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Oncolytic virus (OV) is a replication competent virus that selectively invades cancer cells; as these cells die under the viral burden, the released virus particles proceed to infect other cancer cells. Oncolytic viruses are designed to also be able to stimulate the anticancer immune response. Thus, one may represent an OV by two parameters: its replication potential and its immunogenicity. In this paper we consider a combination therapy with OV and a checkpoint inhibitor, anti-PD-1. We evaluate the efficacy of the combination therapy in terms of the tumor volume at some later time, for example, 6 months from initial treatment. Since T cells kill not only virus-free cancer cells but also virus-infected cancer cells, the following question arises: Does increasing the amount of the checkpoint inhibitor always improve the efficacy? We address this question, by a mathematical model consisting of a system of partial differential equations. We use the model to construct, by simulations, an efficacy map in terms of the doses of the checkpoint inhibitor and the OV injection. We show that there are regions in the map where an increase in the checkpoint inhibitor actually decreases the efficacy of the treatment. We also construct efficacy maps with checkpoint inhibitor vs. the replication potential of the virus that show the same antagonism, namely, an increase in the checkpoint inhibitor may actually decrease the efficacy. These results have implications for clinical trials. [ABSTRACT FROM AUTHOR]

Experimental and mathematical studies in immunology have revealed that the dynamics of the programmed T cell response to vigorous infection can be conveniently modelled using a sigmoidal or a discontinuous immune response function. This paper hypothesizes strong synergies between this existing work and the dynamical behaviour of engineering systems with a variable structure control (VSC) law. These findings motivate the interpretation of the immune system as a variable structure control system. It is shown that dynamical properties as well as conditions to analytically assess the transition from health to disease can be developed for the specific T cell response from the theory of variable structure control. In particular, it is shown that the robustness properties of the specific T cell response as observed in experiments can be explained analytically using a VSC perspective. Further, the predictive capacity of the VSC framework to determine the T cell help required to overcome chronic Lymphocytic Choriomeningitis Virus (LCMV) infection is demonstrated. The findings demonstrate that studying the immune system using variable structure control theory provides a new framework for evaluating immunological dynamics and experimental observations. A modelling and simulation tool results with predictive capacity to determine how to modify the immune response to achieve healthy outcomes which may have application in drug development and vaccine design. [ABSTRACT FROM AUTHOR]

The skin is a very suitable organ for the induction of immune responses to vaccine antigens. Antigen delivery systems to the skin by needle and syringe directly deposit the antigen into the epidermal-dermal compartment, one of the most immunocompetent sites due to the presence of professional antigen-presenting cells aimed at the induction of antigen-specific T cells. In this study, we analyzed the amount of ovalbumin as an antigen delivered to the skin by a microneedle. When ovalbumin protein as an antigen was delivered to the skin of mice using a dissolving microneedle, it induced an immune response through the enhanced proliferation and cytokines production by the splenocytes and lymph nodes. Also, it effectively increased the ovalbumin-specific CD8+ T cell and CD4+ T cell population and induced an ovalbumin-specific CTL response against the graft of ovalbumin-expressing EG7 tumor cells in the immunized mice. Also, we identified the inhibition of tumor growth and prevention of tumor formation in the context of the therapeutic and prophylactic vaccine, respectively through EG-7 tumor mouse model. Finally, these data show the potential of patches as attractive antigen delivery vehicles. [ABSTRACT FROM AUTHOR]

We aimed to explore whether the combination of intradermal DNA vaccination, to boost immune response against melanoma antigens, and immune checkpoint blockade, to alleviate immunosuppression, improves antitumor effectiveness in a murine B16F10 melanoma tumor model. Compared to single treatments, a combination of intradermal DNA vaccination (ovalbumin or gp100 plasmid adjuvanted with IL12 plasmid) and immune checkpoint CTLA-4/PD-1 blockade resulted in a significant delay in tumor growth and prolonged survival of treated mice. Strong activation of the immune response induced by combined treatment resulted in a significant antigen-specific immune response, with elevated production of antigen-specific IgG antibodies and increased intratumoral CD8+ infiltration. These results indicate a potential application of the combined DNA vaccination and immune checkpoint blockade, specifically, to enhance the efficacy of DNA vaccines and to overcome the resistance to immune checkpoint inhibitors in certain cancer types. [ABSTRACT FROM AUTHOR]

Cancer cells harness immune checkpoints such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1) and indoleamine 2,3-dioxygenase 1 (IDO1) to evade immune control. Checkpoint inhibitors have demonstrated durable anti-tumor efficacy in human and preclinical models. Liver toxicity is one of the common immune-related adverse events associated with checkpoint inhibitors (CPIs) and its frequency and severity often increase significantly during CPI combination therapies. We aim to develop a mouse model to elucidate the immune mechanisms of CPI-associated liver toxicity. Co-administration of CTLA-4 blocking antibody, 9D9, and/or an IDO1 inhibitor, epacadostat in wild-type and PD-1-/- mice (to simulate the effect of PD1 blockade) synergistically induced liver injury and immune cell infiltration. Infiltrated cells were primarily composed of CD8+ T cells and positively associated with hepatocyte necrosis. Strikingly, sites of hepatocyte necrosis were frequently surrounded by clusters of mononuclear immune cells. CPI treatments resulted in increased expression of genes associated with hepatocyte cell death, leukocyte migration and T cell activation in the liver. In conclusion, blockade of immune checkpoints PD-1, CTLA-4, and IDO1 act synergistically to enhance T cell infiltration and activity in the liver, leading to hepatocyte death. [ABSTRACT FROM AUTHOR]

T cells are crucial players in obesity-mediated adipose tissue inflammation. We hypothesized that osteopontin (OPN), an inflammatory protein with enhanced activity when proteolytically cleaved, affects the number of viable T cells in adipose tissue and assessed inhibition of the interaction between T cells and thrombin and matrix metalloproteinases-cleaved OPN using antibodies and postimmune sera. Gene expression of T cell markers in adipose tissue from wild-type (wt) and Spp1-/- (OPN deficient) mice was analyzed after 16 weeks of high fat diet (HFD) or low fat diet (LFD) feeding. CD3, CD8 and OPN gene expression in omental adipose tissue from individuals with obesity was measured. OPN-T cell interactions were assessed with a fluorescence-based adhesion assay and blocked with antibodies targeting OPN. Comparison of T cell gene expression in adipose tissue from wt and Spp1-/- mice showed that OPN affected the number of T cells while in humans, levels of OPN correlated with T cell markers in omental adipose tissue. The interaction between T cells and cleaved OPN was blocked by postimmune sera following OPN peptide vaccinations and with monoclonal antibodies. In conclusion, levels of OPN affected the number of T cells in obesity and antibodies against cleaved OPN antagonize OPN-T cell interactions. [ABSTRACT FROM AUTHOR]

Effective vaccine design relies on accurate knowledge of protection against a pathogen, so as to be able to induce relevant and effective protective responses against it. An ideal Human Immunodeficiency virus (HIV) vaccine should induce humoral as well as cellular immune responses to prevent initial infection of host cells or limit early events of viral dissemination. A Phase I HIV-1 prophylactic vaccine trial sponsored by the International AIDS Vaccine Initiative (IAVI) was conducted in India in 2009.The trial tested a HIV-1 subtype C vaccine in a prime-boost regimen, comprising of a DNA prime (ADVAX) and Modified Vaccine Ankara (MVA) (TBC-M4) boost. The trial reported that the vaccine regimen was safe, well tolerated, and resulted in enhancement of HIV-specific immune responses. However, preliminary immunological studies were limited to vaccine-induced IFN-γ responses against the Env and Gag peptides. The present study is a retrospective study to characterize in detail the nature of the vaccine-induced cell mediated immune responses among volunteers, using Peripheral Blood Mononuclear Cells (PBMC) that were archived during the trial. ELISpot was used to measure IFN-γ responses and polyfunctional T cells were analyzed by intracellular multicolor flow cytometry. It was observed that DNA priming and MVA boosting induced Env and Gag specific bi-functional and multi-functional CD4+ and CD8+ T cells expressing IFN-γ, TNF-α and IL-2. The heterologous prime-boost regimen appeared to be slightly superior to the homologous prime-boost regimen in inducing favorable cell mediated immune responses. These results suggest that an in-depth analysis of vaccine-induced cellular immune response can aid in the identification of correlates of an effective immunogenic response, and inform future design of HIV vaccines. [ABSTRACT FROM AUTHOR]

