Yeast Hsp7O genes constitute a multigene family in which at least five of the nine members are heat inducible. Hsp7O RNA levels also vary dramatically during stationary arrest and sporulatien. During growth to stationary phase, SSBI-SSB2 and SSCI RNAs decreased in abundance as cell density increased. In contrast, SSAI-SSA2 RNA levels increased before the diauxic shift and then decreased as cells approach stationary phase. SSA3 RNA was detected only after the diauxic shift and accumulated to high levels as cells entered stationary phase. This accumulation was reversed by addition of glucose. Studies with cyri mutants indicated that SSA3 RNA accumulation is stimulated by decreasing intracellular cyclic AMP concentrations. When cells were incubated in sporulation medium, most Hsp7O RNAs, with the exception of SSAI-SSA2 RNA, decreased in abundance. This finding contrasted with the SSAI-SSA2 pattern observed during growth to stationary phase. SSA3 RNA was not detected during growth in acetate-based medium but accumulated after several hours. SSA3 RNA accumulation was higher in sporulating cells than in nonsporulating cells and was reversed by addition of glucose.

SummaryProstatic acid phosphatase (PAP) is a tumor antigen in prostate cancer and the target of several anti-tumor vaccines in earlier clinical trials. Ultimately, the goal of anti-tumor vaccines is to elicit a sustainable immune response, able to eradicate a tumor, or at least restrain its growth. We have investigated plasmid DNA vaccines and have previously conducted a phase 1 trial in which patients with recurrent prostate cancer were vaccinated with a DNA vaccine encoding PAP. In this study, we investigated the immunologic efficacy of subsequent booster immunizations, and conducted more detailed longitudinal immune analysis, to answer several questions aimed at guiding optimal schedules of vaccine administration for future clinical trials. We report that antigen-specific cytolytic T-cell responses were amplified after immunization in 7 of 12 human leukocyte antigen-A2-expressing individuals, and that multiple immunizations seemed necessary to elicit PAP-specific interferon-γ-secreting immune responses detectable by enzyme-linked immunosorbent spot assay. Moreover, among individuals who experienced a ≥200% increase in prostate-specific antigen doubling time, long-term PAP-specific interferon-γ-secreting T-cell responses were detectable in 6 of 8, but in only 1 of 14 individuals without an observed change in prostate-specific antigen doubling time (P=0.001). Finally, we identified that immune responses elicited could be further amplified by subsequent booster immunizations. These results suggest that future trials using this DNA vaccine, and potentially other anti-tumor DNA vaccines, could investigate ongoing schedules of administration with periodic booster immunizations. Moreover, these results suggest that DNA vaccines targeting PAP could potentially be combined in heterologous immunization strategies with other vaccines to further augment PAP-specific T-cell immunity. Despite the significant effort in developing and evaluating anti-tumor vaccines, there is little consensus as to the "best" antigens to target and the optimal means of targeting these antigens. The prioritization of anti-tumor vaccine antigens, in fact, has been the focus of recent efforts led by the National Cancer Institute. 12 Most vaccine approaches targeting individual antigens have focused on eliciting CD8 + T cells, as these are part of the adaptive arm of the immune system with direct cytolytic activity. Consequently, to specifically elicit CD8 + T cells, most approaches have used antigen-loaded antigen-presenting cellular vaccines [13][14][15] or genetic vaccines using viral vaccines or naked DNA plasmids. [3][4][5]8,10 Of these particular approaches, DNA vaccines are generally believed to be a "weaker" immunization strategy given the absence of a concurrent inflammatory antiviral response, and the low level of in-vivo transfection of antigen-presenting cells that occurs after direct administration. However, we have been particularly interested in DNA vaccines as a simpler means of antigen-specific immunization given our experience i...

