TLR Stimulation Produces IFN-ß as the Primary Driver of IFN Signaling in Nonlymphoid Primary Human Cells.

Several human autoimmune diseases are characterized by increased expression of type 1 IFN-stimulated genes in both the peripheral blood and tissue. The contributions of different type I IFNs to this gene signature are uncertain as the type I IFN family consists of 13 alphas and one each of ß, ε, κ, and ω subtypes. We sought to investigate the contribution of various IFNs to IFN signaling in primary human cell types. We stimulated primary skin, muscle, kidney, and PBMCs from normal healthy human donors with various TLR ligands and measured the expression of type I IFN subtypes and activation of downstream signaling by quantitative PCR. We show that IFNB1 is the dominant type I IFN expressed upon TLR3 and TLR4 stimulation, and its expression profile is associated with subsequent MX1 transcription. Furthermore, using an IFN-ß-specific neutralizing Ab, we show that MX1 expression is inhibited in a dose-dependent manner, suggesting that IFN-ß is the primary driver of IFN-stimulated genes following TLR3 and TLR4 engagement. Stimulation with TLR7/8 and TLR9 ligands induced IFNB1 and IFNA subtypes and MX1 expression only in PBMCs and not in tissue resident cell types. Concordantly, IFN-ß neutralization had no effect on MX1 expression in PBMCs potentially because of the combination of IFNB1 and IFNA expression. Combined, these data highlight the potential role for IFN-ß in driving local inflammatory responses in clinically relevant human tissue types and opportunities to treat local inflammation by targeting IFN-ß.

Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay.

Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2', 3' -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2'-5' and 3'-5' phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a high affinity (KD = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors.

Biological properties of chimeric West Nile viruses.

Recently, we have described a lineage 2 attenuated WN virus suitable for the development of a live WN vaccine. To design vaccine candidates with an improved immunogenicity, we assembled an infectious clone of the NY99 strain and created several chimeric constructs with reciprocal exchanges of structural protein genes between attenuated W956 and virulent NY99 and investigated their biological properties. Our data indicated that, while the growth rates of NY99 and chimeric viruses in tissue culture are determined primarily by properties of the structural proteins, determinants responsible for a highly cytopathic phenotype of NY99 or lack thereof for W956 are located within the nonstructural protein region of the WN genome. The high virulence of NY99 and the attenuated phenotype of W956 were found to be associated with determinants in the nonstructural region. Chimeric viruses carrying the NY99 structural proteins were attenuated in neuroinvasiveness and demonstrated an immunogenicity superior to W956.

Immunogenicity of West Nile virus infectious DNA and its noninfectious derivatives.

The exceptionally high virulence of the West Nile NY99 strain makes its suitability in the development of a live WN vaccine uncertain. The aim of this study is to investigate the immunogenicity of noninfectious virus derivatives carrying pseudolethal mutations, which preclude virion formation without affecting preceding steps of the viral infectious cycle. When administered using DNA immunization, such constructs initiate an infectious cycle but cannot lead to a viremia. While the magnitude of the immune response to a noninfectious replication-competent construct was lower than that of virus or infectious DNA, its overall quality and the protective effect were similar. In contrast, a nonreplicating construct of similar length induced only a marginally detectable immune response in the dose range used. Thus, replication-competent noninfectious constructs derived from infectious DNA may offer an advantageous combination of the safety of noninfectious formulations with the quality of the immune response characteristic of infectious vaccines.

The suitability of yellow fever and Japanese encephalitis vaccines for immunization against West Nile virus.

Seven volunteers involved in flavivirus studies have been immunized with commercial Japanese encephalitis and yellow fever vaccines JE-VAX and YF-VAX. Strong homologous and cross-reactive with West Nile virus (WNV) antibody responses with titers 1:1600 to 1:51200 were found in all donors. All donors developed high levels of yellow fever virus (YFV) and Japanese encephalitis virus (JEV) neutralizing antibodies with titers 1:50 to 1:1600 and 1:20 to 1:640, respectively, and WNV neutralizing antibodies with titers 1:10 to 1:80. In contrast, predominantly YF-specific cell-mediated immunity was detected in all immunized donors. Responses to YFV were long lasting, but the anti-JEV humoral immunity was found to decrease with time. Cross-reactive anti-WNV responses were following the same trend dropping below detectable level at 4 years post-immunization and sharply coming back after booster immunization with the JE vaccine. Thus, immunization with the commercial flavivirus JE vaccine may be beneficial for individuals at high risk of exposure to WNV, such as personnel involved in WN research.