Immunotherapy has fundamentally changed the landscape of cancer treatment. Despite the encouraging results with the checkpoint modulators, response rates vary widely across tumor types, with a majority of patients exhibiting either primary resistance without a significant initial response to treatment or acquired resistance with subsequent disease progression. Hematopoietic progenitor kinase 1 (HPK1) is predominantly expressed in hematopoietic cell linages and serves as a negative regulator in T cells and dendritic cells (DC). While HPK1 gene knockout (KO) studies suggest its role in anti-tumor immune responses, the involvement of kinase activity and thereof its therapeutic potential remain unknown. To investigate the potential of pharmacological intervention using inhibitors of HPK1, we generated HPK1 kinase dead (KD) mice which carry a single loss-of—function point mutation in the kinase domain and interrogated the role of kinase activity in immune cells in the context of suppressive factors or the tumor microenvironment (TME). Our data provide novel findings that HKP1 kinase activity is critical in conferring suppressive functions of HPK1 in a wide range of immune cells including CD4+, CD8+, DC, NK to Tregs, and inactivation of kinase domain was sufficient to elicit robust anti-tumor immune responses. These data support the concept that an HPK1 small molecule kinase inhibitor could serve as a novel agent to provide additional benefit in combination with existing immunotherapies, particularly to overcome resistance to current treatment regimens. [ABSTRACT FROM AUTHOR]

Chemotherapy or chemoradiotherapy conditioning regimens required for bone marrow transplantation (BMT) cause significant morbidity and mortality as a result of insufficient immune surveillance mechanisms leading to increased risks of infection and tumor recurrence. Such conditioning causes host stromal cell injury, impairing restoration of the central (thymus) and peripheral (spleen and lymph node) T cell compartments and slow immune reconstitution. The chemokine, CCL21, produced by host stromal cells, recruits T- and B-cells that provide lymphotoxin mediated instructive signals to stromal cells for lymphoid organogenesis. Moreover, T- and B-cell recruitment into these sites is required for optimal adaptive immune responses to pathogens and tumor antigens. Previously, we reported that CCL21 was markedly reduced in secondary lymphoid organs of transplanted animals. Here, we utilized adenoviral CCL21 gene transduced dendritic cells (DC/CCL21) given by footpad injections as a novel approach to restore CCL21 expression in secondary lymphoid organs post-transplant. CCL21 expression in secondary lymphoid organs reached levels of naïve controls and resulted in increased T cell trafficking to draining lymph nodes (LNs). An increase in both lymphoid tissue inducer cells and the B cell chemokine CXCL13 known to be important in LN formation was observed. Strikingly, only mice vaccinated with DC/CCL21 loaded with bacterial, viral or tumor antigens and not recipients of DC/control adenovirus loaded cells or no DCs had a marked increase in the systemic clearance of pathogens (bacteria; virus) and leukemia cells. Because DC/CCL21 vaccines have been tested in clinical trials for patients with lung cancer and melanoma, our studies provide the foundation for future trials of DC/CCL21 vaccination in patients receiving pre-transplant conditioning regimens. [ABSTRACT FROM AUTHOR]

Leishmania (V.) braziliensis and Leishmania(L.) amazonensis are the most pathogenic agents of American Cutaneous Leishmaniasis in Brazil, causing a wide spectrum of clinical and immunopathological manifestations, including: localized cutaneous leishmaniasis (LCLDTH+/++), borderline disseminated cutaneous leishmaniasis (BDCLDTH±), anergic diffuse cutaneous leishmaniasis (ADCLDTH-), and mucosal leishmaniasis (MLDTH++++). It has recently been demonstrated, however, that while L. (V.) braziliensis shows a clear potential to advance the infection from central LCL (a moderate T-cell hypersensitivity form) towards ML (the highest T-cell hypersensitivity pole), L. (L.) amazonensis drives the infection in the opposite direction to ADCL (the lowest T-cell hypersensitivity pole). This study evaluated by immunohistochemistry the expression of Toll-like receptors (TLRs) 2, 4, and 9 and their relationships with CD4 and CD8 T-cells, and TNF-α, IL-10, and TGF-β cytokines in that disease spectrum. Biopsies of skin and mucosal lesions from 43 patients were examined: 6 cases of ADCL, 5 of BDCL, and 11 of LCL caused byL. (L.) amazonensis; as well as 10 cases of LCL, 4 of BDCL, and 6 of ML caused byL. (V.) braziliensis. CD4+ T-cells demonstrated their highest expression in ML and, in contrast, their lowest in ADCL. CD8+ T-cells also showed their lowest expression in ADCL as compared to the other forms of the disease. TNF-α+showed increased expression from ADCL to ML, while IL-10+and TGF-β+ showed increased expression in the opposite direction, from ML to ADCL. With regards to TLR2, 4, and 9 expressions, strong interactions of TLR2 and 4 with clinical forms associated with L. (V.) braziliensis were observed, while TLR9, in contrast, showed a strong interaction with clinical forms linked to L. (L.) amazonensis. These findings strongly suggest the ability of L. (V.) braziliensis and L. (L.) amazonensis to interact with those TLRs to promote a dichotomous T-cell immune response in ACL. [ABSTRACT FROM AUTHOR]

We investigated immune responses to a lytic cytomegalovirus antigen (CMVpp52), and to a lytic human herpes virus (HHV) 6 antigen (HHV6p41), in systemic lupus erythematosus (SLE) patients and healthy controls (HCs), in order to clarify if the previously established impaired responses to Epstein-Barr virus (EBV) in SLE patients is a general defect in their responses against (all) HHVs. Multiplex Luminex technology results showed a normal induction of five quantified cytokines (interferon γ, interleukin(IL)12, IL17, IL10, and tumor necrosis factor α) in SLE patients compared to HCs upon stimulation with CMVpp52 and HHV6p41. However, flow cytometric results showed a reduced upregulation of the activation marker CD69 on T-cells from SLE patients (n = 17) compared to HCs (n = 17) upon stimulation with CMVpp52, indicating limited or defective CMVpp52-specific T-cells and/or poor antigen-presentation in SLE patients, and thereby possibly decreased control of the CMV infection. In conclusion, the dysfunctional immune response against EBV previously established in SLE patients does not seem to apply to the same degree regarding the immune responses against CMV or HHV6. Results designate that the main contributing HHV agent in development or exacerbation of SLE (in genetically predisposed individuals) is the previously determined uncontrolled EBV infection, and to a lesser extent CMV infection, and probably with no involvement of HHV6 infection. [ABSTRACT FROM AUTHOR]

We propose an agent-based model for the simulation of the autoimmune response in T1D. The model incorporates cell behavior from various rules derived from the current literature and is implemented on a high-performance computing system, which enables the simulation of a significant portion of the islets in the mouse pancreas. Simulation results indicate that the model is able to capture the trends that emerge during the progression of the autoimmunity. The multi-scale nature of the model enables definition of rules or equations that govern cellular or sub-cellular level phenomena and observation of the outcomes at the tissue scale. It is expected that such a model would facilitate in vivo clinical studies through rapid testing of hypotheses and planning of future experiments by providing insight into disease progression at different scales, some of which may not be obtained easily in clinical studies. Furthermore, the modular structure of the model simplifies tasks such as the addition of new cell types, and the definition or modification of different behaviors of the environment and the cells with ease. [ABSTRACT FROM AUTHOR]

Cytotoxic T-lymphocytes (CTLs) play a key role in immunity against cancer; however, the induction of CTL responses with currently available vaccines remains difficult. Because several reports have suggested that pigmentation and immunity might be functionally linked, we investigated whether melanin can act as an adjuvant in vaccines. Short synthetic peptides (8–35 amino acids long) containing T-cell epitopes were mixed with a solution of L-Dopa, a precursor of melanin. The mixture was then oxidized to generate nanoparticles of melanin-bound peptides. Immunization with melanin-bound peptides efficiently triggered CTL responses in mice, even against self-antigens and at a very low dose of peptides (microgram range). Immunization against a tumor antigen inhibited the growth of established tumors in mice, an effect that was abrogated by the depletion of CD8+ lymphocytes. These results demonstrate the efficacy of melanin as a vaccine adjuvant. [ABSTRACT FROM AUTHOR]

Many biopharmaceuticals (BPs) are known to be immunogenic in the clinic, which can result in modified pharmacokinetics, reduced efficacy, allergic reactions and anaphylaxis. During recent years, several technologies to predict immunogenicity have been introduced, but the predictive value is still considered low. Thus, there is an unmet medical need for optimization of such technologies. The generation of T cell dependent high affinity anti-drug antibodies plays a key role in clinical immunogenicity. This study aimed at developing and evaluating a novel in vitro T cell:PBMC assay for prediction of the immunogenicity potential of BPs. To this end, we assessed the ability of infliximab (anti-TNF-α), rituximab (anti-CD20), adalimumab (anti-TNF-α) and natalizumab (anti-α4-integrin), all showing immunogenicity in the clinic, to induce a CD4+ T cells response. Keyhole limpet hemocyanin (KLH) and cytomegalovirus pp65 protein (CMV) were included as neo-antigen and recall antigen positive controls, respectively. By analyzing 26 healthy donors having HLA-DRB1 alleles matching the European population, we calculated the frequency of responding donors, the magnitude of the response, and the frequency of BP-specific T cells, as measured by 3[H]-thymidine incorporation and ELISpot IL-2 secretion. KLH and CMV demonstrated a strong T cell response in all the donors analyzed. The frequency of responding donors to the BPs was 4% for infliximab, 8% for adalimumab, 19% for rituximab and 27% for natalizumab, which is compared to and discussed with their respective observed clinical immunogenicity. This study further complements predictive immunogenicity testing by quantifying the in vitro CD4+ T cell responses to different BPs. Even though the data generated using this modified method does not directly translate to the clinical situation, a high sensitivity and immunogenic potential of most BPs is demonstrated. [ABSTRACT FROM AUTHOR]

In an effort to make affordable vaccines suitable for the regions most affected by HIV-1, we have constructed stable vaccines that express an HIV-1 subtype C mosaic Gag immunogen (BCG-GagM, MVA-GagM and DNA-GagM). Mosaic immunogens have been designed to address the tremendous diversity of this virus. Here we have shown that GagM buds from cells infected and transfected with MVA-GagM and DNA-GagM respectively and forms virus-like particles. Previously we showed that a BCG-GagM prime MVA-GagM boost generated strong cellular immune responses in mice. In this study immune responses to the DNA-GagM and MVA-GagM vaccines were evaluated in homologous and heterologous prime-boost vaccinations. The DNA homologous prime boost vaccination elicited predominantly CD8+ T cells while the homologous MVA vaccination induced predominantly CD4+ T cells. A heterologous DNA-GagM prime MVA-GagM boost induced strong, more balanced Gag CD8+ and CD4+ T cell responses and that were predominantly of an effector memory phenotype. The immunogenicity of the mosaic Gag (GagM) was compared to a naturally occurring subtype C Gag (GagN) using a DNA homologous vaccination regimen. DNA-GagN expresses a natural Gag with a sequence that was closest to the consensus sequence of subtype C viruses sampled in South Africa. DNA-GagM homologous vaccination induced cumulative HIV-1 Gag-specific IFN-γ ELISPOT responses that were 6.5-fold higher than those induced by the DNA-GagN vaccination. Similarly, DNA-GagM vaccination generated 7-fold higher levels of cytokine-positive CD8+ T cells than DNA-GagN, indicating that this subtype C mosaic Gag elicits far more potent immune responses than a consensus-type Gag. Cells transfected and infected with DNA-GagM and MVA-GagM respectively, expressed high levels of GagM and produced budding virus-like particles. Our data indicates that a heterologous prime boost regimen using DNA and MVA vaccines expressing HIV-1 subtype C mosaic Gag is highly immunogenic in mice and warrants further investigation in non-human primates. [ABSTRACT FROM AUTHOR]

Whole malaria sporozoite vaccine regimens are promising new strategies, and some candidates have demonstrated high rates of durable clinical protection associated with memory T cell responses. Little is known about the anatomical distribution of memory T cells following whole sporozoite vaccines, and immunization of nonhuman primates can be used as a relevant model for humans. We conducted a chemoprophylaxis with sporozoite (CPS) immunization in P. knowlesi rhesus monkeys and challenged via mosquito bites. Half of CPS immunized animals developed complete protection, with a marked delay in parasitemia demonstrated in the other half. Antibody responses to whole sporozoites, CSP, and AMA1, but not CelTOS were detected. Peripheral blood T cell responses to whole sporozoites, but not CSP and AMA1 peptides were observed. Unlike peripheral blood, there was a high frequency of sporozoite-specific memory T cells observed in the liver and bone marrow. Interestingly, sporozoite-specific CD4+ and CD8+ memory T cells in the liver highly expressed chemokine receptors CCR5 and CXCR6, both of which are known for liver sinusoid homing. The majority of liver sporozoite-specific memory T cells expressed CD69, a phenotypic marker of tissue-resident memory (TRM) cells, which are well positioned to rapidly control liver-stage infection. Vaccine strategies that aim to elicit large number of liver TRM cells may efficiently increase the efficacy and durability of response against pre-erythrocytic parasites. [ABSTRACT FROM AUTHOR]

Fibroblast activation protein alpha (FAP) is a unique dual peptidase of the S9B serine protease family, being capable of both dipeptidyl peptidase and endopeptidase activities. FAP is expressed at low level in healthy adult organs including the pancreas, cervix, uterus, submaxillary gland and the skin, and highly upregulated in embryogenesis, chronic inflammation and tissue remodelling. It is also expressed by cancer-associated stromal fibroblasts in more than 90% of epithelial tumours. FAP has enzymatic and non-enzymatic functions in the growth, immunosuppression, invasion and cell signalling of tumour cells. FAP deficient mice are fertile and viable with no gross abnormality, but little data exist on the role of FAP in the immune system. FAP is upregulated in association with microbial stimulation and chronic inflammation, but its function in infection remains unknown. We showed that major populations of immune cells including CD4+ and CD8+ T cells, B cells, dendritic cells and neutrophils are generated and maintained normally in FAP knockout mice. Upon intranasal challenge with influenza virus, FAP mRNA was increased in the lungs and lung-draining lymph nodes. Nonetheless, FAP deficient mice showed similar pathologic kinetics to wildtype controls, and were capable of supporting normal anti-influenza T and B cell responses. There was no evidence of compensatory upregulation of other DPP4 family members in influenza-infected FAP-deficient mice. FAP appears to be dispensable in anti-influenza adaptive immunity. [ABSTRACT FROM AUTHOR]

Background: Burn injury initiates an acute inflammatory response that subsequently drives wound repair. However, acute disruption to the immune response is also common, leading to susceptibility to sepsis and increased morbidity and mortality. Despite increased understanding of the impact of burn injury on the immune system in the acute phase, little is known about long-term consequences of burn injury on immune function. This study was established to determine whether burn injury has long-term clinical impacts on patients’ immune responses. Methods: Using a population-based retrospective longitudinal study and linked hospital morbidity and death data from Western Australia, comparative rates of hospitalisation for respiratory infections in burn patients and a non-injured comparator cohort were assessed. In addition, a mouse model of non-severe burn injury was also used in which viral respiratory infection was induced at 4 weeks post-injury using a mouse modified version of the Influenza A virus (H3NN; A/mem/71-a). Results and conclusions: The burn injured cohort contained 14893 adult patients from 1980–2012 after removal of those patients with evidence of smoke inhalation or injury to the respiratory tract. During the study follow-up study a total of 2,884 and 2,625 respiratory infection hospital admissions for the burn and uninjured cohorts, respectively, were identified. After adjusting for covariates, the burn cohort experienced significantly elevated admission rates for influenza and viral pneumonia (IRR, 95%CI: 1.73, 1.27–2.36), bacterial pneumonia (IRR, 95%CI: 2.05, 1.85–2.27) and for other types of upper and lower respiratory infections (IRR, 95% CI: 2.38, 2.09–2.71). In the mouse study an increased viral titre was observed after burn injury, accompanied by a reduced CD8 response and increased NK and NKT cells in the draining lymph nodes. This data suggests burn patients are at long-term increased risk of infection due to sustained modulation of the immune response. [ABSTRACT FROM AUTHOR]

Virus spreading in tissues is determined by virus transport, virus multiplication in host cells and the virus-induced immune response. Cytotoxic T cells remove infected cells with a rate determined by the infection level. The intensity of the immune response has a bell-shaped dependence on the concentration of virus, i.e., it increases at low and decays at high infection levels. A combination of these effects and a time delay in the immune response determine the development of virus infection in tissues like spleen or lymph nodes. The mathematical model described in this work consists of reaction-diffusion equations with a delay. It shows that the different regimes of infection spreading like the establishment of a low level infection, a high level infection or a transition between both are determined by the initial virus load and by the intensity of the immune response. The dynamics of the model solutions include simple and composed waves, and periodic and aperiodic oscillations. The results of analytical and numerical studies of the model provide a systematic basis for a quantitative understanding and interpretation of the determinants of the infection process in target organs and tissues from the image-derived data as well as of the spatiotemporal mechanisms of viral disease pathogenesis, and have direct implications for a biopsy-based medical testing of the chronic infection processes caused by viruses, e.g. HIV, HCV and HBV. [ABSTRACT FROM AUTHOR]

As part of an international effort to develop vaccines for Theileria lestoquardi, we undertook a limited screen to test T. lestoquardi orthologues of antigens recognised by CD8+ T lymphocyte responses against T. annulata and T. parva in cattle. Five MHC defined sheep were immunized by live T. lestoquardi infection and their CD8+ T lymphocyte responses determined. Thirteen T. lestoquardi orthologues of T. parva and T. annulata genes, previously shown to be targets of CD8+ T lymphocyte responses of immune cattle, were expressed in autologous fibroblasts and screened for T cell recognition using an IFNγ assay. Genes encoding T. lestoquardi antigens Tl8 (putative cysteine proteinase, 349 aa) or Tl9 (hypothetical secreted protein, 293 aa) were recognise by T cells from one animal that displayed a unique MHC class I genotype. Antigenic 9-mer peptide epitopes of Tl8 and Tl9 were identified through peptide scans using CD8+ T cells from the responding animal. These experiments identify the first T. lestoquardi antigens recognised by CD8+ T cell responses linked to specific MHC class I alleles. [ABSTRACT FROM AUTHOR]

The monoclonal antibodies ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1) have shown remarkable antitumor activity in an increasing number of cancers. When combined, ipilimumab and nivolumab have demonstrated superior activity in patients with metastatic melanoma (CHECKMATE-067). Here we describe the preclinical development strategy that predicted these clinical results. Synergistic antitumor activity in mouse MC38 and CT26 colorectal tumor models was observed with concurrent, but not sequential CTLA-4 and PD-1 blockade. Significant antitumor activity was maintained using a fixed dose of anti-CTLA-4 antibody with decreasing doses of anti-PD-1 antibody in the MC38 model. Immunohistochemical and flow cytometric analyses confirmed that CD3+ T cells accumulated at the tumor margin and infiltrated the tumor mass in response to the combination therapy, resulting in favorable effector and regulatory T-cell ratios, increased pro-inflammatory cytokine secretion, and activation of tumor-specific T cells. Similarly, in vitro studies with combined ipilimumab and nivolumab showed enhanced cytokine secretion in superantigen stimulation of human peripheral blood lymphocytes and in mixed lymphocyte response assays. In a cynomolgus macaque toxicology study, dose-dependent immune-related gastrointestinal inflammation was observed with the combination therapy; this response had not been observed in previous single agent cynomolgus studies. Together, these in vitro assays and in vivo models comprise a preclinical strategy for the identification and development of highly effective antitumor combination immunotherapies. [ABSTRACT FROM AUTHOR]

Virus vector-based vaccination against tumor-specific antigens remains a promising therapeutic approach to overcome the immune suppressive tumor microenvironment. However, the extent that the desired CD8 T cell response against the targeted tumor antigen is impacted by the CD8 T cell response against the virus vector is unclear. To address this question, we used picornavirus vaccination with Theiler’s murine encephalomyelitis virus (TMEV) as our vector against tumor-expressed ovalbumin (OVA257-264) antigen in both the B16-OVA murine melanoma and GL261-quad cassette murine glioma models. Prior to vaccination, we employed vector silencing to inhibit the CD8 T cell response against the immunodominant TMEV antigen, VP2121-130. We then monitored the resulting effect on the CD8 T cell response against the targeted tumor-specific antigen, ovalbumin. We demonstrate that employing vector silencing in the context of B16-OVA melanoma does not reduce tumor burden or improve survival, while TMEV-OVA vaccination without vector silencing controls tumor burden. Meanwhile, employing vector silencing during picornavirus vaccination against the GL261-quad cassette glioma resulted in a lower frequency of tumor antigen-specific CD8 T cells. The results of this study are relevant to antigen-specific immunotherapy, in that the virus vector-specific CD8 T cell response is not competing with tumor antigen-specific CD8 T cells. Furthermore, vector silencing may have the adverse consequence of reducing the tumor antigen-specific CD8 T cell response, as demonstrated by our findings in the GL261-quad cassette model. [ABSTRACT FROM AUTHOR]

Cell-free virus is a well-recognized and efficient mechanism for the spread of hepatitis B virus (HBV) infection in the liver. Cell-to-cell transmission (CCT) can be a more efficient means of virus propagation. Despite experimental evidence implying CCT occurs in HBV, its relative impact is uncertain. We develop a 3-D agent-based model where each hepatocyte changes its viral state according to a dynamical process driven by cell-free virus infection, CCT and intracellular replication. We determine the relative importance of CCT in the development and resolution of acute HBV infection in the presence of cytolytic (CTL) and non-CTL mechanisms. T cell clearance number is defined as the minimum number of infected cells needed to be killed by each T cell at peak infection that results in infection clearance within 12 weeks with hepatocyte turnover (HT, number of equivalent livers) ≤3. We find that CCT has very little impact on the establishment of infection as the mean cccDNA copies/cell remains between 15 to 20 at the peak of the infection regardless of CCT strength. In contrast, CCT inhibit immune-mediated clearance of acute HBV infection as higher CCT strength requires higher T cell clearance number and increases the probability of T cell exhaustion. An effective non-CTL inhibition can counter these negative effects of higher strengths of CCT by supporting rapid, efficient viral clearance and with little liver destruction. This is evident as the T cell clearance number drops by approximately 50% when non-CTL inhibition is increased from 10% to 80%. Higher CCT strength also increases the probability of the incidence of fulminant hepatitis with this phenomenon being unlikely to arise for no CCT. In conclusion, we report the possibility of CCT impacting HBV clearance and its contribution to fulminant hepatitis. [ABSTRACT FROM AUTHOR]

Malnutrition is commonly associated with increased infectious disease susceptibility and severity. Whereas malnutrition might enhance the incidence of disease as well as its severity, active infection can in turn exacerbate malnutrition. Therefore, in a malnourished individual suffering from a severe infection, it is not possible to determine the contribution of the pre-existing malnutrition and/or the infection itself to increased disease severity. In the current study we focussed on two groups of malnourished, but otherwise healthy individuals: moderately malnourished (BMI: 18.4–16.5) and severely malnourished (BMI <16.5) and compared several immune parameters with those of individuals with a normal BMI (≥18.5). Our results show a similar haematological profile in all three groups, as well as a similar ratio of CD4+ and CD8+ T cells. We found significant correlations between low BMI and increased levels of T helper (Th) 1 (Interferon (IFN)-γ, (interleukin (IL)-2, IL-12), Th2 (IL-4, IL-5, IL-13), as well as IL-10, IL-33 and tumor necrosis factor-α, but not IL-8 or C reactive protein. The activities of arginase, an enzyme associated with immunosuppression, were similar in plasma, peripheral blood mononuclear cells (PBMC) and neutrophils from all groups and no differences in the expression levels of CD3ζ, a marker of T cell activation, were observed in CD4+ and CD8+T cells. Furthermore, whereas the capacity of neutrophils from the malnourished groups to phagocytose particles was not impaired, their capacity to produce reactive oxygen species was impaired. Finally we evaluated the frequency of a subpopulation of low-density neutrophils and show that they are significantly increased in the malnourished individuals. These differences were more pronounced in the severely malnourished group. In summary, our results show that even in the absence of apparent infections, healthy malnourished individuals display dysfunctional immune responses that might contribute to increased susceptibility and severity to infectious diseases. [ABSTRACT FROM AUTHOR]

Myalgic Encephalopathy/Chronic Fatigue Syndrome (ME/CFS) is a disease of unknown etiology. We have previously suggested clinical benefit from B-cell depletion using the monoclonal anti-CD20 antibody rituximab in a randomized and placebo-controlled study. Prolonged responses were then demonstrated in an open-label phase-II study with maintenance rituximab treatment. Using blood samples from patients in the previous two clinical trials, we investigated quantitative changes in T-lymphocyte subsets, in immunoglobulins, and in serum levels of two B-cell regulating cytokines during follow-up. B-lymphocyte activating factor of the tumor necrosis family (BAFF) in baseline serum samples was elevated in 70 ME/CFS patients as compared to 56 healthy controls (p = 0.011). There were no significant differences in baseline serum BAFF levels between patients with mild, moderate, or severe ME/CFS, or between responders and non-responders to rituximab. A proliferation-inducing ligand (APRIL) serum levels were not significantly different in ME/CFS patients compared to healthy controls at baseline, and no changes in serum levels were seen during follow-up. Immunophenotyping of peripheral blood T-lymphocyte subsets and T-cell activation markers at multiple time points during follow-up showed no significant differences over time, between rituximab and placebo groups, or between responders and non-responders to rituximab. Baseline serum IgG levels were significantly lower in patients with subsequent response after rituximab therapy compared to non-responders (p = 0.03). In the maintenance study, slight but significant reductions in mean serum immunoglobulin levels were observed at 24 months compared to baseline; IgG 10.6–9.5 g/L, IgA 1.8–1.5 g/L, and IgM 0.97–0.70 g/L. Although no functional assays were performed, the lack of significant associations of T- and NK-cell subset numbers with B-cell depletion, as well as the lack of associations to clinical responses, suggest that B-cell regulatory effects on T-cell or NK-cell subsets are not the main mechanisms for the observed improvements in ME/CFS symptoms observed in the two previous trials. The modest increase in serum BAFF levels at baseline may indicate an activated B-lymphocyte system in a subgroup of ME/CFS patients. [ABSTRACT FROM AUTHOR]

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells are killer lymphocytes that provide defense against viral infections and tumor transformation. Analogous to that of CTL, interactions of killer-cell immunoglobulin-like receptors (KIR) with specific human leukocyte antigen (HLA) class I ligands calibrate NK cell education and response. Gene families encoding KIRs and HLA ligands are located on different chromosomes, and feature variation in the number and type of genes. The independent segregation of KIR and HLA genes results in variable KIR-HLA interactions in individuals, which may impact disease susceptibility. We tested whether KIR-HLA combinations are associated with Vogt-Koyanagi-Harada (VKH) disease, a bilateral granulomatous panuveitis that has strong association with HLA-DR4. We present a case control study of 196 VKH patients and 209 controls from a highly homogeneous native population of Japan. KIR and HLA class I genes were typed using oligonucleotide hybridization method and analyzed using two-tailed Fisher’s exact probabilities. The incidence of Bx-KIR genotypes was decreased in VKH patients (odds ratio [OR] 0.58, P = 0.007), due primarily to a decrease in centromeric B-KIR motif and its associated KIRs 2DS2, 2DL2, 2DS3, and 2DL5B. HLA-B22, implicated in poor immune response, was increased in VKH (OR = 4.25, P = 0.0001). HLA-Bw4, the ligand for KIR3DL1, was decreased in VKH (OR = 0.59, P = 0.01). The KIR-HLA combinations 2DL2+C1/C2 and 3DL1+Bw4, which function in NK education, were also decreased in VKH (OR = 0.49, P = 0.012; OR = 0.59, P = 0.013). Genotypes missing these two inhibitory KIR-HLA combinations in addition to missing activating KIRs 2DS2 and 2DS3 were more common in VKH (OR = 1.90, P = 0.002). These results suggest that synergistic hyporesponsiveness of NK cells (due to poor NK education along with missing of activating KIRs) and CTL (due to HLA-B22 restriction) fail to mount an effective immune response against viral-infection that may trigger VKH pathogenesis in genetically susceptible individuals, such as HLA-DR4 carriers. [ABSTRACT FROM AUTHOR]

Background: Obesity reflects a chronic inflammatory environment that may contribute to prostate cancer progression and poor treatment outcomes. However, it is not clear which mechanisms drive this association within the tumor microenvironment. The aim of this pilot study was to examine prostatic inflammation via tumor infiltrating lymphocytes and macrophages characterized by obesity and cancer severity. Methods: We studied paraffin-embedded prostatectomy tissue from 99 participants (63 non-obese and 36 obese) from the Study of Clinical Outcomes, Risk and Ethnicity (University of Pennsylvania). Pathologists analyzed the tissue for type and count of lymphocytes and macrophages, including CD3, CD8, FOXP3, and CD68. Pathology data were linked to clinical and demographic variables. Statistical analyses included frequency tables, Kruskal-Wallis tests, Spearman correlations, and multivariable models. Results: We observed positive univariate associations between the number of CD68 cells and tumor grade (p = 0.019). In multivariable analysis, CD8 counts were associated with time to biochemical failure (HR = 1.09, 95% CI = 1.004–1.192, p-value = 0.041.) There were no differences in lymphocytes or macrophages by obesity status or BMI. Conclusions: The number of lymphocytes and macrophages in the tumor microenvironment did not differ by obesity status. However, these inflammation markers were associated with poor prostate cancer outcomes. Further examination of underlying mechanisms that influence obesity-related effects on prostate cancer outcomes is warranted. Such research will guide immunotherapy protocols and weight management as they apply to diverse patient populations and phenotypes. [ABSTRACT FROM AUTHOR]

Objective: Human Immunodeficiency Virus (HIV) and Hepatitis C virus (HCV) co-infection is recognized as a major cause of morbidity and mortality among HIV-1 infected patients. Our understanding of the impact of HIV infection on HCV specific immune responses and liver disease outcome is limited by the heterogeneous study populations with genetically diverse infecting viruses, varying duration of infection and anti-viral treatment. Methods: Viral-specific immune responses in a cohort of 151 HCV mono- and HIV co-infected former plasma donors infected with a narrow source of virus were studied. HCV and HIV specific T cell responses were correlated with clinical data. Results: HIV-1 accelerated liver disease progression and decreased HCV specific T cell immunity. The magnitude of HCV specific T cell responses inversely correlated with lower HCV RNA load and reduced liver injury as assessed by non-invasive markers of liver fibrosis. HIV co-infection reduced the frequency of HCV specific CD4+ T cells with no detectable effect on CD8+ T cells or neutralizing antibody levels. Conclusion: Our study highlights the impact of HIV co-infection on HCV specific CD4+ T cell responses in a unique cohort of patients for both HCV and HIV and suggests a crucial role for these cells in controlling chronic HCV replication and liver disease progression. [ABSTRACT FROM AUTHOR]

Over 90% of HIV/AIDS positive individuals in sub-Saharan Africa are infected with highly heterogeneous HIV-1 subtype C (HIV-1C) viruses. One of the best ways to reduce the burden of this disease is the development of an affordable and effective prophylactic vaccine. Mosaic immunogens are computationally designed to overcome the hurdle of HIV diversity by maximizing the expression of potential T cell epitopes. Mycobacterium bovis BCG ΔpanCD auxotroph and modified vaccinia Ankara (MVA) vaccines expressing HIV-1C mosaic Gag (GagM) were tested in a prime-boost regimen to demonstrate immunogenicity in a mouse study. The BCG-GagM vaccine was stable and persisted 11.5 weeks post vaccination in BALB/c mice. Priming with BCG-GagM and boosting with MVA-GagM elicited higher Gag-specific IFN-γ ELISPOT responses than the BCG-GagM only and MVA-GagM only homologous vaccination regimens. The heterologous vaccination also generated a more balanced and persistent CD4+ and CD8+ T cell Gag-specific IFN-γ ELISPOT response with a predominant effector memory phenotype. A Th1 bias was induced by the vaccines as determined by the predominant secretion of IFN-γ, TNF-α, and IL-2. This study shows that a low dose of MVA (104 pfu) can effectively boost a BCG prime expressing the same mosaic immunogen, generating strong, cellular immune responses against Gag in mice. Our data warrants further evaluation in non-human primates. A low dose vaccine would be an advantage in the resource limited countries of sub-Saharan Africa and India (where the predominating virus is HIV-1 subtype C). [ABSTRACT FROM AUTHOR]

Background: The failure to establish potent anti-HBV T cell responses suggests the absence of an effective innate immune activation. Kupffer cells and liver-infiltrating monocytes/macrophages have an essential role in establishing anti-HBV responses. These cells express the costimulatory molecules CD80 and CD86. CD80 expression on antigen-presenting cells (APCs) induces Th1 cell differentiation, whereas CD86 expression drives the differentiation towards a Th2 profile. The relative expression of CD80, CD86 and PD-L1 on APCs, regulates T cell activation. Few studies investigated CD80 and CD86 expression on KCs and infiltrating monocytes/macrophages in HBV-infected liver and knowledge about the expression of PD-L1 on these cells is controversial. The expression of these molecules together in CD68+ cells has not been explored in HBV-infected livers. Methods: Double staining immunohistochemistry was applied to liver biopsies of HBV-infected and control donors to explore CD80, CD86 and PD-L1 expression in the lobular and portal areas. Results: Chronic HBV infection was associated with increased CD68+CD86+ cell count and percentage in the lobular areas, and no changes in the count and percentage of CD68+CD80+ and CD68+PD-L1+ cells, compared to the control group. While CD68+CD80+ cell count in portal areas correlated with the fibrosis score, CD68+CD80+ cell percentage in lobular areas correlated with the inflammation grade. Conclusion: The upregulation of CD86 but not CD80 and PD-L1 on CD68+ cells in HBV-infected livers, suggests that these cells do not support the induction of potent Th1. Moreover, the expression of CD80 on CD68+ cells correlates with liver inflammation and fibrosis. [ABSTRACT FROM AUTHOR]

Pegylated interferon α-2a (Peg-IFN-α) represents a therapeutic alternative to the prolonged use of nucleos(t)ide analog (NA) in chronic hepatitis B (CHB) infection. The mechanisms leading to a positive clinical outcome remain unclear. As immune responses are critical for virus control, we investigated the effects of Peg-IFN-α on both innate and adaptive immunity, and related it to the clinical evolution. The phenotypic and functional features of the dendritic cells (DCs), natural killer (NK) cells and HBV-specific CD4/CD8 T cells were analyzed in HBeAg-negative CHB patients treated for 48-weeks with NA alone or together with Peg-IFN-α, before, during and up to 2-years after therapy. Peg-IFN-α induced an early activation of DCs, a potent expansion of the CD56bright NK subset, and enhanced the activation and functionality of the CD56dim NK subset. Peg-IFN-α triggered an increase in the frequencies of Th1- and Th17-oriented HBV-specific CD4/CD8 T cells. Peg-IFN-α reversed the unresponsiveness of patients to a specific stimulation. Most of the parameters returned to baseline after the stop of Peg-IFN-α therapy. Peg-IFN-α impacts both innate and adaptive immunity, overcoming dysfunctional immune responses in CHB patients. These modulations were not associated with seroconversion, which questioned the benefit of the add-on Peg-IFN-α treatment. [ABSTRACT FROM AUTHOR]

Natural regulatory T (Treg) cells interfere with multiple functions, which are crucial for the development of strong anti-tumour responses. In a model of 4T1 mammary carcinoma, depletion of CD25+Tregs results in tumour regression in Balb/c mice, but the mechanisms underlying this process are not fully understood. Here, we show that partial Treg depletion leads to the generation of a particular effector CD8 T cell subset expressing CD11c and low level of PD-1 in tumour draining lymph nodes. These cells have the capacity to migrate into the tumour, to kill DCs, and to locally regulate the anti-tumour response. These events are concordant with a substantial increase in CD11b+ resident dendritic cells (DCs) subsets in draining lymph nodes followed by CD8+ DCs. These results indicate that Treg depletion leads to tumour regression by unmasking an increase of DC subsets as a part of a program that optimizes the microenvironment by orchestrating the activation, amplification, and migration of high numbers of fully differentiated CD8+CD11c+PD1lo effector T cells to the tumour sites. They also indicate that a critical pattern of DC subsets correlates with the evolution of the anti-tumour response and provide a template for Treg depletion and DC-based therapy. [ABSTRACT FROM AUTHOR]

T cells are essential players in the defense against infection. By targeting the MHC class I antigen-presenting pathway with peptide-based vaccines, antigen-specific T cells can be induced. However, low immunogenicity of peptides poses a challenge. Here, we set out to increase immunogenicity of influenza-specific CD8+ T cell epitopes. By substituting amino acids in wild type sequences with non-proteogenic amino acids, affinity for MHC can be increased, which may ultimately enhance cytotoxic CD8+ T cell responses. Since preventive vaccines against viruses should induce a broad immune response, we used this method to optimize influenza-specific epitopes of varying dominance. For this purpose, HLA-A*0201 epitopes GILGFVFTL, FMYSDFHFI and NMLSTVLGV were selected in order of decreasing MHC-affinity and dominance. For all epitopes, we designed chemically enhanced altered peptide ligands (CPLs) that exhibited greater binding affinity than their WT counterparts; even binding scores of the high affinity GILGFVFTL epitope could be improved. When HLA-A*0201 transgenic mice were vaccinated with selected CPLs, at least 2 out of 4 CPLs of each epitope showed an increase in IFN-γ responses of splenocytes. Moreover, modification of the low affinity epitope NMLSTVLGV led to an increase in the number of mice that responded. By optimizing three additional influenza epitopes specific for HLA-A*0301, we show that this strategy can be extended to other alleles. Thus, enhancing binding affinity of peptides provides a valuable tool to improve the immunogenicity and range of preventive T cell-targeted peptide vaccines. [ABSTRACT FROM AUTHOR]

Despite our knowledge of the protective role of antibodies passed to infants through breast milk, our understanding of immunity transfer via maternal leukocytes is still limited. To emulate the immunological interface between the mother and her infant while breast-feeding, we used murine pups fostered after birth onto MHC-matched and MHC-mismatched dams. Overall, data revealed that: 1) Survival of breast milk leukocytes in suckling infants is possible, but not significant after the foster-nursing ceases; 2) Most breast milk lymphocytes establish themselves in specific areas of the intestine termed Peyer’s patches (PPs); 3) While most leukocytes in the milk bolus were myeloid cells, the majority of breast milk leukocytes localized to PPs were T lymphocytes, and cytotoxic T cells (CTLs) in particular; 4) These CTLs exhibit high levels of the gut-homing molecules α4β7 and CCR9, but a reduced expression of the systemic homing marker CD62L; 5) Under the same activation conditions, transferred CD8 T cells through breast milk have a superior capacity to produce potent cytolytic and inflammatory mediators when compared to those generated by the breastfed infant. It is therefore possible that maternal CTLs found in breast milk are directed to the PPs to compensate for the immature adaptive immune system of the infant in order to protect it against constant oral infectious risks during the postnatal phase. [ABSTRACT FROM AUTHOR]

Efforts to develop vaccines that can elicit mucosal immune responses in the female genital tract against sexually transmitted infections have been hampered by an inability to measure immune responses in these tissues. The differential expression of adhesion molecules is known to confer site-dependent homing of circulating effector T cells to mucosal tissues. Specific homing molecules have been defined that can be measured in blood as surrogate markers of local immunity (e.g. α4β7 for gut). Here we analyzed the expression pattern of adhesion molecules by circulating effector T cells following mucosal infection of the female genital tract in mice and during a symptomatic episode of vaginosis in women. While CCR2, CCR5, CXCR6 and CD11c were preferentially expressed in a mouse model of Chlamydia infection, only CCR5 and CD11c were clearly expressed by effector T cells during bacterial vaginosis in women. Other homing molecules previously suggested as required for homing to the genital mucosa such as α4β1 and α4β7 were also differentially expressed in these patients. However, CD11c expression, an integrin chain rarely analyzed in the context of T cell immunity, was the most consistently elevated in all activated effector CD8+ T cell subsets analyzed. This molecule was also induced after systemic infection in mice, suggesting that CD11c is not exclusive of genital tract infection. Still, its increase in response to genital tract disorders may represent a novel surrogate marker of mucosal immunity in women, and warrants further exploration for diagnostic and therapeutic purposes. [ABSTRACT FROM AUTHOR]

Binding of pattern recognition receptors (PRRs) by pathogen-associated molecular patterns (PAMPs) activates innate immune responses and contributes to development of adaptive immunity. Simultaneous stimulation of different types of PRRs can have synergistic immunostimulatory effects resulting in enhanced production of molecules that mediate innate immunity such as inflammatory cytokines, antimicrobial peptides, etc. Here, we evaluated the impact of combined stimulation of PRRs from different families on adaptive immunity by generating alum-based vaccine formulations with ovalbumin as a model antigen and the Toll-like receptor 4 (TLR4) agonist MPLA and the Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) agonist MDP adsorbed individually or together on the alum-ovalbumin particles. Multiple in vitro and in vivo readouts of immune system activation all showed that while individual PRR agonists increased the immunogenicity of vaccines compared to alum alone, the combination of both PRR agonists was significantly more effective. Combined stimulation of TLR4 and NOD2 results in a stronger and broader transcriptional response in THP-1 cells compared to individual PRR stimulation. Immunostimulatory composition containing both PRR agonists (MPLA and MDP) in the context of the alum-based ovalbumin vaccine also enhanced uptake of vaccine particles by bone marrow derived dendritic cells (BMDCs) and promoted maturation (up-regulation of expression of CD80, CD86, MHCII) and activation (production of cytokines) of BMDCs. Finally, immunization of mice with vaccine particles containing both PRR agonists resulted in enhanced cellular immunity as indicated by increased proliferation and activation (IFN-γ production) of splenic CD4+ and CD8+ T cells following in vitro restimulation with ovalbumin and enhanced humoral immunity as indicated by higher titers of ovalbumin-specific IgG antibodies. These results indicate that combined stimulation of TLR4 and NOD2 receptors dramatically enhances activation of both the humoral and cellular branches of adaptive immunity and suggests that inclusion of agonists of these receptors in standard alum-based adjuvants could be used to improve the effectiveness of vaccination. [ABSTRACT FROM AUTHOR]

Background: Currently, the pathogenesis of rheumatoid arthritis (RA) is not clearly understood. The LIGHT/HVEM/BTLA co-signaling pathway may be involved in the pathogenesis of RA, although reports on the expression levels of LIGHT, HVEM and BTLA in T lymphocytes from RA patients are limited. Method: In this study, we recruited 30 healthy controls and 21 RA patients. Clinical characteristics were collected for RA patients. The levels of LIGHT, HVEM and BTLA expressed on the surface of circulating T cells of RA patients and healthy controls were measured by flow cytometry. Result: The percentages of CD3+, CD4+ and CD8+ T lymphocytes that expressed BTLA from RA patients were all higher than those of the controls (all p < 0.05), while the percentages of CD3+, CD4+ and CD8+ T lymphocytes that expressed HVEM and LIGHT were all lower than those of the controls (all p < 0.05). The rheumatoid factor and the percentage of HVEM+CD4+ T lymphocytes showed a statistically significant negative correlation in RA patients (r = -0.453, p = 0.039), as did the swollen joint count and the percentage of BTLA+CD8+ T lymphocytes (r = -0.501, p = 0.021). Conclusion: Here, we provide the first report on the increased expression of BTLA in T lymphocytes and on the decreased expression of HVEM and LIGHT in RA patients. BTLA, HVEM and LIGHT might be involved in the pathogenesis of RA and have the potential to be new clinically useful characteristics of RA. [ABSTRACT FROM AUTHOR]

The heat shock protein gp96 elicits specific T cell responses to its chaperoned peptides against cancer and infectious diseases in both rodent models and clinical trials. Although gp96-induced innate immunity, via a subset of Toll like receptors (TLRs), and adaptive immunity, through antigen presentation, are both believed to be important for priming potent T cell responses, direct evidence for the role of gp96-mediated TLR activation related to its functional T cell activation is lacking. Here, we report that gp96 containing mutations in its TLR-binding domain failed to activate macrophages, but peptide presentation was unaffected. Moreover, we found that peptide-specific T cell responses, as well as antitumor T cell immunity induced by gp96, are severely impaired when the TLR-binding domain is mutated. These data demonstrate the essential role of the gp96-TLR interaction in priming T cell immunity and provide further molecular basis for the coupling of gp96-mediated innate with adaptive immunity. [ABSTRACT FROM AUTHOR]

Interferon Regulatory Factor-8 (IRF8) is constitutively expressed in monocytes and B cell lineages and plays important roles in immunity to pathogens and cancer. Although IRF8 expression is induced in activated T cells, the functional relevance of IRF8 in T cell-mediated immunity is not well understood. In this study, we used mice with targeted deletion of Irf8 in T-cells (IRF8KO) to investigate the role of IRF8 in T cell-mediated responses during herpes simplex virus 1 (HSV-1) infection of the eye. In contrast to wild type mice, HSV-1-infected IRF8KO mice mounted a more robust anti-HSV-1 immune response, which included marked expansion of HSV-1-specific CD8+ T cells, increased infiltration of inflammatory cells into the cornea and trigeminal ganglia (TG) and enhanced elimination of virus within the trigeminal ganglion. However, the consequence of the enhanced immunological response was the development of ocular inflammation, limbitis, and neutrophilic infiltration into the cornea of HSV-1-infected IRF8KO mice. Surprisingly, we observed a marked increase in virus-specific memory precursor effector cells (MPEC) in IRF8KO mice, suggesting that IRF8 might play a role in regulating the differentiation of effector CD8+ T cells to the memory phenotype. Together, our data suggest that IRF8 might play a role in restraining excess lymphocyte proliferation. Thus, modulating IRF8 levels in T cells can be exploited therapeutically to prevent immune-mediated ocular pathology during autoimmune and infectious diseases of the eye. [ABSTRACT FROM AUTHOR]

Background: The relationship between host microRNAs (miRNA), viral control and immune response has not yet been elucidated in the field of HIV. The aim of this study was to assess the differential miRNA profile in CD8+ T-cells between HIV-infected individuals who differ in terms of viral replication control and immune response. Methods: miRNA profile from resting and CD3/CD28-stimulated CD8+ T-cells from uninfected individuals (HIV-, n = 11), Elite Controllers (EC, n = 15), Viremic Controllers (VC, n = 15), Viremic Progressors (VP, n = 13) and HIV-infected patients on therapy (ART, n = 14) was assessed using Affymetrix miRNA 3.1 arrays. After background correction, quantile normalization and median polish summarization, normalized data were fit to a linear model. The analysis comprised: resting samples between groups; stimulated samples between groups; and stimulated versus resting samples within each group. Enrichment analyses of the putative target genes were perfomed using bioinformatic algorithms. Results: A downregulated miRNA pattern was observed when resting samples from all infected groups were compared to HIV-. A miRNA downregulation was also observed when stimulated samples from EC, ART and HIV- groups were compared to VP, being hsa-miR-4492 the most downregulated. Although a preferential miRNA downregulation was observed when stimulated samples were compared to the respective resting samples, VP presented a differential miRNA expression pattern. In fact, hsa-miR-155 and hsa-miR-181a were downregulated in VP whereas in the other groups, either an upregulation or no differences were observed after stimulation, respectively. Overall, functional enrichment analysis revealed that the predicted target genes were involved in signal transduction pathways, metabolic regulation, apoptosis, and immune response. Conclusions: Resting CD8+ T-cells do not exhibit a differential miRNA expression between HIV-infected individuals but they do differ from non-infected individuals. Moreover, a specific miRNA pattern is present in stimulated CD8+ T-cells from VP which could reflect a detrimental pattern in terms of CD8+ T-cell immune response. [ABSTRACT FROM AUTHOR]

Strategies to develop a functional cure for HIV infection will likely require boosting of effector T cell responses to eliminate reactivated, latently infected cells. We have recently explored an assay for assessing antigen-specific regulation of T cell proliferation, which was related to clinical progression in untreated patients and to vaccine efficacy in two trials of therapeutic Gag-based vaccines. We here expand the same assay to further investigate regulation mediated by various inhibitory pathways. Peripheral blood mononuclear cells from 26 asymptomatic HIV-infected, antiretroviral therapy-naïve patients were stimulated with Gag and Env overlapping peptide panels for 5 days. Monoclonal antibodies (mAbs) blocking inhibitory mediators interleukin (IL) 10, transforming growth factor (TGF) β, programmed death ligand (PD–L) 1 and herpes virus entry mediator (HVEM) were added to parallel cultures. Functional T cell regulation (FTR) was defined as the difference in proliferation between stimulated cultures with and without blocking mAbs. FTR was detected in 54% of patients. Blockade of IL-10/PD-L1 and IL10/TGF-β detected all cases with Gag- and Env-associated FTR, respectively. In accordance with previous findings, isolated Env FTR was associated with higher plasma HIV RNA and lower CD4 counts, while patients with both Gag and Env FTR also had higher Gag- and Env-specific proliferative CD8+ T cell responses. There was no association between FTR and frequencies of activated regulatory T cells. In conclusion, we observed substantial heterogeneity in FTR between patients, inhibitory pathways and HIV antigens. FTR may help to individualize immunomodulation and warrants further assessment in clinical immunotherapy trials. [ABSTRACT FROM AUTHOR]

A malaria vaccine is a public health priority. In order to produce an effective vaccine, a multistage approach targeting both the blood and the liver stage infection is desirable. The vaccine candidates also need to induce balanced immune responses including antibodies, CD4+ and CD8+ T cells. Protein-based subunit vaccines like RTS,S are able to induce strong antibody response but poor cellular reactivity. Adenoviral vectors have been effective inducing protective CD8+ T cell responses in several models including malaria; nonetheless this vaccine platform exhibits a limited induction of humoral immune responses. Two approaches have been used to improve the humoral immunogenicity of recombinant adenovirus vectors, the use of heterologous prime-boost regimens with recombinant proteins or the genetic modification of the hypervariable regions (HVR) of the capsid protein hexon to express B cell epitopes of interest. In this study, we describe the development of capsid modified Ad5 vectors that express a promiscuous Plasmodium yoelii T helper epitope denominated PyT53 within the hexon HVR2 region. Several regimens were tested in mice to determine the relevance of the hexon modification in enhancing protective immune responses induced by the previously described protein-based multi-stage experimental vaccine PyCMP. A heterologous prime-boost immunization regime that combines a hexon modified vector with transgenic expression of PyCMP followed by protein immunizations resulted in the induction of robust antibody and cellular immune responses in comparison to a similar regimen that includes a vector with unmodified hexon. These differences in immunogenicity translated into a better protective efficacy against both the hepatic and red blood cell stages of P. yoelii. To our knowledge, this is the first time that a hexon modification is used to deliver a promiscuous T cell epitope. Our data support the use of such modification to enhance the immunogenicity and protective efficacy of adenoviral based malaria vaccines. [ABSTRACT FROM AUTHOR]

Introduction: Initial evaluation of a candidate vaccine against HIV includes an assessment of the vaccine’s ability to generate immune responses. However, the dynamics of vaccine-induced immune responses are unclear. We hypothesized that the IFN-γ producing cytotoxic CD8+ (CD8+ IFN-γ+) T cell responses could be predicted by early IL-2 producing CD4+ (CD4+ IL-2+) helper T cell responses, and we evaluated this hypothesis using data from a phase I/II prophylactic HIV vaccine trial. The objective was to assess the dynamics and correlations between CD4+ IL-2+ T cell and CD8+ IFN-γ+ T cell responses after vaccination with a recombinant adenoviral serotype 5 (rAd5) HIV vaccine. Methods: We analyzed data from the HVTN 068 HIV vaccine trial, which evaluated the immunogenicity of two different strategies for prime and boost vaccination (rAd5-rAd5 vaccine versus DNA-rAd5) in 66 healthy volunteers. Spearman correlations between immunogenicity markers across time-points were calculated. CD8+ IFN-γ+ T cell response in the rAd5-rAd5 arm was modeled as a function of CD4+ IL-2+ T cell response and time using mixed effects regression models. Results: Moderate to high correlations (r = 0.48–0.76) were observed in the rAd5-rAd5 arm between the CD4+ IL-2+ T cell response at week 2 and later CD8+ IFN-γ+ T cell responses (weeks 2–52). Regression models confirmed this relationship with a significant association between the two markers: for a 1.0% increase in CD4+ IL-2+ T cells at week 2 post-prime, a 0.3% increase in CD8+ IFN-γ+ T cell responses across subsequent time points, including post-boost time points, was observed (p<0.01). Conclusion: These results suggest an early and leading role of CD4+ T cells in the cellular response to the rAd5-rAd5 vaccine and in particular the stimulation of cytotoxic CD8+ T cell responses. These results could inform better timing of CD4+ T cell measurements in future clinical trials. [ABSTRACT FROM AUTHOR]

Tumor-infiltrating lymphocytes (TIL) has been associated with improved survival in cancer patients. Within the tumor microenvironment, regulatory cells and expression of co-inhibitory immune checkpoint molecules can lead to the inactivation of TIL. Hence, there is a need to develop strategies that disrupt these negative regulators to achieve robust anti-tumor immune responses. We evaluated the blockade of immune checkpoints and their effect on T cell infiltration and function. We examined the ability of TIL to induce tumor-specific immune responses in vitro and in vivo. TIL isolated from tumor bearing mice were tumor-specific and expressed co-inhibitory immune checkpoint molecules. Administration of monoclonal antibodies against immune checkpoints led to a significant delay in tumor growth. However, anti-PD-L1 antibody treated mice had a significant increase in T cell infiltration and IFN-γ production compared to other groups. Adoptive transfer of in vitro expanded TIL from tumors of anti-PD-L1 antibody treated mice led to a significant delay in tumor growth. Blockade of co-inhibitory immune checkpoints could be an effective strategy to improve TIL infiltration and function. [ABSTRACT FROM AUTHOR]

Dengue virus purified inactivated vaccines (PIV) are highly immunogenic and protective over the short term, but may be poor at inducing cell-mediated immune responses and long-term protection. The dengue nonstructural protein 3 (NS3) is considered the main target for T-cell responses during viral infection. The amino (N)-terminal protease and the carboxy (C)-terminal helicase domains of DENV-2 NS3 were expressed in E. coli and analyzed for their immune-potentiating capacity. Mice were immunized with DENV-2 PIV with and without recombinant NS3 protease or NS3 helicase proteins, and NS3 proteins alone on days 0, 14 and 28. The NS3 helicase but not the NS3 protease was effective in inducing T-cell responses quantified by IFN-γ ELISPOT. In addition, markedly increased total IgG antibody titer against virus antigen was seen in mice immunized with the PIV/NS3 helicase combination in the ELISA, as well as increased neutralizing antibody titer measured by the plaque reduction neutralization test. These results indicate the potential immunogenic properties of the NS3 helicase protein and its use in a dengue vaccine formulation. [ABSTRACT FROM AUTHOR]

Background: Islet transplantation may potentially cure type 1 diabetes mellitus (T1DM). However, immune rejection, especially that induced by the alloreactive T-cell response, remains a restraining factor for the long-term survival of grafted islets. Programmed death ligand-1 (PD-L1) is a negative costimulatory molecule. PD-L1 deficiency within the donor heart accelerates allograft rejection. Here, we investigate whether PD-L1 deficiency in donor islets reduces allograft survival time. Methods: Glucose Stimulation Assays were performed to evaluate whether PD-L1 deficiency has detrimental effects on islet function. Islets isolated from PDL1-deficient mice or wild- type (WT) mice (C57BL/6j) were implanted beneath the renal capsule of streptozotocin (STZ)-induced diabetic BALB/c mice. Blood glucose levels and graft survival time after transplantation were monitored. Moreover, we analyzed the residual islets, infiltrating immune cells and alloreactive cells from the recipients. Results: PD-L1 deficiency within islets does not affect islet function. However, islet PD-L1 deficiency increased allograft rejection and was associated with enhanced inflammatory cell infiltration and recipient T-cell alloreactivity. Conclusions: This is the first report to demonstrate that PD-L1 deficiency accelerated islet allograft rejection and regulated recipient alloimmune responses. [ABSTRACT FROM AUTHOR]

While a plethora of data describes the essential role of systemic CD8+ T cells in the control of SIV replication little is known about the local in situ CD8+ T cell immune responses against SIV at the intact tissue level, due to technical limitations. In situ staining, using GagCM9 Qdot 655 multimers, were here combined with laser capture microdissection to detect and collect SIV Gag CM9 specific CD8+ T cells in lymph node tissue from SIV infected rhesus macaques. CD8+ T cells from SIV infected and uninfected rhesus macaques were also collected and compared to the SIV GagCM9 specific CD8+ T cells. Illumina bead array and transcriptional analyses were used to assess the transcriptional profiles and the three different CD8+ T cell populations displayed unique transcriptional patterns. This pilot study demonstrates that rapid and specific immunostaining combined with laser capture microdissection in concert with transcriptional profiling may be used to elucidate phenotypic differences between CD8+ T cells in SIV infection. Such technologies may be useful to determine differences in functional activities of HIV/SIV specific T cells. [ABSTRACT FROM AUTHOR]

It is widely anticipated that a prophylactic vaccine may be needed to control the HIV/AIDS epidemic worldwide. Despite over two decades of research, a vaccine against HIV-1 remains elusive, although a recent clinical trial has shown promising results. Recent studies have focused on highly conserved domains within HIV-1 such as the membrane proximal external region (MPER) of the envelope glycoprotein, gp41. MPER has been shown to play critical roles in mucosal transmission of HIV-1, though this peptide is poorly immunogenic on its own. Here we provide evidence that plant-produced HIV-1 enveloped virus-like particles (VLPs) consisting of Gag and a deconstructed form of gp41 comprising the MPER, transmembrane, and cytoplasmic domains (Dgp41) provides an effective platform to display MPER for use as an HIV vaccine candidate. Prime-boost strategies combining systemic and mucosal priming with systemic boosting using two different vaccine candidates (VLPs and CTB-MPR—a fusion of MPER and the B-subunit of cholera toxin) were investigated in BALB/c mice. Serum antibody responses against both the Gag and gp41 antigens were elicited when systemically primed with VLPs. These responses could be recalled following systemic boosting with VLPs. In addition, mucosal priming with VLPs allowed for a boosting response against Gag and gp41 when boosted with either candidate. Importantly, the VLPs also induced Gag-specific CD4 and CD8 T-cell responses. This report on the immunogenicity of plant-based Gag/Dgp41 VLPs may represent an important milestone on the road towards a broadly efficacious and inexpensive subunit vaccine against HIV-1. [ABSTRACT FROM AUTHOR]

Objective: Detailed studies of correlation between HIV-M.tb co-infection and hierarchy declines of CD8+/CD4+ T-cell counts and IFN-γ responses have not been done. We conducted case-control studies to address this issue. Methods: 164 HIV-1-infected individuals comprised of HIV-1+ATB, HIV-1+LTB and HIV-1+TB- groups were evaluated. Immune phenotyping and complete blood count (CBC) were employed to measure CD4+ and CD8+ T-cell counts; T.SPOT.TB and intracellular cytokine staining (ICS) were utilized to detect ESAT6, CFP10 or PPD-specific IFN-γ responses. Results: There were significant differences in median CD4+ T-cell counts between HIV-1+ATB (164/μL), HIV-1+LTB (447/μL) and HIV-1+TB- (329/μL) groups. Hierarchy low CD4+ T-cell counts (<200/μL, 200-500/μL, >500/μL) were correlated significantly with active TB but not M.tb co-infection. Interestingly, hierarchy low CD8+ T-cell counts were not only associated significantly with active TB but also with M.tb co-infection (P<0.001). Immunologically, HIV-1+ATB group showed significantly lower numbers of ESAT-6-/CFP-10-specific IFN-γ+ T cells than HIV-1+LTB group. Consistently, PPD-specific IFN-γ+CD4+/CD8+ T effector cells in HIV-1+ATB group were significantly lower than those in HIV-1+LTB group (P<0.001). Conclusions: Hierarchy low CD8+ T-cell counts and effector function in HIV-1-infected individuals are correlated with both M.tb co-infection and active TB. Hierarchy low CD4+ T-cell counts and Th1 effector function in HIV-1+ individuals are associated with increased frequencies of active TB, but not M.tb co-infection. [ABSTRACT FROM AUTHOR]

Immunotherapy approaches using checkpoint blockade, alone, or in combination with tumor antigen vaccination, or adoptive cell transfer, are emerging as promising approaches for the treatment of non-small cell lung cancer (NSCLC). In preparation for upcoming combined immunotherapy approaches in NSCLC, here, we have assessed spontaneous immune responses to XAGE-1b, a tumor specific antigen of the Cancer Testis Antigen group that has been previously reported to be spontaneously immunogenic in the Japanese population, in a cohort of Caucasian patients with NSCLC. We found spontaneous serological responses to XAGE-1b in 9% of the patients. Importantly, these responses were limited to, and represented 13% of, patients with adenocarcinoma tumors, the most frequent histological subtype, for which immunotherapy approaches are under development. Using a set of overlapping peptides spanning the entire XAGE-1b protein, and in support of the serological data, we detected significant XAGE-1b specific CD4+ T cell responses in all XAGE-1b seropositive patients and identified several CD4+ T cell epitopes. Altogether, our results support the relevance of the XAGE-1b antigen in Caucasians NSCLC patients with adenocarcinoma, and the implementation of future immunotherapies exploiting the high immunogenicity of the antigen in this patient population. [ABSTRACT FROM AUTHOR]