An Industry Perspective on Dengue Drug Discovery and Development.

Dengue is the most important mosquito-borne viral disease in the world, representing a major unmet medical need and a growing public health concern. The disease imposes a heavy burden to the affected individuals, to the health care systems, and to the economies of endemic countries. Vector control is the most widespread tool to curb dengue epidemics, but has been insufficient. Therefore, additional means such as vaccines and antivirals are required to aid in a coordinated response. The discovery and development of small molecule dengue virus inhibitors as a tool to prevent and/or treat dengue disease faces major hurdles in combining pan-serotypic efficacy, safety, and optimal drug-like properties. Moreover, the financial return of dengue drug projects may not compensate for the initial investment in research and development. This review article addresses the efforts undertaken to face the dengue epidemics, focusing on antiviral drug development. The dengue drug research and development process is described in detail and a dengue antiviral target product profile is proposed. The article discusses collaborations between the different players in the research field: government and government-sponsored organizations, pharmaceutical and biotechnology companies, academia, and non-profit and philanthropic organizations. Public-private partnerships are proposed as a model to boost dengue research and development towards an approved antiviral drug in the near future.

Synthetic Peptides Elicit Humoral Response against Porcine Reproductive and Respiratory Syndrome Virus in Swine.

The aim of this study was to analyze the immunogenic response elicited in swine by two synthetic peptides derived from GP5 to understand the role of lineal B epitopes in the humoral and B-cell-mediated response against the porcine reproductive and respiratory syndrome virus (PRRSV). For inoculation, twenty-one-day-old pigs were allocated into six groups: control, vehicle, vaccinated (Ingelvac-PRRSV, MLV®), non-vaccinated and naturally infected, GP5-B and GP5-B3. At 2 days post-immunization (dpi), the GP5-B3 peptide increased the serum concentrations of cytokines associated with activate adaptive cellular immunity, IL-1ß (1.15 ± 1.15 to 10.17 ± 0.94 pg/mL) and IL-12 (323.8 ± 23.3 to 778.5 ± 58.11 pg/mL), compared to the control group. The concentration of IgGs anti-GP5-B increased in both cases at 21 and 42 dpi compared to that at 0 days (128.3 ± 8.34 ng/mL to 231.9 ± 17.82 and 331 ± 14.86 ng/mL), while IgGs anti-GP5-B3 increased at 21 dpi (105.1 ± 19.06 to 178 ± 15.09 ng/mL) and remained at the same level until 42 dpi. Also, antibody-forming/Plasma B cells (CD2+/CD21-) increased in both cases (9.85 ± 0.7% to 13.67 ± 0.44 for GP5-B and 15.72 ± 1.27% for GP5-B3). Furthermore, primed B cells (CD2-/CD21+) from immunized pigs showed an increase in both cases (9.62 ± 1.5% to 24.51 ± 1.3 for GP5-B and 34 ± 2.39% for GP5-B3) at 42 dpi. Conversely the naïve B cells from immunized pigs decreased compared with the control group (8.84 ± 0.63% to 6.25 ± 0.66 for GP5-B and 5.78 ± 0.48% for GP5-B3). Importantly, both GP5-B and GP5-B3 peptides exhibited immunoreactivity against serum antibodies from the vaccinated group, as well as the non-vaccinated and naturally infected group. In conclusion, GP5-B and GP5-B3 peptides elicited immunogenicity mediated by antigen-specific IgGs and B cell activation.

Response of lymphocytes from pigs naturally infected with porcine respiratory disease complex at 3 different stages of development.

The objective of this study was to analyze the response of lymphocytes from pigs naturally infected with porcine respiratory disease complex (PRDC) at 3 different stages of development. Porcine respiratory disease complexes were isolated from 2 groups: The infected group, consisting of pigs with PRDC and no vaccination against any virus (n = 24), and the control group, consisting of vaccinated and noninfected piglets (n = 24). Both groups were sampled at 3 stages of development: Weaning (WEA) (n = 8), initiation (INI) (n = 8), and growth (GRO) (n = 8). The PRDC status was confirmed by serological testing against porcine circovirus type 2 (PCV-2), porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (H1N1), and Mycoplasma hyopneumoniae. PCV-2+ cells were quantified by flow cytometry. Weight gain was registered at each stage. PCV-2+ cells, CD4+ cells, monocytes and lymphocytes populations were measured. Gene expression in CD4+ cells was quantified for interferon-γ (IFN-γ), GATA binding protein 3 (GATA3), T-box transcription factor (T-bet), interleukin-10 (IL-10), and IL-4. Control piglets gained approximately 35% more weight than those infected with PRDC. Specifically, PCV-2+ cells were detected in piglets from the infected group in the following proportions: WEA ≤ INI ≤ GRO. In infected piglets, the CD4+ count increased at WEA and decreased at GRO, CD4+ expression profile showed an overexpression of T-bet at INI and GRO, and the expression of IFN-γ was lower at WEA and GRO. In contrast, IL-4 was overexpressed at all 3 stages. GATA3 was overexpressed at INI and GRO. The infected piglets showed lymphopenia and less CD4+ cells. CD4+ cells showed a different expression profile than the control group, in which IFN-γ was less expressed, whereas IL-4 and T-bet were overexpressed.

