There is no licensed human vaccine currently available for Rift Valley Fever Virus (RVFV), a Category A high priority pathogen and a serious zoonotic threat. While neutralizing antibodies targeting the viral glycoproteins are protective, they appear late in the course of infection, and may not be induced in time to prevent a natural or bioterrorism-induced outbreak. Here we examined the immunogenicity of RVFV nucleocapsid (N) protein as a CD8+ T cell antigen with the potential for inducing rapid protection after vaccination. HLA-A*0201 (A2)-restricted epitopic determinants were identified with N-specific CD8+ T cells from eight healthy donors that were primed with dendritic cells transduced to express N, and subsequently expanded in vitro by weekly re-stimulations with monocytes pulsed with 59 15mer overlapping peptides (OLPs) across N. Two immunodominant epitopes, VT9 (VLSEWLPVT, N121–129) and IL9 (ILDAHSLYL, N165–173), were defined. VT9- and IL9-specific CD8+ T cells identified by tetramer staining were cytotoxic and polyfunctional, characteristics deemed important for viral control in vivo. These peptides induced specific CD8+ T cell responses in A2-transgenic mice, and more importantly, potent N-specific CD8+ T cell reactivities, including VT9- and IL9-specific ones, were mounted by mice after a booster vaccination with the live attenuated RVF MP-12. Our data suggest that the RVFV N protein is a potent human T cell immunogen capable of eliciting broad, immunodominant CD8+ T cell responses that are potentially protective. Understanding the immune responses to the nucleocapsid is central to the design of an effective RVFV vaccine irrespective of whether this viral protein is effective as a stand-alone immunogen or only in combination with other RVFV antigens.

This study evaluated bacteriophages 1)X174, T7, PRD1, and 4)6 as possible surrogates for pathogenic human viruses to challenge barrier materials and demonstrated some important factors for their use. Chemical incompatibility with test material was demonstrated when lipid-enveloped 1)6 was inactivated by an aqueous eluate of vinyl gloves, but 0.5% calf serum protected (D6 from the eluate. Low concentrations (2%) of calf serum also prevented the exaggerated binding of the bacteriophages to filters. Recovery of viruses from surfaces decreased with increasing time before recovery. Penetration through punctures displayed different types of kinetics. The combined data indicate that (i) some bacteriophages may serve as surrogate viruses, (ii) experimental conditions determine whether a particular virus is appropriate as a challenge, and (iii) 1)X174 is an excellent choice as a surrogate virus to test barrier materials. The data further indicate that before barrier materials are challenged with viruses, adequate tests should be performed to ensure that the virus is compatible with the test material and test conditions, so that meaningful data will result.

Budding yeast cells interpret shallow pheromone gradients from cells of the opposite mating type, polarize their growth toward the pheromone source, and fuse at the chemotropic growth site. We previously proposed a deterministic, gradient-sensing model that explains how yeast cells switch from the intrinsically positioned default polarity site (DS) to the gradient-aligned chemotropic site (CS) at the plasma membrane. Because phosphorylation of the mating-specific Gβ subunit is thought to be important for this process, we developed a biosensor that bound to phosphorylated but not unphosphorylated Gβ and monitored its spatiotemporal dynamics to test key predictions of our gradient-sensing model. In mating cells, the biosensor colocalized with both Gβ and receptor reporters at the DS and then tracked with them to the CS. The biosensor concentrated on the leading side of the tracking Gβ and receptor peaks and was the first to arrive and stop tracking at the CS. Our data showed that the concentrated localization of phosphorylated Gβ correlated with the tracking direction and final position of the G protein and receptor, consistent with the idea that gradient-regulated phosphorylation and dephosphorylation of Gβ contributes to gradient sensing. Cells expressing a nonphosphorylatable mutant form of Gβ exhibited defects in gradient tracking, orientation toward mating partners, and mating efficiency.

MHC-E, a non-classical MHC molecule, restricted CD8 T-cell responses have been associated with protection in an SIV/rhesus macaque model. The biological relevance of HLA-E restricted CD8 T-cell responses in HIV infection however remains unknown. In this study, CD8 T cells responding to HIV-1 Gag peptides presented by HLA-E were analyzed. Using in-vitro assays, we observed HLA-E restricted T-cell responses to what we believe to be a newly identified subdominant Gag-KL9 as well as a well-described immuno-dominant Gag-KF11 epitope in T-cell lines derived from chronically HIV-infected patients and also primed from healthy donors. Blocking of the HLA-E/KF11 binding by the B7 signal peptide resulted in decreased CD8 T-cell responses. KF11 presented via HLA-E in HIV infected cells was recognized by antigen specific CD8 T cells. Importantly, bulk CD8 T cells obtained from HIV infected individuals recognized infected cells via HLA-E presentation. Ex-vivo analyses at the epitope level showed a higher responder frequency of HLA-E restricted responses to KF11 compared to KL9.Taken together, our findings of HLA-E restricted HIV specific immune responses offer intriguing and possibly paradigm shifting insights into factors that contribute to the immuno-dominance of CD8 T-cell responses in HIV infection.

Filters with well-defined holes were used to determine the effective diameters in buffer of human inmunodeficiency virus type 1, herpes simplex virus type 1, and four bacteriophages (+X174, T7, PRD1, and +6), which may serve as surrogate viruses for testing barrier materials. Bacteriophages +6 and PRD1 most closely model human immunodeficiency virus type 1 in filtration size.