Stimulating immune responses to fight cancer: Basic biology and mechanisms

Chronic inflammation is now recognized as a major cause of malignant disease. In concert with various mechanisms (including DNA instability), hypoxia and activation of inflammatory bioactive lipid pathways and pro-inflammatory cytokines open the doorway to malignant transformation and proliferation, angiogenesis, and metastasis in many cancers. A balance between stimulatory and inhibitory signals regulates the immune response to cancer. These include inhibitory checkpoints that modulate the extent and duration of the immune response and may be activated by tumor cells. This contributes to immune resistance, especially against tumor antigen-specific T-cells. Targeting these checkpoints is an evolving approach to cancer immunotherapy, designed to foster an immune response. The current focus of these trials is on the programmed cell death protein 1 (PD-1) receptor and its ligands (PD-L1, PD-L2) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Researchers have developed anti-PD-1 and anti-PDL-1 antibodies that interfere with the ligands and receptor and allow the tumor cell to be recognized and attacked by tumor-infiltrating T-cells. These are currently being studied in lung cancer. Likewise, CTLA-4 inhibitors, which have had success treating advanced melanoma, are being studied in lung cancer with encouraging results.

Src Homology 3-interacting Domain of Rv1917c of Mycobacterium tuberculosis Induces Selective Maturation of Human Dendritic Cells by Regulating PI3K-MAPK-NF-kappa B Signaling and Drives Th2 Immune Responses

Mycobacterium tuberculosis, an etiological agent of pulmonary tuberculosis, causes significant morbidity and mortality worldwide. Pathogenic mycobacteria survive in the host by subverting host innate immunity. Dendritic cells (DCs) are professional antigen-presenting cells that are vital for eliciting immune responses to infectious agents, including pathogenic mycobacteria. DCs orchestrate distinct Th responses based on the signals they receive. In this perspective, deciphering the interactions of the proline-glutamic acid/proline-proline-glutamic acid (PE/PPE) family of proteins of M. tuberculosis with DCs assumes significant pathophysiological attributes. In this study, we demonstrate that Rv1917c (PPE34), a representative member of the proline-proline-glutamic-major polymorphic tandem repeat family, interacts with TLR2 and triggers functional maturation of human DCs. Signaling perturbations implicated a critical role for integrated cross-talk among PI3K-MAPK and NF-kappa B signaling cascades in Rv1917c-induced maturation of DCs. However, this maturation of DCs was associated with a secretion of high amounts of anti-inflammatory cytokine IL-10, whereas Th1-polarizing cytokine IL-12 was not induced. Consistent with these results, Rv1917c-matured DCs favored secretion of IL-4, IL-5, and IL-10 from CD4(+) T cells and contributed to Th2-skewed cytokine balance ex vivo in healthy individuals and in patients with pulmonary tuberculosis. Interestingly, the Rv1917c-skewed Th2 immune response involved induced expression of cyclooxygenase-2 (COX-2) in DCs. Taken together, these results indicate that Rv1917c facilitates a shift in the ensuing immunity toward the Th2 phenotype and could aid in immune evasion by mycobacteria.

Immune responses to hemagglutinin-neuraminidase protein of peste des petits ruminants virus expressed in transgenic peanut plants in sheep

Peste des petits ruminants (PPR) is an acute, highly contagious disease of small ruminants caused by a morbillivirus, Peste des petits ruminants virus (PPRV). The disease is prevalent in equatorial Africa, the Middle East, and the Indian subcontinent. A live attenuated vaccine is in use in some of the countries and has been shown to provide protection for at least three years against PPR. However, the live attenuated vaccine is not robust in terms of thermotolerance. As a step towards development of a heat stable subunit vaccine, we have expressed a hemagglutinin-neuraminidase (HN) protein of PPRV in peanut plants (Arachis hypogea) in a biologically active form, possessing neuraminidase activity. Importantly. HN protein expressed in peanut plants retained its immunodominant epitopes in their natural conformation. The immunogenicity of the plant derived HN protein was analyzed in sheep upon oral immunization. Virus neutralizing antibody responses were elicited upon oral immunization of sheep in the absence of any mucosal adjuvant. In addition, anti-PPRV-HN specific cell-mediated immune responses were also detected in mucosally immunized sheep. (C) 2010 Elsevier B.V. All rights reserved.

