Polarized Dendritic Cells for Tumor Immunotherapy

An elusive objective for tumor immunologists has been the development of an effective tumor vaccine capable of inducing potent immune responses to eliminate established tumors and induce long-term protective antitumor immunity. Dendritic cells (DCs) are potent inducers of immunity and represent a promising tool for the purpose of immune-based tumor therapies. However, DC-based vaccines have enjoyed only limited success in clinical trials, probably due to the use of immature/intermediate mature DCs that maintain tolerance during the steady state, or to the use of non-polarized DCs which lack the proper cytokine production that favors cellular immune responses needed to eliminate established tumors. The failure of past tumor vaccines demonstrates a need to examine and enhance immunization strategies on multiple levels. The underlying hypothesis for these studies was that combining a DC1 polarization signal with an effective antigen-loading strategy will result in enhanced tumor immunotherapy. Our first aim was to compare cytosolic and membrane-bound antigen presentation of tumor-derived proteins by DCs following three different antigen-loading strategies; coculture of DCs and tumor cells, feeding DCs with tumor lysate, and fusion of DCs and tumor cells. We demonstrated that both DC-tumor coculture and fusion result in a higher level of tumor-derived peptide presentation compared to feeding DCs with tumor lysate. Our second aim was to develop a murine DC1 polarization model to evaluate DC1-based tumor immunotherapy. Herein, we described the synergistic affect of TLR3 and TLR9 ligation on IL-12p70 production by murine DCs, characterizing the timing and exhaustion of IL-12p70 production. Furthermore, we examined the ability of polarized DCs to stimulate T cell proliferation and cytokine secretion in response to a model antigen in vitro. For our third aim we examined the capacity of DC1s to stimulate immune responses to a model antigen as well as native tumor antigens in vivo and tested the therapeutic effect of tumor-loaded DC1 vaccines. These studies demonstrate the ability of antigen-loaded polarized DCs to induce strong Th1-mediated anti-tumor immunity characterized by tumor infiltrating CD4+ T cells and macrophages, but not CD8+ T cells, resulting in tumor growth inhibition

Gas Pulsation Control Using a Shunt Pulsation Trap

Gas pulsations commonly exist in HVACR, energy and automotive industry. They are believed to be a major source for system inefficiency, vibrations, noises and fatigue failures. It has been widely accepted that gas pulsations mainly take place at the discharge side of a positive displacement (PD) type compressor such as a screw, scroll or internal combustion engine. The pulsation magnitudes, ranging from a fraction to a few bars, are especially significant at off-design conditions of either under-compression (UC) or over-compression (OC). Traditionally, a serial pulsation dampener, often a reactive type silencer, is connected after the compressor or engine discharge. It is capable of reducing pressure pulsation by 10 plus fold; or 20 plus dB. However these dampeners are bulky and impose sizable back pressure losses. This paper discusses an alternative method, a shunt pulsation trap (SPT), using a parallel configuration, which tackles the gas pulsations before the compressor or engine discharge. The SPT method is based on the shock tube theory [1] that characterizes the nature of gas pulsation as a composition of non-linear waves, compression wave (CW) and expansion wave (EW) and induced fluid flow (IFF). The theory also predicts that the dominant source of gas pulsation is a direct result from the sudden discharging process under UC or OC condition. Two prototypes, 75 HP and 350 HP Roots type blowers, were built and tested with and without SPT. The experimental results partially validate the new theory and also indicate a 10 plus fold; or 20 plus dB pulsation reduction by using SPT under different load and speed conditions without suffering any back pressure losses

Gas Pulsation Control by a Shunt Pulsation Trap with Perforated Tubes and an Optional Absorptive Silencer

This paper is a continuing work from the same authors on the same topic of gas pulsation control using a shunt pulsation trap method by Huang (2012a, 2014). Traditionally, a serial pulsation dampener/muffler, often a reactive type, is connected AFTER the discharge of a positive displacement compressor or engine. It is capable of reducing pressure pulsation level up to 10-20 folds or 20-40 dB but is bulky and suffers sizable back pressure losses. It has been demonstrated in the previous theoretical and experimental investigations by Huang (2012a, 2014) that gas pulsations can be as effectively controlled by an alternative method, shunt pulsation trap (SPT) of a parallel configuration, which is more compact and tackles the gas pulsations BEFORE the compressor or engine discharge, hence not suffering any back pressure losses. However, it is observed that the dampening element inside SPT by a perforated plate (p-plate) induces a severe “drum effect†with enhanced vibration and noise. This paper investigates experimentally the effect of a new dampening element inside the SPT by using circular perforated tubes (p-tubes) with minimum trap volume. It is found that p-tubes can effectively resolve the adverse “drum effect†of p-plate with improved pulsation/vibration/noise reduction. Furthermore, the use of an absorption type silencer is beneficial to further reduce noise level by 10-15 dB in addition to the p-tubes under Exhauster and Blower modes. However, this type of silencer will not have the sizable back pressure drop associated with reaction type silencers and therefore will not affect system efficiency. The integration of absorptive silencer into a p-tube based SPT would provide an optimal design choice for size/weight and pulsation/noise reduction, energy saving and for improving equipment fatigue life. The potentials for applying the SPT to screw and scroll compressors are enormous in the future since a significant portion of their operation is under off-design conditions of either an under-compression (UC) or over-compression (OC)

