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We have previously reported two single-agent phase I trials, evaluating the dose or schedule, of a DNA vaccine (pTVG-HP) encoding prostatic acid phosphatase (PAP) administered with GM-CSF as the adjuvant. These were in patients with PSA-recurrent, radiographically non-metastatic, prostate cancer (PCa). We report here the long-term safety and overall survival of these patients. Specifically, 22 patients with non-metastatic, castration-sensitive PCa (nmCSPC) were treated with pTVG-HP, 100–1500 µg, administered over 12 weeks and followed for 15 y. 17 patients with non-metastatic castration-resistant PCa (nmCRPC) were treated with 100 µg pTVG-HP with different schedules of administration over 1 y and followed for 5 y. No adverse events were detected in long-term follow-up from either trial that were deemed possibly related to vaccination. Patients with nmCSPC had a median overall survival of 12.3 y, with 5/22 (23%) alive at 15 y. 8/22 (36%) died due to prostate cancer with a median survival of 11.0 y, and 9/22 (41%) died of other causes. Patients with nmCRPC had a median overall survival of 4.5 y, with 8/17 (47%) alive at 5 y. The presence of T-cells specific for the PAP target antigen was detectable in 6/10 (60%) individuals with nmCSPC, and 3/5 (60%) individuals with nmCRPC, many years after immunization. The detection of immune responses to the vaccine target years after immunization suggests durable immunity can be elicited in patients using a DNA vaccine encoding a tumor-associated antigen.Trial Registration: NCT00582140 and NCT00849121

Tissue-specific antigens can serve as targets for adoptive T cell transfer-based cancer immunotherapy. Recognition of tumor by T cells is mediated by interaction between peptide-major histocompatibility complexes (pMHCs) and T cell receptors (TCRs). Revealing the identity of peptides bound to MHC is critical in discovering cognate TCRs and predicting potential toxicity. We performed multimodal immunopeptidomic analyses for human prostatic acid phosphatase (PAP), a well-recognized tissue antigen. Three physical methods, including mild acid elution, coimmunoprecipitation, and secreted MHC precipitation, were used to capture a thorough signature of PAP on HLA-A*02:01. Eleven PAP peptides that are potentially A*02:01-restricted were identified, including five predicted strong binders by NetMHCpan 4.0. Peripheral blood mononuclear cells (PBMCs) from more than 20 healthy donors were screened with the PAP peptides. Seven cognate TCRs were isolated which can recognize three distinct epitopes when expressed in PBMCs. One TCR shows reactivity toward cell lines expressing both full-length PAP and HLA-A*02:01. Our results show that a combined multimodal immunopeptidomic approach is productive in revealing target peptides and defining the cloned TCR sequences reactive with prostatic acid phosphatase epitopes.

Semen prostatic acid phosphatase (PAP) has been proposed as an endogenous ligand for dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN), which plays a critical immuno-modulating role in maintaining homeostasis in the female reproductive tracts. In the current study, we assumed that semen PAP bears a set of fucosylated and mannosylated glycans, which may mediate the efficient binding of PAP to DC-SIGN. To investigate this hypothesis, we developed ELISA assays using Galanthus nivalis and Lotus tetragonolobus lectins capable of binding mannose-containing glycans or LewisX and LewisY motifs, respectively. In our assay with Galanthus nivalis, we detected that the relative reactivity of PAP mannose-presenting glycans in the normozoospermic idiopathic group was significantly higher than in the asthenozoospermic, oligozoospermic and oligoasthenozoospermic groups. Simultaneously, we observed slight differences in the relative reactivities of PAP glycans with Lotus tetragonolobus lectin among groups of patients with abnormal semen parameters. Subsequently, we examined whether DC-SIGN interacts with seminal plasma PAP glycans, and we detected a significantly higher relative reactivity in the normozoospermic group compared to the oligozoospermic group. Finally, we concluded that the significantly aberrant abundance of mannosylated functional groups of PAP among patients with semen disorders can suggest that PAP may thereby be engaged in modulating the immune response and promoting a tolerogenic response to male antigens in the female reproductive system. [ABSTRACT FROM AUTHOR]

Key message: PAP-FcK and PSA-FcK prostate cancer antigenic proteins transiently co-expressed in plant induce their specific humoral immune responses in mice. Prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) have been considered as immunotherapeutic antigens for prostate cancer. The use of a single antigenic agent is unlikely to be effective in eliciting immunotherapeutic responses due to the heterogeneous and multifocal nature of prostate cancer. Thus, multiple antigens have been combined to enhance their anti-cancer effects. In the current study, PSA and PAP were fused to the crystallizable region (Fc region) of immunoglobulin G1 and tagged with KDEL, the endoplasmic reticulum (ER) retention signal motif, to generate PSA-FcK and PAP-FcK, respectively, and were transiently co-expressed in Nicotiana benthamiana. Western blot analysis confirmed the co-expression of PSA-FcK and PAP-FcK (PSA-FcK + PAP-FcK) with a 1:3 ratios in the co-infiltrated plants. PSA-FcK, PAP-FcK, and PSA-FcK + PAP-FcK proteins were successfully purified from N. benthamiana by protein A affinity chromatography. ELISA showed that anti-PAP and anti-PSA antibodies successfully detected PAP-FcK and PSA-FcK, respectively, and both detected PSA-FcK + PAP-FcK. Surface plasmon resonance (SPR) analysis confirmed the binding affinity of the plant-derived Fc fusion proteins to FcγRI/CD64. Furthermore, we also confirmed that mice injected with PSA-FcK + PAP-FcK produced both PSA- and PAP-specific IgGs, demonstrating their immunogenicity. This study suggested that the transient plant expression system can be applied to produce the dual-antigen Fc fusion protein (PSA-FcK + PAP-FcK) for prostate cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Semen prostatic acid phosphatase (PAP) has been proposed as an endogenous ligand for dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN), which plays a critical immuno-modulating role in maintaining homeostasis in the female reproductive tracts. In the current study, we assumed that semen PAP bears a set of fucosylated and mannosylated glycans, which may mediate the efficient binding of PAP to DC-SIGN. To investigate this hypothesis, we developed ELISA assays using Galanthus nivalis and Lotus tetragonolobus lectins capable of binding mannose-containing glycans or LewisX and LewisY motifs, respectively. In our assay with Galanthus nivalis, we detected that the relative reactivity of PAP mannose-presenting glycans in the normozoospermic idiopathic group was significantly higher than in the asthenozoospermic, oligozoospermic and oligoasthenozoospermic groups. Simultaneously, we observed slight differences in the relative reactivities of PAP glycans with Lotus tetragonolobus lectin among groups of patients with abnormal semen parameters. Subsequently, we examined whether DC-SIGN interacts with seminal plasma PAP glycans, and we detected a significantly higher relative reactivity in the normozoospermic group compared to the oligozoospermic group. Finally, we concluded that the significantly aberrant abundance of mannosylated functional groups of PAP among patients with semen disorders can suggest that PAP may thereby be engaged in modulating the immune response and promoting a tolerogenic response to male antigens in the female reproductive system.

Amyloid fibrils, dubbed SEVI (semen-derived enhancer of virus infection), contribute to the spread of HIV infection. The main components of SEVI are the fragments of prostatic acid phosphatase (PAP): PAP248-286 and PAP85-120. SEVI captures the viral particles and further stimulates their attachment to the target cells, thereby boosting viral fusion and infection. To study the oligomers of SEVI-forming peptides, we used molecular modeling, which is a powerful tool that has been applied in a great variety of studies on SEVI, and an advanced accelerated sampling method of metadynamics. Based on the obtained molecular dynamics data, it was shown that PAP248-286 has a horseshoe shape with bends in the regions of amino acid residues A274 and N269 in the dimeric state. It was suggested that the horseshoe shape might lead in the fibrillation process to the steric zipper model formation, which is typical of amyloids. It was confirmed that the process of fibril formation of PAP248-286 starts with a pairwise parallel arrangement of the peptide helical regions.

Purpose: To investigate the effect of virgin coconut oil, lauric acid and myristic acid on serum and prostatic androgens in testosterone-induced prostatic hyperplasia. Methods: Benign prostatic hyperplasia (BPH) was induced in the animals by repeated subcutaneous injection of testosterone propionate (5 mg/kg) at the inguinal region once a day for 28 days. Thereafter, BPH was treated for 56 days by oral administration of virgin coconut oil, lauric acid and myristic acid. Following the treatment period, the rats were sacrificed and blood samples were collected through cardiac puncture for biochemical analysis. Results: Virgin coconut oil, lauric acid and myristic acid led to a significant reduction (p < 0.05) in serum prostatic acid phosphatase (PAcP), prostate specific antigen (PSA) and dihydrotestosterone (DHT) levels, and also in prostatic DHT level. Conclusion: This study provides evidence that virgin coconut oil, lauric acid and myristic acid may be useful in the management of BPH because they exerted some anti-proliferative effects in the development and progression of BPH. Therefore, coconut may be a potential functional food for the management of BPH patients because it is rich in both lauric and myristic acid. However, further investigations, including clinical trials are required to buttress this. [ABSTRACT FROM AUTHOR]

This study included the isolation and study of prostatic acid phosphatese (PAP) from the serum of prostate cancer patients, fifty blood samples were collected from patients attending the Oncology and Atomic Medicine Hospital in Mosul city of Mosul for the period from October to March of 2019, their ages ranged from 50 years and over, and 50 blood samples were collected from apparently healthy persons of equal ages as a control group. The results showed that there was a significant increase in the activity of prostatic acid phosphatese in the serum of the patients with prostate cancer since the enzyme's activity rate reached in the patients' blood serum was 31.41±2.25IU/L, compared to a control group 5.55±0.24IU/L and at a probability level p d" 0.05, considered a predictor of prostate cancer. The PAP was isolated using different biotechnologies. Two protein bands were isolated using gel filtration technology to the protein precipitate resulting from the precipitation process with ammonium sulfate. Peak A showed high activity of the PAP, and the molecular weight was estimated using gel filtration and it was within the limits of 98400±103 Daltons. The study determined to find the optimal condition for the effectivenss of PAP and the maximum activity was as follows using 60 microgram/ml of partially purified enzyme and pH 4.6, with areaction time 15 minutes and at a temperature of 60°C at a concentration of 50 micromole of the parent substance paranitro phenyl phosphate (PNPP) as substrate. The maximum activity value (Vmax) and Michaelis-Menton constant Km were determined using Lineweaved-Burk plot, which was equal to 47.6 IU/L and 29.9 mmole, respectively. The impact of some chemical compounds on the effectiveness of PAP was investigated and a conclusion was made that tartaric acid operates as a competitive inhibitor at the 2 mmole concentration. [ABSTRACT FROM AUTHOR]

We have previously reported two single-agent phase I trials, evaluating the dose or schedule, of a DNA vaccine (pTVG-HP) encoding prostatic acid phosphatase (PAP) administered with GM-CSF as the adjuvant. These were in patients with PSA-recurrent, radiographically non-metastatic, prostate cancer (PCa). We report here the long-term safety and overall survival of these patients. Specifically, 22 patients with non-metastatic, castration-sensitive PCa (nmCSPC) were treated with pTVG-HP, 100-1500 µg, administered over 12 weeks and followed for 15 y. 17 patients with non-metastatic castration-resistant PCa (nmCRPC) were treated with 100 µg pTVG-HP with different schedules of administration over 1 y and followed for 5 y. No adverse events were detected in long-term follow-up from either trial that were deemed possibly related to vaccination. Patients with nmCSPC had a median overall survival of 12.3 y, with 5/22 (23%) alive at 15 y. 8/22 (36%) died due to prostate cancer with a median survival of 11.0 y, and 9/22 (41%) died of other causes. Patients with nmCRPC had a median overall survival of 4.5 y, with 8/17 (47%) alive at 5 y. The presence of T-cells specific for the PAP target antigen was detectable in 6/10 (60%) individuals with nmCSPC, and 3/5 (60%) individuals with nmCRPC, many years after immunization. The detection of immune responses to the vaccine target years after immunization suggests durable immunity can be elicited in patients using a DNA vaccine encoding a tumor-associated antigen. Trial Registration : NCT00582140 and NCT00849121.

Purpose of Review: Despite significant progress, patients with metastatic prostate cancer continue to have poor prognosis. Immunotherapy has revolutionized cancer care for many tumor types but has a limited role in the treatment of prostate cancer. This review discusses the promise of immunotherapy in prostate cancer treatment with an emphasis on emerging therapeutic targets. Recent Findings: Most prostate tumors have low tumor mutational burden and lack immunogenicity, representing significant hurdles to induction of anti-tumor immunity. However, recent research centered on deciphering key mechanisms of immune resistance in the prostate tumor microenvironment has led to the discovery of a range of new treatment targets. These discoveries are currently being translated into innovative immunotherapy clinical trials for patients with prostate cancer. Recent progress includes early evidence of activity for these novel approaches and the identification of potential predictive biomarkers of response. Summary: Novel treatment strategies using new antigen-directed therapies, drugs targeting the immunosuppressive tumor microenvironment, and combination immunotherapy therapies show great potential and are currently in clinical development. In addition, a deeper understanding of predictors of response and resistance to immunotherapy in prostate cancer is allowing for a more personalized approach to therapy. [ABSTRACT FROM AUTHOR]

Abstract Background Prostatic acid phosphatase (PAP) is a prostate tumor antigen, and the target of the only FDA-approved anti-tumor vaccine, sipuleucel-T. We have previously reported in two clinical trials that a DNA vaccine encoding PAP (pTVG-HP) could elicit PAP-specific, Th1-biased T cells in patients with PSA-recurrent prostate cancer. In the current pilot trial we sought to evaluate whether this vaccine could augment PAP-specific immunity when used as a booster to immunization with sipuleucel-T in patients with metastatic, castration-resistant prostate cancer (mCRPC). Methods Eigthteen patients with mCRPC were randomized to receive sipuleucel-T alone or followed by intradermal immunization with pTVG-HP DNA vaccine. Patients were followed for time to progression, and immune monitoring was conducted at defined intervals. Results Overall, patients were followed for a median of 24 months. 11/18 patients completed treatments as per protocol. No treatment-associated events > grade 2 were observed. Th1-biased PAP-specific T-cell responses were detected in 11/18 individuals, and were not statistically different between study arms. Higher titer antibody responses to PAP were detectable in patients who received pTVG-HP booster immunizations. Median time to progression was less than 6 months and not statistically different between study arms. The median overall survival for all patients was 28 months. Conclusions These findings suggest that prime-boost vaccination can augment and diversify the type of immunity elicited with anti-tumor vaccination in terms of T-cell and humoral immunity. Future studies will explore DNA as priming immunization rather than a booster immunization. Trial registration NCT01706458.

The current study included estimation of Acid Phosphatase (AP) and Prostatic Acid Phosphatase (PAP) activity in the sera of Breast Cancer patient̓s. The study included (58) samples as satisfactory sample of women with breast cancer who are treated at (Breast cancer Center) in Azadi teaching hospital in Kirkuk, age ranged from (17-80) years old, and (58) samples from healthy people (control group) aged between (20-80) years old. The results showed significal elevation in the AP and PAP activity in breast cancer patients compared with control group. From the results above concluded that it is possible to use the enzyme prostatic acid phosphatase PAP as an indicator of breast cancer .