Pattern recognition and cellular immune responses to novel Mycobacterium tuberculosis-antigens in individuals from Belarus

Background: Tuberculosis (TB) is an enduring health problem worldwide and the emerging threat of multidrug resistant (MDR) TB and extensively drug resistant (XDR) TB is of particular concern. A better understanding of biomarkers associated with TB will aid to guide the development of better targets for TB diagnosis and for the development of improved TB vaccines. Methods: Recombinant proteins (n = 7) and peptide pools (n = 14) from M. tuberculosis (M.tb) antigens associated with M.tb pathogenicity, modification of cell lipids or cellular metabolism, were used to compare T cell immune responses defined by IFN-gamma production using a whole blood assay (WBA) from i) patients with TB, ii) individuals recovered from TB and iii) individuals exposed to TB without evidence of clinical TB infection from Minsk, Belarus. Results: We identified differences in M.tb target peptide recognition between the test groups, i.e. a frequent recognition of antigens associated with lipid metabolism, e.g. cyclopropane fatty acyl phospholipid synthase. The pattern of peptide recognition was broader in blood from healthy individuals and those recovered from TB as compared to individuals suffering from pulmonary TB. Detection of biologically relevant M.tb targets was confirmed by staining for intracellular cytokines (IL-2, TNF-alpha and IFN-gamma) in T cells from non-human primates (NHPs) after BCG vaccination. Conclusions: PBMCs from healthy individuals and those recovered from TB recognized a broader spectrum of M.tb antigens as compared to patients with TB. The nature of the pattern recognition of a broad panel of M.tb antigens will devise better strategies to identify improved diagnostics gauging previous exposure to M.tb; it may also guide the development of improved TB-vaccines.

Differential cellular recognition pattern to M. tuberculosis targets defined by IFN-gamma and IL-17 production in blood from TB plus patients from Honduras as compared to health care workers: TB and immune responses in patients from Honduras

Background: A better understanding of the quality of cellular immune responses directed against molecularly defined targets will guide the development of TB diagnostics and identification of molecularly defined, clinically relevant M.tb vaccine candidates. Methods: Recombinant proteins (n = 8) and peptide pools (n = 14) from M. tuberculosis (M.tb) targets were used to compare cellular immune responses defined by IFN-gamma and IL-17 production using a Whole Blood Assay (WBA) in a cohort of 148 individuals, i.e. patients with TB + (n = 38), TB- individuals with other pulmonary diseases (n = 81) and individuals exposed to TB without evidence of clinical TB (health care workers, n = 29). Results: M.tb antigens Rv2958c (glycosyltransferase), Rv2962c (mycolyltransferase), Rv1886c (Ag85B), Rv3804c (Ag85A), and the PPE family member Rv3347c were frequently recognized, defined by IFN-gamma production, in blood from healthy individuals exposed to M.tb (health care workers). A different recognition pattern was found for IL-17 production in blood from M.tb exposed individuals responding to TB10.4 (Rv0288), Ag85B (Rv1886c) and the PPE family members Rv0978c and Rv1917c. Conclusions: The pattern of immune target recognition is different in regard to IFN-gamma and IL-17 production to defined molecular M.tb targets in PBMCs from individuals frequently exposed to M.tb. The data represent the first mapping of cellular immune responses against M.tb targets in TB patients from Honduras.

Epigenetics and miRNA during bacteria-induced host immune responses

Various cellular processes including the pathogen-specific immune responses, host-pathogen interactions and the related evasion mechanisms rely on the ability of the immune cells to be reprogrammed accurately and in many cases instantaneously. In this context, the exact functions of epigenetic and miRNA-mediated regulation of genes, coupled with recent advent in techniques that aid such studies, make it an attractive field for research. Here, we review examples that involve the epigenetic and miRNA control of the host immune system during infection with bacteria. Interestingly, many pathogens utilize the epigenetic and miRNA machinery to modify and evade the host immune responses. Thus, we believe that global epigenetic and miRNA mapping of such host-pathogen interactions would provide key insights into their cellular functions and help to identify various determinants for therapeutic value.