Regulatory T cells play important roles in cancer development and progression by limiting the generation of innate and adaptive anti-tumor immunity. We hypothesized that in addition to natural CD4+CD25+ Tregs and myeloid-derived suppressor cells, tumor antigen-specific regulatory T cells interfere with the detection of anti-tumor immunity following immunotherapy. Using samples from prostate cancer patients immunized with a DNA vaccine encoding prostatic acid phosphatase (PAP) and a trans-vivo delayed type hypersensitivity (tvDTH) assay, we found that the detection of PAP-specific effector responses following immunization was prevented by the activity of PAP-specific regulatory cells. These regulatory cells were CD8+CTLA-4+, and their suppression was relieved by blockade of CTLA-4, but not IL-10 or TGF-β. Moreover, antigen-specific CD8+ regulatory T cells were detected prior to immunization in the absence of PAP-specific effector responses. These PAP-specific CD8+CTLA-4+ suppressor T cells expressed IL-35, which was decreased following blockade of CTLA-4, and inhibition of either CTLA-4 or IL-35 reversed PAP-specific suppression of tvDTH response. PAP-specific CD8+CTLA-4+ T cells also suppressed T-cell proliferation in an IL-35-dependent, contact-independent fashion. Taken together, these findings suggest a novel population of CD8+CTLA-4+ IL-35-secreting tumor antigen-specific regulatory T cells arise spontaneously in some prostate cancer patients, persist during immunization, and can prevent the detection of antigen-specific effector responses by an IL-35-dependent mechanism.

BACKGROUND We have previously reported that a DNA vaccine encoding prostatic acid phosphatase (PAP) could elicit PAP-specific T cells in patients with early recurrent prostate cancer. In the current pilot trial we sought to evaluate whether prolonged immunization with regular booster immunizations, or “personalized” schedules of immunization determined using real-time immune monitoring, could elicit persistent, antigen-specific T cells, and whether treatment was associated with changes in PSA doubling time (PSA DT). METHODS 16 patients with castration-resistant, non-metastatic prostate cancer received six immunizations at two-week intervals, and then either quarterly (Arm 1) or as determined by multi-parameter immune monitoring (Arm 2). RESULTS Patients were on study a median of 16 months; four received 24 vaccinations. Only one event associated with treatment > grade 2 was observed. 6/16 (38%) remained metastasis-free at 2 years. PAP-specific T cells were elicited in 12/16 (75%), predominantly of a Th1 phenotype, which persisted in frequency and phenotype for at least one year. IFNγ-secreting T-cell responses measured by ELISPOT were detectable in 5/13 individuals at one year, and this was not statistically different between study arms. The overall median fold change in PSA DT from pre-treatment to post-treatment was 1.6 (range 0.6–7.0, p=0.036). CONCLUSIONS Repetitive immunization with a plasmid DNA vaccine was safe and elicited Th1-biased antigen-specific T cells that persisted over time. Modifications in the immunization schedule based on real-time immune monitoring did not increase the frequency of patients developing effector and memory T-cell responses with this DNA vaccine.

Retroviruses encode cis-acting RNA nuclear export elements that override nuclear retention of intron-containing viral mRNAs including the full-length, unspliced genomic RNAs (gRNAs) packaged into assembling virions. The HIV-1 Rev-response element (RRE) recruits the cellular nuclear export receptor CRM1 (also known as exportin-1/XPO1) using the viral protein Rev, while simple retroviruses encode constitutive transport elements (CTEs) that directly recruit components of the NXF1(Tap)/NXT1(p15) mRNA nuclear export machinery. How gRNA nuclear export is linked to trafficking machineries in the cytoplasm upstream of virus particle assembly is unknown. Here we used long-term (>24 h), multicolor live cell imaging to directly visualize HIV-1 gRNA nuclear export, translation, cytoplasmic trafficking, and virus particle production in single cells. We show that the HIV-1 RRE regulates unique, en masse, Rev- and CRM1-dependent “burst-like” transitions of mRNAs from the nucleus to flood the cytoplasm in a non-localized fashion. By contrast, the CTE derived from Mason-Pfizer monkey virus (M-PMV) links gRNAs to microtubules in the cytoplasm, driving them to cluster markedly to the centrosome that forms the pericentriolar core of the microtubule-organizing center (MTOC). Adding each export element to selected heterologous mRNAs was sufficient to confer each distinct export behavior, as was directing Rev/CRM1 or NXF1/NXT1 transport modules to mRNAs using a site-specific RNA tethering strategy. Moreover, multiple CTEs per transcript enhanced MTOC targeting, suggesting that a cooperative mechanism links NXF1/NXT1 to microtubules. Combined, these results reveal striking, unexpected features of retroviral gRNA nucleocytoplasmic transport and demonstrate roles for mRNA export elements that extend beyond nuclear pores to impact gRNA distribution in the cytoplasm.