Abstract Background Immunotherapies have demonstrated clinical benefit for many types of cancers, however many patients do not respond, and treatment-related adverse effects can be severe. Hence many efforts are underway to identify treatment predictive biomarkers. We have reported the results of two phase I trials using a DNA vaccine encoding prostatic acid phosphatase (PAP) in patients with biochemically recurrent prostate cancer. In both trials, persistent PAP-specific Th1 immunity developed in some patients, and this was associated with favorable changes in serum PSA kinetics. In the current study, we sought to determine if measures of antigen-specific or antigen non-specific immunity were present prior to treatment, and associated with subsequent immune response, to identify possible predictive immune biomarkers. Methods Patients who developed persistent PAP-specific, IFNγ-secreting immune responses were defined as immune “responders.” The frequency of peripheral T cell and B cell lymphocytes, natural killer cells, monocytes, dendritic cells, myeloid derived suppressor cells, and regulatory T cells were assessed by flow cytometry and clinical laboratory values. PAP-specific immune responses were evaluated by cytokine secretion in vitro, and by antigen-specific suppression of delayed-type hypersensitivity to a recall antigen in an in vivo SCID mouse model. Results The frequency of peripheral blood cell types did not differ between the immune responder and non-responder groups. Non-responder patients tended to have higher PAP-specific IL-10 production pre-vaccination (p = 0.09). Responder patients had greater preexisting PAP-specific bystander regulatory responses that suppressed DTH to a recall antigen (p = 0.016). Conclusions While our study population was small (n = 38), these results suggest that different measures of antigen-specific tolerance or regulation might help predict immunological outcome from DNA vaccination. These will be prospectively evaluated in an ongoing randomized, phase II trial.

Specialized microdomains which have cholesterol-rich membrane regions contain transient receptor potential vanilloid subtype 1 (TRPV1) are involved in pain development. Our previous studies have demonstrated that the depletion of prostatic acid phosphatase (PAP) – a membrane-bound ectonucleotidase ­– and disordered adenosine signaling reduce the antinociceptive effect. The role of membrane integrity in the PAP-mediated antinociceptive effect in small-fiber neuropathy remains unclear, especially with respect to whether TRPV1 and PAP are colocalized in the same microdomain which is responsible for PAP-mediated antinociception. Immunohistochemistry was conducted on the dorsal root ganglion to identify the membrane compositions, and pharmacological interventions were conducted using methyl-β-cyclodextrin (MβC) – a membrane integrity disruptor that works by depleting cholesterol – in pure small-fiber neuropathy with resiniferatoxin (RTX). Immunohistochemical evidence indicated that TRPV1 and PAP were highly colocalized with flotillin 1 (66.7%±9.7%) and flotillin 2 (73.7%±6.0%), which reside in part in the microdomain. MβC mildly depleted PAP, which maintained the ability to hydrolyze phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and delayed the development of mechanical allodynia. MβC treatment had no role in thermal transduction and neuronal injury following RTX neuropathy. In summary, this study demonstrated the following: (1) membrane cholesterol depletion preserves PAP-mediated antinociception through PI(4,5)P2 hydrolysis and (2) pain hypersensitivity that develops after TRPV1(+) neuron depletion-mediated neurodegeneration following RTX neuropathy is attributable to the downregulation of PAP analgesic signaling.

Semen prostatic acid phosphatase (PAP) has been proposed as an endogenous ligand for dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN), which plays a critical immuno-modulating role in maintaining homeostasis in the female reproductive tracts. In the current study, we assumed that semen PAP bears a set of fucosylated and mannosylated glycans, which may mediate the efficient binding of PAP to DC-SIGN. To investigate this hypothesis, we developed ELISA assays using Galanthus nivalis and Lotus tetragonolobus lectins capable of binding mannose-containing glycans or LewisX and LewisY motifs, respectively. In our assay with Galanthus nivalis, we detected that the relative reactivity of PAP mannose-presenting glycans in the normozoospermic idiopathic group was significantly higher than in the asthenozoospermic, oligozoospermic and oligoasthenozoospermic groups. Simultaneously, we observed slight differences in the relative reactivities of PAP glycans with Lotus tetragonolobus lectin among groups of patients with abnormal semen parameters. Subsequently, we examined whether DC-SIGN interacts with seminal plasma PAP glycans, and we detected a significantly higher relative reactivity in the normozoospermic group compared to the oligozoospermic group. Finally, we concluded that the significantly aberrant abundance of mannosylated functional groups of PAP among patients with semen disorders can suggest that PAP may thereby be engaged in modulating the immune response and promoting a tolerogenic response to male antigens in the female reproductive system.

