia syndrome phlebovirus (SFTS phlebovirus; SFTSV) of the Phenuiviridae family of bunyaviruses which also includes significant emerging pathogens in both Asia and the USA (Heartland virus, HRTV). To date, over 2500 SFTSV cases have been reported in China, 172 cases in South Korea and 96 cases in Japan, with case fatality rates ranging from 8.2% to 32.6%. The annual incidence of reporting is increasing in all endemic countries, e.g. in China where 53 cases were reported in 2010, compared to 676 in 2013. To address the threat posed by novel, emerging bunyaviruses, scientists at the MRC-University of Glasgow Centre for Virus Research (CVR) have developed a "reverse genetics" system with which SFTSV can be manipulated, and which permit the rational design of rationally attenuated virus vaccine candidates. Such reverse genetics systems enable the rapid recovery of replication competent bunyaviruses from plasmids encoding cDNA copies of the viral genomic RNA. This technology facilitates the mapping of epitopes targeted by the humoral and cellular immune response as well as the identification and manipulation of virally-encoded virulence factors. The NSs proteins of many bunyaviruses are recognised as virulence factors due to their ability to counteract the mammalian innate immune responses. Hence, these reverse genetics-based strategies offer a realistic means to develop rational vaccine candidates for emerging bunyaviral diseases, as well as the development of viruses that help to further elucidate the immune responses mounted to infection and viral pathogenesis. We have now generated a recombinant NSs-deletant SFTSV (called SFTSVdelNSs) which is unable to antagonise type I interferon responses mounted in response to infection. This facilitates the studies into the role of innate immune responses in controlling SFTSV, as well as in vivo tissue tropism and pathogenesis in absence of the virulence factor NSs, leading to the evaluation of such potentially attenuated viruses as vaccine candidates. The strengths of reverse genetics-based approaches to producing recombinant bunyaviruses extends beyond the production of attenuated virus vaccine candidates. The system also allows the development and production of virus-like particles (VLPs). Both recombinant viruses and VLPs can be tested for their ability to induce immune responses in immunised animals as well as protective vaccine candidates. This proposal will bring together CVR and Beijing Institutes of Life Science (BIOLS) researchers to elucidate host immune responses to SFTSV infection and pathogenesis, the development of recombinant virus vaccine candidates and tools to further study SFTSV.

Aim 1. To elucidate the contribution of SFTSV NSs to in vivo tissue tropism and pathogenesis. We will also assess a recombinant SFTSV (lacking NSs; SFTSVdelNSs) as vaccine candidate in comparison to VLPs.

Aim 2. To produce novel neutralising antibodies against SFTSV for basic research and as potential therapeutic agents. We will investigate antibody responses using a novel SFTS viruses expressing reporter genes to study immune responses in both patient sera and infected animals. This will allow us to define the breadth of the neutralising antibody responses and to map antigenic determinants on the virion. Antigenic variation and escape from neutralisation of the SFTSV Gn structural protein (known target for antibody responses) will be investigated through mutational analysis.

The project will also include training in reverse genetics technology for the partner BIOLS.

The global incidence of tick-borne disease is increasing annually, yet there is a fundamental lack of research into developing vaccine candidates for this emerging group of pathogens.

This proposal will focus on severe fever with thrombocytopenia syndrome phlebovirus (SFTSV), a member of the Phenuiviridae family of bunyaviruses. This tick-borne virus was first identified in China, and since then in other parts of Asia where it causes human disease. Our overarching ambition is to characterise interactions with the host as well as humoral and innate immune responses to this virus and a recently developed recombinant SFTSV engineered to lack expression of the virally-encoded virulence factor, NSs (SFTSVdelNSs).

To achieve our goals, we will conduct in vitro and in vivo studies that can be divided into two specific aims, with the first aim to (i) characterise the pathogenesis and tissue tropism of recombinant viruses and assess the efficacy of replication competent live-virus (specifically SFTSVdelNSs) versus virus-like particles as immunogens and (ii) assess the neutralisation capabilities of antibody responses mounted to infection in both vaccinated animals and infected patient sera.

The second aim will also lead to the production of a new panel of neutralising monoclonal antibodies for research and potentually therapeutic purposes. Such tools will allow us to map antigenic determinants on the viral particle. We will also analyse how viruses can escape neutralisation through mutagenic analysis of the viral Gn glycoprotein (a known antibody target) by using our unique reverse genetics system and structural analysis; this information will be useful for design of vaccines.

CVR will also use this opportunity to train scientists from the project partner BIOLS in China in the development and applications of bunyavirus/SFTSV reverse genetics technologies.

SFTS phlebovirus (SFTSV) is an emerging tick-borne bunyavirus and public health problem in China, the key partner country for this project between the Beijing Institutes of Life Science at the Chinese Academy of Sciences (BIOLS) and the MRC-University of Glasgow Centre for Virus Research (CVR).
SFTSV infections of humans are frequently lethal and can lead to haemorrhagic fever as well as neural complications such as encephalitis. As this pathogenic human virus was relatively recently identified in China (and subsequently in other parts of Asia), there are important gaps in our knowledge of the biology of SFTSV infections, including virus-host interactions and immune responses. This has dramatically hindered the development of vaccines and therapeutics to treat SFTS disease.

In this project, we propose to (a) study the comparative pathogenesis and immune response of wild type and recombinant SFTS viruses (e.g. viruses lacking the interferon antagonist NSs; SFTSVdelNSs) and its potential as an attenuated virus vaccine (in comparison to virus-like particles); (b) assess antibody responses to SFTSV and identify neutralising epitopes and characterise potential virus escape mutants using novel reporter virus systems as well as reverse genetics/structural biology and (c) develop antibodies to SFTSV that can be useful for basic research and as potential therapeutics. This will be combined with training of staff from our partner BIOLS to transfer knowledge, reagents and skills between the UK and China. The most relevant areas of impact are summarised below:

(1) A key impact is on fundamental virology as we study areas of virus-host interactions and immune responses that are novel and poorly understood for SFTSV (and similar bunyaviruses). We will also develop tools that are relevant for this project, but also other scientists who aim to study this and related viruses. Our data will be published and reagents made available to institutions qualified to obtain material.

(2) We believe that knowledge generated throughout the proposed scientific investigations will allow us to assess whether SFTSVdelNSs (and virus-like particles) can potentially be used as vaccine candidate. If a positive outcome is achieved, further development of attenuated viruses in clinical trials could benefit the people in affected areas in China, and possibly beyond. Moreover, experimental tools to determine and measure antibody responses to SFTSV (including mutations that may prevent virus escape mutants that can then be included in vaccine compositions) have applications beyond what we propose here, from vaccine design to diagnostics in human patients to investigations of SFTSV reservoir(s). The novel antibodies which we aim to produce in the context of this project also have the potential to support basic research/diagnostics and may lead towards therapeutics. Thus, in the long term there is the potential for a broad societal impact of this research.

(3) The third impact area is transfer knowledge and staff training. CVR has unique experience in SFTSV studies through the reverse genetics system we have developed, allowing the engineering of this virus (for example through the creation of virulence factor deficient viruses, as proposed here). CVR will train the BIOLS-based postdoctoral fellow in bunyavirus/SFTSV reverse genetics and transfer expertise and reagents so that such experiments can be carried out by the Chinese partner in the future. Given the strong interest in SFTSV research in China and elsewhere in Asia, this has the potential to transform studies on SFTSV in a positive manner and allow future projects to be entirely designed in affected countries.

Overall, we believe that we have designed a logical flow of work that combines scientific studies while remaining mindful of the societal impact of our work, all of which this group of collaborators is very well set up to do.