Immune responses against hepatitis C virus genotype 3a virus-like particles in mice: A novel VLP prime-adenovirus boost strategy

Chronic hepatitis C virus (HCV) infection represents a major health threat to global population. In India, approximately 15-20% of cases of chronic liver diseases are caused by HCV infection. Although, new drug treatments hold great promise for HCV eradication in infected individuals, the treatments are highly expensive. A vaccine for preventing or treating HCV infection would be of great value, particularly in developing countries. Several preclinical trials of virus-like particle (VLP) based vaccine strategies are in progress throughout the world. Previously, using baculovirus based system, we have reported the production of hepatitis C virus-like particles (HCV-LPs) encoding structural proteins for genotype 3a, which is prevalent in India. In the present study, we have generated HCV-LPs using adenovirus based system and tried different immunization strategies by using combinations of both kinds of HCV-LPs with other genotype 3a-based immunogens. HCV-LPs and peptides based ELISAs were used to evaluate antibody responses generated by these combinations. Cell-mediated immune responses were measured by using T-cell proliferation assay and intracellular cytokine staining. We observed that administration of recombinant adenoviruses expressing HCV structural proteins as final booster enhances both antibody as well as T-cell responses. Additionally, reduction of binding of VLP and JFH1 virus to human hepatocellular carcinoma cells demonstrated the presence of neutralizing antibodies in immunized sera. Taken together, our results suggest that the combined regimen of VLP followed by recombinant adenovirus could more effectively inhibit HCV infection, endorsing the novel vaccine strategy. (C) 2015 Elsevier Ltd. All rights reserved.

In vivo regulation of the Vi antigen in Salmonella and induction of immune responses with an in vivo-inducible promoter.

Salmonella enterica serovar Typhi, the agent of typhoid fever in humans, expresses the surface Vi polysaccharide antigen that contributes to virulence. However, Vi expression can also be detrimental to some key steps of S. Typhi infectivity, for example, invasion, and Vi is the target of protective immune responses. We used a strain of S. Typhimurium carrying the whole Salmonella pathogenicity island 7 (SPI-7) to monitor in vivo Vi expression within phagocytic cells of mice at different times after systemic infection. We also tested whether it is possible to modulate Vi expression via the use of in vivo-inducible promoters and whether this would trigger anti-Vi antibodies through the use of Vi-expressing live bacteria. Our results show that Vi expression in the liver and spleen is downregulated with the progression of infection and that the Vi-negative population of bacteria becomes prevalent by day 4 postinfection. Furthermore, we showed that replacing the natural tviA promoter with the promoter of the SPI-2 gene ssaG resulted in sustained Vi expression in the tissues. Intravenous or oral infection of mice with a strain of S. Typhimurium expressing Vi under the control of the ssaG promoter triggered detectable levels of all IgG subclasses specific for Vi. Our work highlights that Vi is downregulated in vivo and provides proof of principle that it is possible to generate a live attenuated vaccine that induces Vi-specific antibodies after single oral administration.

Immune Responses to Six Synthetic Peptides of Capsid Protein Immune Responses to Six Synthetic Peptides of Capsid Protein

Many B cell epitopes within p24 of human immunodeficiency virus type 1 (HIV-1) were identified, while most of them were determined by using murine monoclonal antibodies reacting with overlapping peptides of p24. Therefore these epitopes may not represent the actual epitopes recognized by the HIV-1 infected individuals. In the present study, immune responses of 67 HIV-1 positive sera from Yunnan Province, China to five peptides on p24 of HIV-1 and one of HIV-2 were analyzed. All of 67 sera did not recognize peptide GA-12 on HIV-1 and peptide AG-23 on HIV-2, which indicated that GA-12 was not human B cell epitope and AG-23 did not cross-react with HIV-1 positive serum. Except 13 sera (19.4%), all remaining sera did not recognize peptides NI-15, DR-16, DC-22 and PS-18, which indicated that these four peptides represented B cell linear epitopes of HIV-1 p24 in some HIV-1 infected individuals but not the immuno-dominant epitopes in most individuals. Cellular & Molecular Immunology. 2005;2(4):289-293.

The effects of CpG-C oligodeoxynucleotides on innate immune responses in Eriocheir sinensis (H. Milne-Edwards, 1853)

CpG oligodeoxynucleotides (ODNs) can stimulate the immune system, and therefore are widely used as a therapeutic vaccination and immune adjuvant in human. In the present study, CpG-C, a combination of A- and B-class ODN, was injected into Chinese mitten crab Eriocheir sinensis at three doses (0.1, 1 and 10 mu g crab-1), and the reactive oxygen species (ROS) levels, activities of total intracellular phenoloxidase (PO) and lysozyme-like activities, the mRNA transcripts of EsproPO, EsCrustin and EsALF were assayed to evaluate its modulating effects on the immune system of crab. The ROS levels in all treated and control groups were significantly increased from 6 to 24 h, except that ROS in 0.1 mu g CpG-C-treated crabs was comparable to that of the blank at 6 h. The PO activity was significantly enhanced and EsproPO transcripts were down-regulated (P < 0.01) at 6 h after the injection of 0.1 mu g CpG-C, with no significant changes in the other dosage treatments. The lysozyme-like activities and EsCrustin transcripts in the CpG-C-treatment groups were significantly higher than those of controls. The mRNA expression of EsALF remained almost constant in all the groups during the treatment. These results collectively suggested that CpG-C could activate the immune responses of E. sinensis, and might be used as a novel immunostimulant for disease control in crabs.

The effects of CpG-C oligodeoxynucleotides on innate immune responses in Eriocheir sinensis (H. Milne-Edwards, 1853)

CpG oligodeoxynucleotides (ODNs) can stimulate the immune system, and therefore are widely used as a therapeutic vaccination and immune adjuvant in human. In the present study, CpG-C, a combination of A- and B-class ODN, was injected into Chinese mitten crab Eriocheir sinensis at three doses (0.1, 1 and 10 mu g crab-1), and the reactive oxygen species (ROS) levels, activities of total intracellular phenoloxidase (PO) and lysozyme-like activities, the mRNA transcripts of EsproPO, EsCrustin and EsALF were assayed to evaluate its modulating effects on the immune system of crab. The ROS levels in all treated and control groups were significantly increased from 6 to 24 h, except that ROS in 0.1 mu g CpG-C-treated crabs was comparable to that of the blank at 6 h. The PO activity was significantly enhanced and EsproPO transcripts were down-regulated (P < 0.01) at 6 h after the injection of 0.1 mu g CpG-C, with no significant changes in the other dosage treatments. The lysozyme-like activities and EsCrustin transcripts in the CpG-C-treatment groups were significantly higher than those of controls. The mRNA expression of EsALF remained almost constant in all the groups during the treatment. These results collectively suggested that CpG-C could activate the immune responses of E. sinensis, and might be used as a novel immunostimulant for disease control in crabs.

The immune responses in Chinese mitten crab Eriocheir sinensis challenged with double-stranded RNA

Double-stranded RNA (dsRNA) is a virus-associated molecular pattern which induces antiviral innate immune responses and RNA interference (RNAi) in mammals. In invertebrates, RNAi phenomenon has been widely studied, but dsRNA-induced innate immune response is seldom reported. In the present study, two different dsRNAs specific for green fluorescent protein (GFP) and the putative D1 protein of photosystem II (NoPSD) from Nannochloropsis oculata, were employed to challenge Chinese mitten crab Eriocheir sinensis. The temporal changes of phenoloxidase (PO), acid phosphatase (ACP), superoxide dismutase (SOD) and malondialdehyde (MDA) content, as well as the mRNA expression of some immune-related genes were examined in order to estimate the effect of dsRNAs on the innate immunity of E. sinensis. The activities of PO, ACP and SOD significantly increased after dsRNA treatment, whereas malondialdehyde (MDA) content did not change significantly. Among the examined genes, only the mRNA expression of EsALF, an antibacterial peptide in E. sinensis, was significantly up-regulated (about 5 fold, P < 0.05) at 12 h after dsRNA treatment, while no significant expression changes were observed among the other immune genes. The increase of PO, ACP and SOD activities, and mRNA expression level of EsALF after dsRNA stimulation indicate that phenoloxidase, hydrolytic enzyme, antioxidation and EsALF were involved in dsRNA-induced innate immunity, suggesting that broad-spectrum immune responses could be induced by dsRNA in E. sinensis. (C) 2009 Elsevier Ltd. All rights reserved.

Physiological and immune responses of zhikong scallop Chlamys farreri to the acute viral necrobiotic virus infection

The Zhikong Scallop, Chlamys farreri, is one of the most Important bivalve mollusks cultured in northern China However, mass mortality of the cultured C farreri has posed a serious threat to the maricultural Industry in recent years. Acute Viral Necrobiotic Virus (AVNV) is believed as an important etiological agent causing the scallop mass mortalities To understand the mechanism behind the AVNV associated scallop disease and mortality, we assessed the physiological and immune responses of C farreri to the virus infection using oxygen consumption rate, ammonium-nitrogen excretion rate, hemocyte copper, zinc superoxide dismutase gene expression, and plasma superoxide dismutase activity and alkaline phosphatase activity as indicators Scallops challenged by AVNV at 25 C developed typical disease signs 2 days after virus injection Before the disease manifested, scallop oxygen consumption and NH4+-N excretion rates rose and then fell back. Real-time PCR revealed that the hemocyte cytosol Cu, Zn SOD gene expression was upregulated followed by recovery The plasma SOD activity, however, augmented consistently following virus injection Moreover, plasma AKP activity first lowered and then elevated gradually to the highest level at 24 h post virus injection Scallops challenged by AVNV at 17 degrees C neither developed notable disease nor showed obvious responses that could be associated with the virus infection. While the results suggested a correlation between the elevated seawater temperature and the AVNV infection associated C farreri mortalities, they also indicated that the viral infection provoked multiple physiological and immune responses in the host scallops (C) 2010 Elsevier Ltd All rights reserved

Immune responses of the scallop Chlamys farreri after air exposure to different temperatures

The present study examined the influence of air exposure at different temperatures: a common perturbation associated with aquaculture handling practices, on immune responses in zhikong scallop Chlamys farreri. Scallops were exposed to air for 2 h, 6 h, 12 h and 24 h at 5 degrees C, 17 degrees C and 25 degrees C respectively. Thereafter, a recovery period of 24 h at 17 degrees C was applied. Haemocyte mortality, phagocytosis and reactive oxygen species (ROS) production of haemocytes, acid phosphatase (ACP) and superoxide dismutase (SOD) activity in haemocyte lysates were chosen as immumomarkers of anoxic stress. The results showed that an increase of haemocyte mortality and a decrease of phagocytosis and ACP activity were observed after 2 h of air exposure for all temperatures tested. Moreover, a significant increase of ROS production occurred following 2 h of air exposure at 25 degrees C and 24 h of air exposure at 17 degrees C. Significant differences were also observed in haemocyte mortality, percentage of phagocytic cells and ACP and SOD activity depending on the temperature of air exposure. Finally, after 24 h of recovery at 17 degrees C, percentage of phagocytic haemocytes and ACP activity did not return to initial values. ROS production was significantly higher than before the recovery period and initial values for scallops subjected to air exposure at 5 degrees C. In our study, scallops showed a relative low anoxia tolerance under a high temperature. All the scallops air exposed to 25 degrees C died after the 6 h sampling. In conclusion, air exposure associated to aquaculture practices was demonstrated to strongly affect functional immune activities of scallop haemocytes, and high temperature air exposure caused reduced survival of scallops. (c) 2007 Elsevier B.V. All rights reserved.