DISSECT

EC contribution

: € 2 375 892

Duration

: 36 months

Starting date

: 01/10/2004

Instrument

: STREP

Keywords

: SARS, vaccines, antivirals, immunotherapy, Coronavirus

Project Number

: SP22-CT-2004-511060

Web-site

: http://www.cnb.csic.es/~webcoron/EUprojectdissect

Results:

A collection of viral proteins expressed mainly in baculoviruses and plants has been obtained. This collection includes proteins S, S1, S2, M, E, N, 3a, 6, and 7a. To increase the immunogenicity of these proteins, virus-like particles (VLPs) formation was achieved by the co-expression in the baculovirus system of three proteins: M, E, and S. These VLPs induced a neutralising immune response to SARS-CoV and elicited protection in a mice model.

Several plant expression systems based on Plum pox potyvirus (PPV) and Potato virus X (PVX) have been developed to express at low cost SARS-CoV antigens. SARS-CoV S, S1, E, M, N, 3a proteins alone or linked to a cell compartment retention signal, to increase expression, were also obtained. In addition, several transgenic lines expressing constitutively tagged S, S1, M, N and 3a proteins were produced. Furthermore, expression of SARS-CoV antigens in plants, with modified glycosylation systems mimicking the mammalian glycosylation pattern, has been obtained.

Electron microscopy of the recombinant SARS-CoV engineered. Left panel: viral factory of the parental virulent virus (SARS-CoV). Right panel: viral factory of the attenuated deletion mutant vaccine candidate (SARS-CoV-E)

The first recombinant vaccine for SARS-CoV has been constructed. The efficacy of this vaccine has been shown in two animal model systems, and has provided protection against homologous and heterologous viruses. The morphogenesis of the vaccine virus missing protein E is shown in comparison with that of the parental full-length virus (Figure 1). To assemble a recombinant vaccine for SARS-CoV, an infectious cDNA of SARS-CoV was constructed using a bacterial artificial chromosome (BAC).

From this cDNA, a replicon was derived that is very useful for screening anti-virals without the need to use infectious virus. E, 6, 7a, 7b, 8a, 8b and 9b proteins were not essential for virus replication in vitro or in vivo. In a transgenic mice model highly sensitive to the virus, the DISSECT partners have shown that a SARSCoV E protein deletion mutant was attenuated, whereas removal of genes 6 to 9b reduced very little pathogenicity. A virus mutant missing all these seven genes provided protection against SARS-CoV.

The attenuated rSARS-CoV engineered is an excellent starting point for the production of a chemically inactivated vaccine. This type of vaccine will include two safety guards, the chemical inactivation, and the attenuated phenotype, in case the chemical inactivation is incomplete inside a virus aggregate.

The partners have shown that inactivated SARS-CoV vaccines protect macaques and ferrets against an intratracheal challenge with SARS-CoV, and that the induction of neutralising antibodies may suffice to protect against SARSCoV.

The inflammatory response in SARS-CoV infected vaccinated and nave macaques was analysed. A wide range of cytokines including IL-1, IL-6, IL-8 and IFNs, several chemokines such as monocyte chemotactic protein genes like CCL8, CCL7, and also CCL11 (eotaxin), a chemotactic protein for eosinophils were identified. These results provided a rationale for using live attenuated vaccines.

Both ferret and macaque have been developed as animal models for SARS-CoV. Polyvalent antibodies of human origin protect against SARS in macaques. In addition to the description of pegylated interferon alpha as an antiviral, two other antivirals have been identified: IL-4 and interferon-gamma. The mechanism of action of IL-4 includes a reduction in the expression of the ACE-2 receptor of SARS-CoV.

The identification of genes that may contribute to SARS-CoV virulence was studied as their removal may lead to safer SARS-CoV vaccines. The role of accessory 3, 6, and 8 proteins in virus host interaction has been determined. 3a protein is O-glycosylated, and that interacts with the viral M protein. ORF 8 acquired a deletion early after SARS-CoV jumped from the animal reservoir to the human population and this deletion was found in all later human isolates. The 29nt deletion plays a role in the efficient spread or the high pathogenicity of SARS-CoV in the human population. Protein 6 was involved in virus replication and virulence. The consortium has shown that IFN-a inhibits SARS-CoV replication in macaques and has identified IL-8 as one of the pathogenic cytokines induced at high levels in macaques.

These studies emphasised that IFN production inversely correlated with IL-8 induction. As IL-8 is a central mediator of inflammatory responses, including acute respiratory distress and acute lung injury, modulation of its expression by the IFNs may reveal an important new intervention strategy to suppress pathogenic responses.

A new genomic approach to diagnose SARSCoV, based on microarray technology, is being developed, which is independent of hybridisation techniques, and uses primer extension (APEX) technology.

Potential applications:

1. diagnosis and protection of the population in case of SARS re-emergence by vaccination;
2. therapeutic strategies for SARS, including antivirals and immunotherapy.

Coordinator:
Prof. Luis Enjuanes

Partners:

Collaborators: