Host cell killing by the West Nile Virus NS2B–NS3 proteolytic complex: NS3 alone is sufficient to recruit caspase-8-based apoptotic pathway

AbstractThe West Nile Virus (WNV) non-structural proteins 2B and 3 (NS2B–NS3) constitute the proteolytic complex that mediates the cleavage and processing of the viral polyprotein. NS3 recruits NS2B and NS5 proteins to direct protease and replication activities. In an effort to investigate the biology of the viral protease, we cloned cDNA encoding the NS2B–NS3 proteolytic complex from brain tissue of a WNV-infected dead crow, collected from the Lower Merion area (Merion strain). Expression of the NS2B–NS3 gene cassette induced apoptosis within 48 h of transfection. Electron microscopic analysis of NS2B–NS3-transfected cells revealed ultra-structural changes that are typical of apoptotic cells including membrane blebbing, nuclear disintegration and cytoplasmic vacuolations. The role of NS3 or NS2B in contributing to host cell apoptosis was examined. NS3 alone triggers the apoptotic pathways involving caspases-8 and -3. Experimental results from the use of caspase-specific inhibitors and caspase-8 siRNA demonstrated that the activation of caspase-8 was essential to initiate apoptotic signaling in NS3-expressing cells. Downstream of caspase-3 activation, we observed nuclear membrane ruptures and cleavage of the DNA-repair enzyme, PARP in NS3-expressing cells. Nuclear herniations due to NS3 expression were absent in the cells treated with a caspase-3 inhibitor. Expression of protease and helicase domains themselves was sufficient to trigger apoptosis generating insight into the apoptotic pathways triggered by NS3 from WNV

Unique Th1/Th2 Phenotypes Induced during Priming and Memory Phases by Use of Interleukin-12 (IL-12) or IL-28B Vaccine Adjuvants in Rhesus Macaques ▿

Adjuvant compounds are usually included in vaccinations in order to bolster total vaccine-specific responses or to tailor an immune response toward a desired endpoint, such as the production of gamma interferon or an increase in antibody titers. While most adjuvants are studied in regard to their impact on vaccine-specific responses during and just after the vaccination period, a detailed analysis of how adjuvants skew the Th1/Th2 axis at more distant time points is not often undertaken. In the current study, we present data that suggests that adjuvants differ in their relative abilities to bolster and skew immune responses in the short term compared with more distant time points. To that end, we have employed interleukin-12 (IL-12) and IL-28B as adjuvants for DNA vaccination of rhesus macaques. While both adjuvants were able to bolster Th1-biased responses, our analysis shows that this skewing was achieved through different mechanisms. Moreover, analysis 3 months after the final immunization revealed the activity of the IL-12 adjuvant to be short lived, while the IL-28B adjuvant continued to exert its influence on the immune system. Taken together, these data suggest that the scientific and medical communities would benefit from a more detailed analysis of adjuvant function, including the determination of long-term influences of administered adjuvants

IL-28B/IFN-λ3 Drives Granzyme B Loading and Significantly Increases CTL Killing Activity in Macaques

Type III/λ interferons (IFNs) were discovered less than a decade ago and are still in the process of being characterized. Although previous studies have focused on the function of IFN-λ3 (also known as interleukin (IL)-28B) in a small animal model, it is unknown whether these functions would translate to a larger, more relevant model. Thus in the present study, we have used DNA vaccination as a method of studying the influence of IFN-λ3 on adaptive immune responses in rhesus macaques. Results of our study show for the first time that IFN-λ3 has significant influence on antigen-specific CD8+ T-cell function, especially in regards to cytotoxicity. Peripheral CD8+ T cells from animals that were administered IFN-λ3 showed substantially increased cytotoxic responses as gauged by CD107a and granzyme B coexpression as well as perforin release. Moreover, CD8+ T cells isolated from the mesenteric lymph nodes (MLN) of animals receiving IFN-λ3 loaded significant amounts of granzyme B upon extended antigenic stimulation and induced significantly more granzyme B-mediated cell death of peptide pulsed targets. These data suggest that IFN-λ3 is a potent effector of the immune system with special emphasis on CD8+ T-cell killing functions which warrants further study as a possible immunoadjuvant