TLR2 axis on peripheral blood mononuclear cells regulates inflammatory responses to non-infectious immature dengue virus particles.

Severe dengue virus (DENV) infection is characterized by exacerbated inflammatory responses that lead to endothelial dysfunction and plasma leakage. We have recently demonstrated that Toll-like receptor 2 (TLR2) on blood monocytes senses DENV infection leading to endothelial activation. Here, we report that non-infectious immature DENV particles, which are released in large numbers by DENV-infected cells, drive endothelial activation via the TLR2 axis. We show that fully immature DENV particles induce a rapid, within 6 hours post-infection, inflammatory response in PBMCs. Furthermore, pharmacological blocking of TLR2/TLR6/CD14 and/or NF-kB prior to exposure of PBMCs to immature DENV reduces the initial production of inter alia TNF-α and IL-1ß by monocytes and prevents endothelial activation. However, prolonged TLR2 block induces TNF-α production and leads to exacerbated endothelial activation, indicating that TLR2-mediated responses play an important role not only in the initiation but also the resolution of inflammation. Altogether, these data indicate that the maturation status of the virus has the potential to influence the kinetics and extent of inflammatory responses during DENV infection.

Nano-multilamellar lipid vesicles loaded with a recombinant form of the chikungunya virus E2 protein improve the induction of virus-neutralizing antibodies.

Chikungunya virus (CHIKV) is responsible for a self-limited illness that can evolve into long-lasting painful joint inflammation. In this study, we report a novel experimental CHIKV vaccine formulation of lipid nanoparticles loaded with a recombinant protein derived from the E2 structural protein. This antigen fragment, designated ∆E2.1, maintained the antigenicity of the native viral protein and was specifically recognized by antibodies induced in CHIKV-infected patients. The antigen has been formulated into nanoparticles consisting of nano-multilamellar vesicles (NMVs) combined with the adjuvant monophosphoryl lipid A (MPLA). The vaccine formulation demonstrated a depot effect, leading to controlled antigen release, and induced strong antibody responses significantly higher than in mice immunized with the purified protein combined with the adjuvant. More relevantly, E2-specific antibodies raised in mice immunized with ∆E2.1-loaded NMV-MPLA neutralized CHIKV under in vitro conditions. Taken together, the results demonstrated that the new nanoparticle-based vaccine formulation represents a promising approach for the development of effective anti-CHIKV vaccines.

Association of high pressure and alkaline condition for solubilization of inclusion bodies and refolding of the NS1 protein from zika virus.

BACKGROUND: Proteins in inclusion bodies (IBs) present native-like secondary structures. However, chaotropic agents at denaturing concentrations, which are widely used for IB solubilization and subsequent refolding, unfold these secondary structures. Removal of the chaotropes frequently causes reaggregation and poor recovery of bioactive proteins. High hydrostatic pressure (HHP) and alkaline pH are two conditions that, in the presence of low level of chaotropes, have been described as non-denaturing solubilization agents. In the present study we evaluated the strategy of combination of HHP and alkaline pH on the solubilization of IB using as a model an antigenic form of the zika virus (ZIKV) non-structural 1 (NS1) protein. RESULTS: Pressure-treatment (2.4 kbar) of NS1-IBs at a pH of 11.0 induced a low degree of NS1 unfolding and led to solubilization of the IBs, mainly into monomers. After dialysis at pH 8.5, NS1 was refolded and formed soluble oligomers. High (up to 68 mg/liter) NS1 concentrations were obtained by solubilization of NS1-IBs at pH 11 in the presence of arginine (Arg) with a final yield of approximately 80% of total protein content. The process proved to be efficient, quick and did not require further purification steps. Refolded NS1 preserved biological features regarding reactivity with antigen-specific antibodies, including sera of ZIKV-infected patients. The method resulted in an increase of approximately 30-fold over conventional IB solubilization-refolding methods. CONCLUSIONS: The present results represent an innovative non-denaturing protein refolding process by means of the concomitant use of HHP and alkaline pH. Application of the reported method allowed the recovery of ZIKV NS1 at a condition that maintained the antigenic properties of the protein.

Antibodies are not required to a protective immune response against dengue virus elicited in a mouse encephalitis model.

Generating neutralizing antibodies have been considered a prerequisite to control dengue virus (DENV) infection. However, T lymphocytes have also been shown to be important in a protective immune state. In order to investigate the contribution of both humoral and cellular immune responses in DENV immunity, we used an experimental model in which a non-lethal DENV2 strain (ACS46) is used to intracranially prime Balb/C mice which develop protective immunity against a lethal DENV2 strain (JHA1). Primed mice generated envelope-specific antibodies and CD8(+) T cell responses targeting mainly non-structural proteins. Immune sera from protected mice did not confer passive protection to naïve mice challenged with the JHA1 strain. In contrast, depletion of CD4(+) and CD8(+) T lymphocytes significantly reduced survival of ACS46-primed mice challenged with the JHA1 strain. Collectively, results presented in this study show that a cellular immune response targeting non-structural proteins are a promising way in vaccine development against dengue.