A Membrane-Embedded Amino Acid Couples the SpoIIQ Channel Protein to Anti-Sigma Factor Transcriptional Repression during Bacillus subtilis Sporulation.

UNLABELLED: SpoIIQ is an essential component of a channel connecting the developing forespore to the adjacent mother cell during Bacillus subtilis sporulation. This channel is generally required for late gene expression in the forespore, including that directed by the late-acting sigma factor σ(G) Here, we present evidence that SpoIIQ also participates in a previously unknown gene regulatory circuit that specifically represses expression of the gene encoding the anti-sigma factor CsfB, a potent inhibitor of σ(G) The csfB gene is ordinarily transcribed in the forespore only by the early-acting sigma factor σ(F) However, in a mutant lacking the highly conserved SpoIIQ transmembrane amino acid Tyr-28, csfB was also aberrantly transcribed later by σ(G), the very target of CsfB inhibition. This regulation of csfB by SpoIIQ Tyr-28 is specific, given that the expression of other σ(F)-dependent genes was unaffected. Moreover, we identified a conserved element within the csfB promoter region that is both necessary and sufficient for SpoIIQ Tyr-28-mediated inhibition. These results indicate that SpoIIQ is a bifunctional protein that not only generally promotes σ(G)activity in the forespore as a channel component but also specifically maximizes σ(G)activity as part of a gene regulatory circuit that represses σ(G)-dependent expression of its own inhibitor, CsfB. Finally, we demonstrate that SpoIIQ Tyr-28 is required for the proper localization and stability of the SpoIIE phosphatase, raising the possibility that these two multifunctional proteins cooperate to fine-tune developmental gene expression in the forespore at late times. IMPORTANCE: Cellular development is orchestrated by gene regulatory networks that activate or repress developmental genes at the right time and place. Late gene expression in the developing Bacillus subtilis spore is directed by the alternative sigma factor σ(G) The activity of σ(G)requires a channel apparatus through which the adjacent mother cell provides substrates that generally support gene expression. Here we report that the channel protein SpoIIQ also specifically maximizes σ(G)activity as part of a previously unknown regulatory circuit that prevents σ(G)from activating transcription of the gene encoding its own inhibitor, the anti-sigma factor CsfB. The discovery of this regulatory circuit significantly expands our understanding of the gene regulatory network controlling late gene expression in the developing B. subtilis spore.

Functional macroautophagy induction by influenza A virus without a contribution to major histocompatibility complex class II-restricted presentation.

Major histocompatibility complex (MHC) class II-presented peptides can be derived from both exogenous (extracellular) and endogenous (biosynthesized) sources of antigen. Although several endogenous antigen-processing pathways have been reported, little is known about their relative contributions to global CD4(+) T cell responses against complex antigens. Using influenza virus for this purpose, we assessed the role of macroautophagy, a process in which cytosolic proteins are delivered to the lysosome by de novo vesicle formation and membrane fusion. Influenza infection triggered productive macroautophagy, and autophagy-dependent presentation was readily observed with model antigens that naturally traffic to the autophagosome. Furthermore, treatments that enhance or inhibit macroautophagy modulated the level of presentation from these model antigens. However, validated enzyme-linked immunospot (ELISpot) assays of influenza-specific CD4(+) T cells from infected mice using a variety of antigen-presenting cells, including primary dendritic cells, revealed no detectable macroautophagy-dependent component. In contrast, the contribution of proteasome-dependent endogenous antigen processing to the global influenza CD4(+) response was readily appreciated. The contribution of macroautophagy to the MHC class II-restricted response may vary depending upon the pathogen.

Exosome-driven antigen transfer for MHC class II presentation facilitated by the receptor binding activity of influenza hemagglutinin.

The mechanisms underlying MHC class I-restricted cross-presentation, the transfer of Ag from an infected cell to a professional APC, have been studied in great detail. Much less is known about the equivalent process for MHC class II-restricted presentation. After infection or transfection of class II-negative donor cells, we observed minimal transfer of a proteasome-dependent "class I-like" epitope within the influenza neuraminidase glycoprotein but potent transfer of a classical, H-2M-dependent epitope within the hemagglutinin (HA) glycoprotein. Additional experiments determined transfer to be exosome-mediated and substantially enhanced by the receptor binding activity of incorporated HA. Furthermore, a carrier effect was observed in that incorporated HA improved exosome-mediated transfer of a second membrane protein. This route of Ag presentation should be relevant to other enveloped viruses, may skew CD4(+) responses toward exosome-incorporated glycoproteins, and points toward novel vaccine strategies.

Human papillomavirus (HPV) infection and vaccination: A cross-sectional study of college students' knowledge, awareness, and attitudes in Villanova, PA.

