Immune response to a model shared placenta/tumor-associated antigen reduces cancer risk in parous mice.

During human pregnancy, paternally inherited antigens expressed by the fetal-placental unit can elicit expansion of antigen-specific CD8+ T cells. These cells can persist for years as memory T cells, but their effects on long-term maternal health are unknown. Shared placenta/tumor-associated antigens are expressed by placenta and tumors, but are minimally expressed or absent in normal adult tissues. We hypothesized that maternal T cells elicited against these antigens can alter risk of cancers expressing the same antigen after pregnancy, and tested this in mice using chicken ovalbumin (OVA) as a surrogate shared placenta/tumor antigen. Hemizygous OVA transgenic males were bred to wild-type C57BL/6 females (H2b haplotype) such that the fetuses inherited and expressed OVA. Maternal OVA/H2Kb-specific CD8+ T cells became detectable during gestation, and persisted in some animals for up to 24 weeks. To determine whether these cells might influence growth of OVA-expressing tumors in OVA-bred females, E.G7-OVA thymoma cells were inoculated subcutaneously in OVA-bred, wild-type bred, and virgin females, and monitored for growth. OVA-bred mice had prolonged survival as compared to virgin mice and the progression of tumors was delayed in comparison to wild-type bred and virgin females. Thus, paternally inherited OVA antigen elicited a CD8+ T cell response during pregnancy that was associated with delayed growth of OVA-expressing tumors following pregnancy. These data suggest a possible role of antigen-specific T cells in protecting parous females against tumors bearing shared placenta/tumor antigens.

Crystallization and preliminary X-ray analysis of Ebola VP35 interferon inhibitory domain mutant proteins.

VP35 is one of seven structural proteins encoded by the Ebola viral genome and mediates viral replication, nucleocapsid formation and host immune suppression. The C-terminal interferon inhibitory domain (IID) of VP35 is critical for dsRNA binding and interferon inhibition. The wild-type VP35 IID structure revealed several conserved residues that are important for dsRNA binding and interferon antagonism. Here, the expression, purification and crystallization of recombinant Zaire Ebola VP35 IID mutants R312A, K319A/R322A and K339A in space groups P6(1)22, P2(1)2(1)2(1) and P2(1), respectively, are described. Diffraction data were collected using synchrotron sources at the Advanced Light Source and the Advanced Photon Source.

Structural and functional characterization of Reston Ebola virus VP35 interferon inhibitory domain.

Ebolaviruses are causative agents of lethal hemorrhagic fever in humans and nonhuman primates. Among the filoviruses characterized thus far, Reston Ebola virus (REBOV) is the only Ebola virus that is nonpathogenic to humans despite the fact that REBOV can cause lethal disease in nonhuman primates. Previous studies also suggest that REBOV is less effective at inhibiting host innate immune responses than Zaire Ebola virus (ZEBOV) or Marburg virus. Virally encoded VP35 protein is critical for immune suppression, but an understanding of the relative contributions of VP35 proteins from REBOV and other filoviruses is currently lacking. In order to address this question, we characterized the REBOV VP35 interferon inhibitory domain (IID) using structural, biochemical, and virological studies. These studies reveal differences in double-stranded RNA binding and interferon inhibition between the two species. These observed differences are likely due to increased stability and loss of flexibility in REBOV VP35 IID, as demonstrated by thermal shift stability assays. Consistent with this finding, the 1.71-A crystal structure of REBOV VP35 IID reveals that it is highly similar to that of ZEBOV VP35 IID, with an overall backbone r.m.s.d. of 0.64 A, but contains an additional helical element at the linker between the two subdomains of VP35 IID. Mutations near the linker, including swapping sequences between REBOV and ZEBOV, reveal that the linker sequence has limited tolerance for variability. Together with the previously solved ligand-free and double-stranded-RNA-bound forms of ZEBOV VP35 IID structures, our current studies on REBOV VP35 IID reinforce the importance of VP35 in immune suppression. Functional differences observed between REBOV and ZEBOV VP35 proteins may contribute to observed differences in pathogenicity, but these are unlikely to be the major determinant. However, the high level of similarity in structure and the low tolerance for sequence variability, coupled with the multiple critical roles played by Ebola virus VP35 proteins, highlight the viability of VP35 as a potential target for therapeutic development.

Structural basis for dsRNA recognition and interferon antagonism by Ebola VP35.

Ebola viral protein 35 (VP35), encoded by the highly pathogenic Ebola virus, facilitates host immune evasion by antagonizing antiviral signaling pathways, including those initiated by RIG-I-like receptors. Here we report the crystal structure of the Ebola VP35 interferon inhibitory domain (IID) bound to short double-stranded RNA (dsRNA), which together with in vivo results reveals how VP35-dsRNA interactions contribute to immune evasion. Conserved basic residues in VP35 IID recognize the dsRNA backbone, whereas the dsRNA blunt ends are 'end-capped' by a pocket of hydrophobic residues that mimic RIG-I-like receptor recognition of blunt-end dsRNA. Residues critical for RNA binding are also important for interferon inhibition in vivo but not for viral polymerase cofactor function of VP35. These results suggest that simultaneous recognition of dsRNA backbone and blunt ends provides a mechanism by which Ebola VP35 antagonizes host dsRNA sensors and immune responses.