Glioblastoma (GBM) is the brain tumor with the worst prognosis composed of a cell subpopulation called Glioblastoma Stem-like Cells (GSCs) responsible for tumor recurrence mediated by cell invasion. GSCs persist in a hypoxic microenvironment which promotes extracellular adenosine production and activation of the A3 Adenosine Receptor (A3AR), therefore, the aim of this study was to determine the role of extracellular adenosine and A3AR on GSCs invasion under hypoxia. GSCs were obtained from a U87MG cell line and primary cultures of GBM patients, and then incubated under normoxia or hypoxia. Gene expression was evaluated by RNAseq, RT-qPCR, and western blot. Cell migration was measured by spreading and transwell boyden chamber assays; cell invasion was evaluated by Matrigel-coated transwell, ex vivo brain slice, and in vivo xenograft assays. The contribution of A3AR on cell migration/invasion was evaluated using the A3AR antagonist, MRS1220. Extracellular adenosine production was higher under hypoxia than normoxia, mainly by the catalytic action of the prostatic acid phosphatase (PAP), promoting cell migration/invasion in a HIF-2-dependent process. A3AR blockade decreased cell migration/invasion and the expression of Epithelial-Mesenchymal Transition markers. In conclusion, high levels of extracellular adenosine production enhance cell migration/invasion of GSCs, through HIF-2/PAP-dependent activation of A3AR under hypoxia. [ABSTRACT FROM AUTHOR]

Background. Sipuleucel T, an autologous cell-based vaccine targeting prostatic acid phosphatase (PAP), has demonstrated efficacy for the treatment of advanced prostate cancer. DNA vaccines encoding PAP and live attenuated Listeria vaccines have entered clinical trials for patients with prostate cancer, and have advantages in terms of eliciting predominantly Th1-biased immunity. In this study, we investigated whether the immunogenicity and anti-tumor efficacy of a DNA and Listeria vaccine, each encoding PAP, could be enhanced by using them in a heterologous prime/boost approach. Methods. Transgenic mice expressing HLA-A2.01 and HLA-DRB1*0101 were immunized alone or with a heterologous prime/boost strategy. Splenocytes were evaluated for MHC class I and II-restricted, PAP-specific immune responses by IFNγ ELISPOTs. Anti-tumor activity to a syngeneic, PAP-expressing tumor line was evaluated. Results. PAP-specific cellular immunity and anti-tumor activity were elicited in mice after immunization with DNA- or listeria-based vaccines. Greater CD4+ and CD8+ responses, and anti-tumor responses, were elicited when mice were immunized first with DNA and boosted with Listeria, but not when administered in the opposite order. This was found to be dependent on CD4+ T cells elicited with DNA priming, and was not due to inflammatory signals by Listeria itself or due to B cells serving as antigen-presenting cells for DNA during priming. Conclusions. Heterologous prime/boost vaccination using DNA priming with Listeria boosting may provide better anti-tumor immunity, similar to many reports evaluating DNA priming with vaccines targeting foreign microbial antigens. These findings have implications for the design of future clinical trials.