Human papillomaviruses are major causative agents of multiple cancers including cervical, vulvar, penile, anal, and oropharyngeal cancers. Almost all sexually active individuals are exposed to HPV in their lifetime and although not all HPV genotypes are capable of causing cancers, several high-risk subtypes widely circulate. Several HPV vaccines have been developed and successfully utilized to limit the spread of these viruses and reduce rates of associated cancers. Despite their success, HPV vaccination rates in the United States remain low. Studies estimate the highest prevalence of HPV in the United States is among college students. This makes college students an important target for interventions that promote HPV vaccination and prevention. To this end, we were interested in investigating the relationship between low HPV vaccine uptake and attitudes and awareness about HPV vaccination among college aged students. We designed a survey to assess knowledge and perception of HPV and HPV vaccination that could help identify correlations between this knowledge and vaccination status. Overall, the data suggest that factors beyond basic knowledge about HPV infections, such as vaccine safety and social acceptance of vaccination, may have important impacts on vaccination rates. More robust education in these areas, supplemented with education about the benefits of HPV vaccination could be utilized to improve vaccination rates.

A novel immunization approach for dengue infection based on conserved T cell epitopes formulated in calcium phosphate nanoparticles.

Dengue virus (DV) is the etiologic agent of dengue fever, the most significant mosquito-borne viral disease in humans. Most DV vaccine approaches are focused on generating antibody mediated responses; one such DV vaccine is approved for use in humans but its efficacy is limited. While it is clear that T cell responses play important role in DV infection and subsequent disease manifestations, fewer studies are aimed at developing vaccines that induce robust T cells responses. Potent T cell based vaccines require 2 critical components: the identification of specific T cell stimulating MHC associated peptides, and an optimized vaccine delivery vehicle capable of simultaneously delivering the antigens and any required adjuvants. We have previously identified and characterized DV specific HLA-A2 and -A24 binding DV serotypes conserved epitopes, and the feasibility of an epitope based vaccine for DV infection. In this study, we build on those previous studies and describe an investigational DV vaccine using T cell epitopes incorporated into a calcium phosphate nanoparticle (CaPNP) delivery system. This study presents a comprehensive analysis of functional immunogenicity of DV CaPNP/multipeptide formulations in vitro and in vivo and demonstrates the CaPNP/multipeptide vaccine is capable of inducing T cell responses against all 4 serotypes of DV. This synthetic vaccine is also cost effective, straightforward to manufacture, and stable at room temperature in a lyophilized form. This formulation may serve as an effective candidate DV vaccine that protects against all 4 serotypes as either a prophylactic or therapeutic vaccine.

MHC class I antigen presentation and implications for developing a new generation of therapeutic vaccines.

Major histocompatibility complex class I (MHC-I) presented peptide epitopes provide a 'window' into the changes occurring in a cell. Conventionally, these peptides are generated by proteolysis of endogenously synthesized proteins in the cytosol, loaded onto MHC-I molecules, and presented on the cell surface for surveillance by CD8(+) T cells. MHC-I restricted processing and presentation alerts the immune system to any infectious or tumorigenic processes unfolding intracellularly and provides potential targets for a cytotoxic T cell response. Therefore, therapeutic vaccines based on MHC-I presented peptide epitopes could, theoretically, induce CD8(+) T cell responses that have tangible clinical impacts on tumor eradication and patient survival. Three major methods have been used to identify MHC-I restricted epitopes for inclusion in peptide-based vaccines for cancer: genetic, motif prediction and, more recently, immunoproteomic analysis. Although the first two methods are capable of identifying T cell stimulatory epitopes, these have significant disadvantages and may not accurately represent epitopes presented by a tumor cell. In contrast, immunoproteomic methods can overcome these disadvantages and identify naturally processed and presented tumor associated epitopes that induce more clinically relevant tumor specific cytotoxic T cell responses. In this review, we discuss the importance of using the naturally presented MHC-I peptide repertoire in formulating peptide vaccines, the recent application of peptide-based vaccines in a variety of cancers, and highlight the pros and cons of the current state of peptide vaccines.

MHC Class I Presented T Cell Epitopes as Potential Antigens for Therapeutic Vaccine against HBV Chronic Infection.

Approximately 370 million people worldwide are chronically infected with hepatitis B virus (HBV). Despite the success of the prophylactic HBV vaccine, no therapeutic vaccine or other immunotherapy modality is available for treatment of chronically infected individuals. Clearance of HBV depends on robust, sustained CD8(+) T activity; however, the limited numbers of therapeutic vaccines tested have not induced such a response. Most of these vaccines have relied on peptide prediction algorithms to identify MHC-I epitopes or characterization of T cell responses during acute infection. Here, we took an immunoproteomic approach to characterize MHC-I restricted epitopes from cells chronically infected with HBV and therefore more likely to represent the true targets of CD8(+) T cells during chronic infection. In this study, we identified eight novel MHC-I restricted epitopes derived from a broad range of HBV proteins that were capable of activating CD8(+) T cells. Furthermore, five of the eight epitopes were able to bind HLA-A2 and A24 alleles and activated HBV specific T cell responses. These epitopes also have potential as new tools to characterize T cell immunity in chronic HBV infection and may serve as candidate antigens for a therapeutic vaccine against HBV infection.