L'objectif de cette étude était d'analyser la réponse des lymphocytes de porcs naturellement infectés par le complexe respiratoire porcin (PRDC) à trois stades de développement différents. Des PRDC ont été isolés à partir de deux groupes : le groupe infecté, composé de porcs atteints de PRDC et non vaccinés contre un virus (n = 24), et le groupe témoin, composé de porcelets vaccinés et non infectés (n = 24). Les deux groupes ont été échantillonnés à trois stades de développement : sevrage (WEA) (n = 8), initiation (INI) (n = 8) et croissance (GRO) (n = 8). Le statut de PRDC a été confirmé par des tests sérologiques contre le circovirus porcin de type 2 (PCV-2), le virus du syndrome reproducteur et respiratoire porcin (PRRSV), le virus de la grippe porcine (H1N1) et Mycoplasma hyopneumoniae. Les cellules PCV-2+ ont été quantifiées par cytométrie en flux. Un gain de poids a été enregistré à chaque étape. Les populations de cellules PCV-2+, de cellules CD4+, de monocytes et de lymphocytes ont été mesurées. L'expression génique dans les cellules CD4+ a été quantifiée pour l'interféron-γ (IFN-γ), la protéine de liaison GATA 3 (GATA3), le facteur de transcription T-box (T-bet), l'interleukine-10 (IL-10) et l'IL-4. Les porcelets témoins ont pris environ 35 % de poids en plus que ceux infectés par le PRDC. Plus précisément, des cellules PCV-2+ ont été détectées chez les porcelets du groupe infecté dans les proportions suivantes : WEA ≤ INI ≤ GRO. Chez les porcelets infectés, le nombre de CD4+ a augmenté à WEA et diminué à GRO, le profil d'expression de CD4+ a montré une surexpression de T-bet à INI et GRO, et l'expression d'IFN-γ était plus faible à WEA et GRO. En revanche, l'IL-4 était surexprimée aux trois stades. GATA3 était surexprimé à INI et GRO. Les porcelets infectés présentaient une lymphopénie et moins de cellules CD4+. Les cellules CD4+ ont montré un profil d'expression différent de celui du groupe témoin, dans lequel l'IFN-γ était moins exprimé, tandis que l'IL-4 et le T-bet étaient surexprimés.(Traduit par Docteur Serge Messier).

Glycoprotein 5-Derived Peptides Induce a Protective T-Cell Response in Swine against the Porcine Reproductive and Respiratory Syndrome Virus.

We analyzed the T-cell responses induced by lineal epitopes of glycoprotein 5 (GP5) from PRRSV to explore the role of this protein in the immunological protection mediated by T-cells. The GP5 peptides were conjugated with a carrier protein for primary immunization and booster doses. Twenty-one-day-old pigs were allocated into four groups (seven pigs per group): control (PBS), vehicle (carrier), PTC1, and PTC2. Cytokine levels were measured at 2 days post-immunization (DPI) from serum samples. Cytotoxic T-lymphocytes (CTLs, CD8+) from peripheral blood were quantified via flow cytometry at 42 DPI. The cytotoxicity was evaluated by co-culturing primed lymphocytes with PRRSV derived from an infectious clone. The PTC2 peptide increased the serum concentrations of pro-inflammatory cytokines (i.e., TNF-α, IL-1ß, IL-8) and cytokines that activate the adaptive cellular immunity associated with T-lymphocytes (i.e., IL-4, IL-6, IL-10, and IL-12). The concentration of CTLs (CD8+) was significantly higher in groups immunized with the peptides, which suggests a proliferative response in this cell population. Primed CTLs from immunized pigs showed cytolytic activity in PRRSV-infected cells in vitro. PTC1 and PTC2 peptides induced a protective T-cell-mediated response in pigs immunized against PRRSV, due to the presence of T epitopes in their sequences.

Characterization of a dengue NS4B inhibitor originating from an HCV small molecule library.

Dengue is the most important mosquito-transmitted viral disease and a major global health concern. Over the last decade, dengue virus (DENV) drug discovery and development has intensified, however, this has not resulted in approved DENV-specific antiviral treatments yet. DENV and hepatitis C virus (HCV) belong to the same Flaviviridae family and, in contrast to DENV, antiviral treatments for HCV have been licensed. Therefore, applying the knowledge gained on anti-HCV drugs may foster the discovery and development of dengue antiviral drugs. Here, we screened a library of compounds with established anti-HCV activity in a DENV-2 sub-genomic replicon inhibition assay and selected compounds with single-digit micromolar activity. These compounds were advanced into a hit-to-lead medicinal chemistry program resulting in lead compound JNJ-1A, which inhibited the DENV-2 sub-genomic replicon at 0.7 µM, in the absence of cytotoxicity. In addition, JNJ-1A showed equipotent antiviral activity against DENV serotypes 1, 2, and 4. In vitro resistance selection experiments with JNJ-1A induced mutation T108I in non-structural protein 4B (NS4B), pointing towards a mechanism of action linked to this protein. Collectively, we described the discovery and characterization of a novel DENV inhibitor potentially targeting NS4B.