Background: Klebsiella pneumoniae is an opportunistic pathogen and many strains are multidrug resistant. KPC is one of the most problematic resistance mechanisms, as it confers resistance to most β-lactams, including carbapenems. A promising platform technology for treating infections caused by MDR pathogens is the nucleic acid-like synthetic oligomers that silence bacterial gene expression by an antisense mechanism. Objectives: To test a peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) in a mouse model of K. pneumoniae infection. Methods: PPMOs were designed to target various essential genes of K. pneumoniae and screened in vitro against a panel of diverse strains. The most potent PPMOs were further tested for their bactericidal effects in broth cultures and in established biofilms. Finally, a PPMO was used to treat mice infected with a KPC-expressing strain. Results: The most potent PPMOs targeted acpP, rpmB and ftsZ and had MIC75s of 0.5, 4 and 4 μM, respectively. AcpP PPMOs were bactericidal at 1-2 x MIC and reduced viable cells and biofilm mass in established biofilms. In a mouse pneumonia model, therapeutic intranasal treatment with -30 mg/kg AcpP PPMO improved survival by 89% and reduced bacterial burden in the lung by -3 logs. Survival was proportional to the dose of AcpP PPMO. Delaying treatment by 2, 8 or 24 h post-infection improved survival compared with control groups treated with PBS or scrambled sequence (Scr) PPMOs. Conclusions: PPMOs have the potential to be effective therapeutic agents against KPC-expressing, MDR K. pneumoniae. [ABSTRACT FROM AUTHOR]

Background: Immunotherapies have demonstrated clinical benefit for many types of cancers, however many patients do not respond, and treatment-related adverse effects can be severe. Hence many efforts are underway to identify treatment predictive biomarkers. We have reported the results of two phase I trials using a DNA vaccine encoding prostatic acid phosphatase (PAP) in patients with biochemically recurrent prostate cancer. In both trials, persistent PAP-specific Th1 immunity developed in some patients, and this was associated with favorable changes in serum PSA kinetics. In the current study, we sought to determine if measures of antigen-specific or antigen non-specific immunity were present prior to treatment, and associated with subsequent immune response, to identify possible predictive immune biomarkers. Methods: Patients who developed persistent PAP-specific, IFNγ-secreting immune responses were defined as immune “responders.” The frequency of peripheral T cell and B cell lymphocytes, natural killer cells, monocytes, dendritic cells, myeloid derived suppressor cells, and regulatory T cells were assessed by flow cytometry and clinical laboratory values. PAP-specific immune responses were evaluated by cytokine secretion in vitro, and by antigen-specific suppression of delayed-type hypersensitivity to a recall antigen in an in vivo SCID mouse model. Results: The frequency of peripheral blood cell types did not differ between the immune responder and non-responder groups. Non-responder patients tended to have higher PAP-specific IL-10 production pre-vaccination (p = 0.09). Responder patients had greater preexisting PAP-specific bystander regulatory responses that suppressed DTH to a recall antigen (p = 0.016). Conclusions: While our study population was small (n = 38), these results suggest that different measures of antigen-specific tolerance or regulation might help predict immunological outcome from DNA vaccination. These will be prospectively evaluated in an ongoing randomized, phase II trial. [ABSTRACT FROM AUTHOR]

In this study, prostatic acid phosphatase (PAP), which is overexpressed in human prostate cancer cells, was cloned to be fused to the IgM constant fragment (Fc) for enhancing immunogenicity and expressed in transgenic tobacco plants. Then, the transgenic plants were propagated by in vitro tissue subculture. Gene insertion and expression of the recombinant PAP-IgM Fc fusion protein were confirmed in each tested the first, second, and third subculture generations (SG1, SG2, and SG3, respectively). Transcription levels were constantly maintained in the SG1; SG2, and SG3 leaf section (top, middle, and base ). The presence of the PAP-IgM Fc gene was also confirmed in each leaf section in all tested subculture generations. RNA expression was confirmed in all subculture generations using real-time PCR and quantitative real-time PCR. PAP-IgM Fc protein expression was confirmed in all leaves of the SG1, SG2, and SG3 recombinant transgenic plants by using quantitative western blotting and chemiluminescence immunoassays. These results demonstrate that the recombinant protein was stably expressed for several generations of in vitro subculture. Therefore, transgenic plants can be propagated using in vitro tissue subculture for the production of recombinant proteins. [ABSTRACT FROM AUTHOR]