HIV-1 replication requires myriad interactions between cellular proteins and the viral unspliced RNA. These interactions are important in archetypal RNA processes such as transcription and translation as well as for more specialized functions including alternative splicing and packaging of unspliced genomic RNA into virions. We present here a hybridization capture strategy for purification of unspliced full-length HIV RNA-protein complexes preserved in vivo by formaldehyde crosslinking, and coupled with mass spectrometry to identify HIV RNA-protein interactors in HIV-1 infected cells. One hundred eighty-nine proteins were identified to interact with unspliced HIV RNA including Rev and Gag/Gag-Pol, 24 host proteins previously shown to bind segments of HIV RNA, and over 90 proteins previously shown to impact HIV replication. Further analysis using siRNA knockdown techniques against several of these proteins revealed significant changes to HIV expression. These results demonstrate the utility of the approach for the discovery of host proteins involved in HIV replication. Additionally, because this strategy only requires availability of 30 nucleotides of the HIV-RNA for hybridization with a capture oligonucleotide, it is readily applicable to any HIV system of interest regardless of cell type, HIV-1 virus strain, or experimental perturbation.

Human APOBEC3H (A3H) is a single-stranded DNA cytosine deaminase that inhibits HIV-1. Seven haplotypes (I–VII) and four splice variants (SV154/182/183/200) with differing antiviral activities and geographic distributions have been described, but the genetic and mechanistic basis for variant expression and function remains unclear. Using a combined bioinformatic/experimental analysis, we find that SV200 expression is specific to haplotype II, which is primarily found in sub-Saharan Africa. The underlying genetic mechanism for differential mRNA splicing is an ancient intronic deletion [del(ctc)] within A3H haplotype II sequence. We show that SV200 is at least fourfold more HIV-1 restrictive than other A3H splice variants. To counteract this elevated antiviral activity, HIV-1 protease cleaves SV200 into a shorter, less restrictive isoform. Our analyses indicate that, in addition to Vif-mediated degradation, HIV-1 may use protease as a  counter-defense mechanism against A3H in >80% of sub-Saharan African populations.

Human Immunodeficiency Virus (HIV) type 1 uses a −1 programmed ribosomal frameshift (−1 PRF) event to translate its enzymes from the same transcript used to encode the virus’ structural proteins. The frequency of this event is highly regulated, and significant deviation from the normal 5–10% frequency has been demonstrated to decrease viral infectivity. Frameshifting is primarily regulated by the Frameshift Stimulatory Signal RNA (FSS-RNA), a thermodynamically stable, highly conserved stem loop that has been proposed as a therapeutic target. We describe the design, synthesis, and testing of a series of N-methyl peptides able to bind the HIV-1 FSS RNA stem loop with low nanomolar affinity and high selectivity. Surface plasmon resonance (SPR) data indicates increased affinity is a reflection of a substantially enhanced on rate. Compounds readily penetrate cell membranes and inhibit HIV infectivity in a pseudotyped virus assay. Viral infectivity inhibition correlates with compound-dependent changes in the ratios of Gag and Gag-Pol in virus particles. As the first compounds with both single digit nanomolar affinities for the FSS RNA and an ability to inhibit HIV in cells, these studies support the use of N-methylation for enhancing the affinity, selectivity, and bioactivity of RNA-binding peptides.