Dengue virus specific dual HLA binding T cell epitopes induce CD8+ T cell responses in seropositive individuals.

Dengue virus infects an estimated 300 million people each year and even more are at risk of becoming infected as the virus continues to spread into new areas. Despite the increase in viral prevalence, no anti-viral medications or vaccines are approved for treating or preventing infection. CD8+ T cell responses play a major role in viral clearance. Therefore, effective vaccines that induce a broad, multi-functional T cell response with substantial cross-reactivity between all virus serotypes can have major impacts on reducing infection rates and infection related complications. Here, we took an immunoproteomic approach to identify novel MHC class I restricted T cell epitopes presented by dengue virus infected cells, representing the natural and authentic targets of the T cell response. Using this approach we identified 4 novel MHC-I restricted epitopes: 2 with the binding motif for HLA-A24 molecules and 2 with both HLA-A2 and HLA-A24 binding motifs. These peptides were able to activate CD8+ T cell responses in both healthy, seronegative individuals and in seropositive individuals who have previously been infected with dengue virus. Importantly, the dual binding epitopes activated pre-existing T cell precursors in PBMCs obtained from both HLA-A2+ and HLA-A24+ seropositive individuals. Together, the data indicate that these epitopes are immunologically relevant T cell activating peptides presented on infected cells during a natural infection and therefore may serve as candidate antigens for the development of effective multi-serotype specific dengue virus vaccines.

In vivo immunogenicity of Tax(11-19) epitope in HLA-A2/DTR transgenic mice: implication for dendritic cell-based anti-HTLV-1 vaccine.

Viral oncoprotein Tax plays key roles in transformation of human T-cell leukemia virus (HTLV-1)-infected T cells leading to adult T-cell leukemia (ATL), and is the key antigen recognized during HTLV-associated myelopathy (HAM). In HLA-A2+ asymptomatic carriers as well as ATL and HAM patients, Tax(11-19) epitope exhibits immunodominance. Here, we evaluate CD8 T-cell immune response against this epitope in the presence and absence of dendritic cells (DCs) given the recent encouraging observations made with Phase 1 DC-based vaccine trial for ATL. To facilitate these studies, we first generated an HLA-A2/DTR hybrid mouse strain carrying the HLA-A2.1 and CD11c-DTR genes. We then studied CD8 T-cell immune response against Tax(11-19) epitope delivered in the absence or presence of Freund's adjuvant and/or DCs. Overall results demonstrate that naturally presented Tax epitope could initiate an antigen-specific CD8T cell response in vivo but failed to do so upon DC depletion. Presence of adjuvant potentiated Tax(11-19)-specific response. Elevated serum IL-6 levels coincided with depletion of DCs whereas decreased TGF-ß was associated with adjuvant use. Thus, Tax(11-19) epitope is a potential candidate for the DC-based anti-HTLV-1 vaccine and the newly hybrid mouse strain could be used for investigating DC involvement in human class-I-restricted immune responses.

In vitro derivation of interferon-γ producing, IL-4 and IL-7 responsive memory-like CD4(+) T cells.

CD4(+) memory is critical for successful protection against pathogenic challenge. As such, understanding the heterogeneity of cells that arise and survive after initial stimulation of naïve CD4(+) T cells will aid in the design of more successful vaccines. In previous studies, in vivo experimental systems have been extensively used to generate functional memory responses by lymphocytes. Here, we have attempted to develop an in vitro experimental system to generate memory CD4(+) T lymphocytes. CD4(+) T cells stimulated through the antigen receptor complex were examined for their memory-like characteristics after 3 weeks of cell culture. A subset of surviving cells expressed high levels of CD44 and low levels of CD45RB (CD44(hi)CD45(lo)), a phenotype that is similar to bonafide memory CD4(+) T cells. In vitro generated memory-like CD4(+) T cells secreted higher levels of IFN-γ, with rapid kinetics, upon re-stimulation than their naïve counterparts. In addition, these memory-like CD4(+) T cells did not produce either IL-2 or IL-4 but readily proliferated when cultured in the presence of IL-7 and IL-4. These observations suggest that CD4(+) cells surviving the expansion phase of immune response produce a Th1-signature cytokine and retain responsiveness to IL-4, a Th-2 cytokine, as well as to a well described survival factor, interleukin-7.