Immune Modulation through 4-1BB Enhances SIV Vaccine Protection in Non-Human Primates against SIVmac251 Challenge

Costimulatory molecules play a central role in the development of cellular immunity. Understanding how costimulatory pathways can be directed to positively influence the immune response may be critical for the generation of an effective HIV vaccine. Here, we evaluated the ability of intravenous administration of a blocking monoclonal antibody (mAb) directed against the negative costimulatory molecule CTLA-4, and an agonist mAb directed against the positive costimulatory molecule 4-1BB, either alone or in combination, to augment intramuscular SIV DNA immunizations. We then tested the ability these of these responses to impact a high-dose SIVmac251 challenge. Following immunization, the groups infused with the anti-4-1BB mAb exhibited enhanced IFN-γ responses compared to the DNA vaccine only group. Interestingly, although CTLA-4 blockade alone did not enhance IFN-γ responses it did increase the proliferative capacity of the CD4+ and CD8+ T cells. The combination of both mAbs enhanced the magnitude of the polyfunctional CD8+ T cell response. Following challenge, the group that received both mAbs exhibited a significant, ∼2.0 log, decrease in plasma viral load compared to the naïve group the included complete suppression of viral load in some animals. Furthermore, the use of the CTLA-4 blocking antibody resulted in significantly higher viral loads during chronic infection compared to animals that received the 4-1BB mAb, likely due to the higher CD4+ T cell proliferative responses which were driven by this adjuvant following immunization. These novel studies show that these adjuvants induce differential modulation of immune responses, which have dramatically different consequences for control of SIV replication, suggesting important implications for HIV vaccine development

A 2-Dose AERAS-402 Regimen Boosts CD8+ Polyfunctionality in HIV-Negative, BCG-Vaccinated Recipients

Despite the widespread use of BCG, tuberculosis (TB) remains a global threat. Existing vaccine candidates in clinical trials are designed to replace or boost BCG which does not provide satisfying long-term protection. AERAS-402 is a replication-deficient Ad35 vaccine encoding a fusion protein of the M. tuberculosis (Mtb) antigens 85A, 85B, and TB10.4. The present phase I trial assessed the safety and immunogenicity of AERAS-402 in participants living in India – a highly TB-endemic area. Healthy male participants aged 18–45 years with a negative QuantiFERON-TB Gold in-tube test (QFT) were recruited. Enrolled participants (n=12) were randomized 2:1 to receive two intramuscular injections of either AERAS-402 (3 x 1010 viral particles [vp]); (n=8) or placebo (n=4) on study days 0 and 28. Safety and immunogenicity parameters were evaluated for up to 182 days post the second injection. Immunogenicity was assessed by a flow cytometry-based intracellular cytokine staining (ICS) assay and transcriptional profiling. The latter was examined using dual-color-Reverse-Transcriptase-Multiplex-Ligation-dependent-Probe-Amplification (dc-RT MLPA) assay. AERAS-402 was well tolerated, and no vaccine-related serious adverse events were recorded. The vaccine-induced CD8+ T-cell responses were dominated by cells co-expressing IFN-γ, TNF-α, and IL-2 (“polyfunctional” cells) and were more robust than CD4+ T-cell responses. Five genes (CXCL10, GNLY, IFI35, IL1B and PTPRCv2) were differentially expressed between the AERAS-402-group and the placebo group, suggesting vaccine-induced responses. Further, compared to pre-vaccination, three genes (CLEC7A, PTPRCv1 and TAGAP) were consistently up-regulated following two doses of vaccination in the AERAS-402-group. No safety concerns were observed for AERAS-402 in healthy Indian adult males. The vaccine-induced predominantly polyfunctional CD8+ T cells in response to Ag85B, humoral immunity, and altered gene expression profiles in peripheral blood mononuclear cells (PBMCs) indicative of activation of various immunologically relevant biological pathways.publishedVersio

The frequencies of IFNγ+IL2+TNFα+ PPD-specific CD4+CD45RO+ T-cells correlate with the magnitude of the QuantiFERON® gold in-tube response in a prospective study of healthy Indian adolescents

Background: QuantiFERON-TB Gold In-Tube (QFT) is an IFNγ-release assay used in the diagnosis of Mycobacterium tuberculosis (MTB) infection. The risk of TB progression increases with the magnitude of the MTB-specific IFNγ-response. QFT reversion, also associated with low Tuberculin Skin Test responses, may therefore represent a transient immune response with control of M. tuberculosis infection. However, studies at the single cell level have suggested that the quality (polyfunctionality) of the T-cell response is more important than the quantity of cytokines produced. Objective: To explore the quality and/or magnitude of mycobacteria-specific T-cell responses associated with QFT reversion and persistent QFT-positivity. Methods: Multi-color flowcytometry on prospectively collected peripheral blood mononuclear cells was applied to assess mycobacteria-specific T-cell responses in 42 QFT positive Indian adolescents of whom 21 became QFT negative (reverters) within one year. Ten QFT consistent negatives were also included as controls. Results: There was no difference in the qualitative PPD-specific CD4+ T-cell response between QFT consistent positives and reverters. However, compared with QFT consistent positives, reverters displayed lower absolute frequencies of polyfunctional (IFNγ+IL2+TNFα+) CD4+ T-cells at baseline, which were further reduced to the point where they were not different to QFT negative controls one year later. Moreover, absolute frequencies of these cells correlated well with the magnitude of the QFT-response. Conclusion: Whereas specific polyfunctional CD4+ T-cells have been suggested to protect against TB progression, our data do not support that higher relative or absolute frequencies of PPD-specific polyfunctional CD4+ T-cells in peripheral blood can explain the reduced risk of TB progression observed in QFT reverters. On the contrary, absolute frequencies of these cells correlated with the QFT-response, suggesting that this readout reflects antigenic load

The Cross-Species Mycobacterial Growth Inhibition Assay (MGIA) Project, 2010-2014.

The development of a functional biomarker assay in the tuberculosis (TB) field would be widely recognized as a major advance in efforts to develop and to test novel TB vaccine candidates efficiently. We present preliminary studies using mycobacterial growth inhibition assays (MGIAs) to detect Mycobacterium bovis BCG vaccine responses across species, and we extend this work to determine whether a standardized MGIA can be applied in characterizing new TB vaccines. The comparative MGIA studies reviewed here aimed to evaluate robustness, reproducibility, and ability to reflect in vivo responses. In doing so, they have laid the foundation for the development of a MGIA that can be standardized and potentially qualified. A major challenge ahead lies in better understanding the relationships between in vivo protection, in vitro growth inhibition, and the immune mechanisms involved. The final outcome would be a MGIA that could be used with confidence in TB vaccine trials. We summarize data arising from this project, present a strategy to meet the goals of developing a functional assay for TB vaccine testing, and describe some of the challenges encountered in performing and transferring such assays

CD4+ T cell cytokine responses to the DAR-901 booster vaccine in BCG-primed adults:A randomized, placebo-controlled trial

<div><p>Background</p><p>DAR-901 is an inactivated whole cell tuberculosis booster vaccine, prepared using a new scalable, broth-grown method from the master cell bank of SRL172, a vaccine previously shown to prevent tuberculosis. This study examined whether DAR-901 (a) induces CD4+ T cell cytokine profiles previously proposed as correlates of protection and (b) has a specific vaccine-induced immunological signature compared to BCG or placebo.</p><p>Methods</p><p>We analysed CD4+ T cell cytokine immune responses from 10 DAR-901 recipients, 9 BCG recipients and 9 placebo recipients from the Phase I DAR-901 MDES trial. In that study, HIV-negative, IGRA-negative participants with prior BCG immunization were randomized (double-blind) to receive three intradermal injections of DAR-901 or saline placebo or two injections of saline placebo followed by an intradermal injection of BCG. Antigen-specific functional and phenotypic CD4+ T cell responses along with effector phenotype of responder cells were measured by intracellular cytokine staining.</p><p>Results</p><p>DAR-901 recipients exhibited increased DAR-901 antigen-specific polyfunctional or bifunctional T cell responses compared to baseline. Vaccine specific CD4+ IFNγ, IL2, TNFα and any cytokine responses peaked at 7 days post-dose 3. Th1 responses predominated, with most responder cells exhibiting a polyfunctional effector memory phenotype. BCG induced greater CD4+ T cell responses than placebo while the more modest DAR-901 responses did not differ from placebo. Neither DAR-901 nor BCG induced substantial or sustained Th17 /Th22 cytokine responses.</p><p>Conclusion</p><p>DAR-901, a TB booster vaccine grown from the master cell bank of SRL 172 which was shown to prevent TB, induced low magnitude polyfunctional effector memory CD4+ T cell responses. DAR-901 responses were lower than those induced by BCG, a vaccine that has been shown ineffective as a booster to prevent tuberculosis disease. These results suggest that induction of higher levels of CD4+ cytokine stimulation may not be a critical or pre-requisite characteristic for candidate TB vaccine boosters.</p><p>Trial registration</p><p>ClinicalTrials.gov <a href="https://clinicaltrials.gov/ct2/show/NCT02063555" target="_blank">NCT02063555</a>.</p></div