Immunization with HIV protease peptides linked to syngeneic erythrocytes

New potent vaccine adjuvants are desirable for increasing the efficacy of novel vaccine modalities such as DNA and peptides. We therefore tested if syngeneic erythrocytes could serve as delivery vectors for selected HIV peptides and compared the potency of these constructs to immunization with peptides in phosphate buffered saline or in incomplete Freunds adjuvant. Immunization of mice with peptides in a low dose (5 ng) coupled to erythrocytes induced a weak immune response in mice. These peptides alone (5 μg) gave no immune responses, while formulating the peptides (50 μg) in IFA induced strong homologous immunity as well as prominent cross reactivity to a related mutant epitope. Thus, vaccine delivery using syngeneic erythrocytes, although attractive for clinical use, might be of limited value due to the low amount of antigen that can be loaded per erythrocyte

Biology of advanced uveal melanoma and next steps for clinical therapeutics.

Uveal melanoma is the most common intraocular malignancy although it is a rare subset of all melanomas. Uveal melanoma has distinct biology relative to cutaneous melanoma, with widely divergent patient outcomes. Patients diagnosed with a primary uveal melanoma can be stratified for risk of metastasis by cytogenetics or gene expression profiling, with approximately half of patients developing metastatic disease, predominately hepatic in location, over a 15-yr period. Historically, no systemic therapy has been associated with a clear clinical benefit for patients with advanced disease, and median survival remains poor. Here, as a joint effort between the Melanoma Research Foundation\u27s ocular melanoma initiative, CURE OM and the National Cancer Institute, the current understanding of the molecular and immunobiology of uveal melanoma is reviewed, and on-going laboratory research into the disease is highlighted. Finally, recent investigations relevant to clinical management via targeted and immunotherapies are reviewed, and next steps in the development of clinical therapeutics are discussed

Distinct signatures of the immune responses in low risk versus high risk neuroblastoma

<p>Abstract</p> <p>Background</p> <p>Over 90% of low risk (LR) neuroblastoma patients survive whereas less than 30% of high risk (HR) patients are long term survivors. Age (children younger than 18 months old) is associated with LR disease. Considering that adaptive immune system is well developed in older children, and that T cells were shown to be involved in tumor escape and progression of cancers, we sought to determine whether HR patients may tend to show a signature of adaptive immune responses compared to LR patients who tend to have diminished T-cell responses but an intact innate immune response.</p> <p>Methods</p> <p>We performed microarray analysis of RNA extracted from the tumor specimens of HR and LR patients. Flow cytometry was performed to determine the cellular constituents in the blood while multiplex cytokine array was used to detect the cytokine profile in patients' sera. A HR tumor cell line, SK-N-SH, was also used for detecting the response to IL-1β, a cytokines which is involved in the innate immune responses.</p> <p>Results</p> <p>Distinct patterns of gene expression were detected in HR and LR patients indicating an active T-cell response and a diminished adaptive immune response, respectively. A diminished adaptive immune response in LR patients was evident by higher levels of IL-10 in the sera. In addition, HR patients had lower levels of circulating myeloid derived suppressor cells (MDSC) compared with a control LR patient. LR patients showed slightly higher levels of cytokines of the innate immune responses. Treatment of the HR tumor line with IL-1β induced expression of cytokines of the innate immune responses.</p> <p>Conclusions</p> <p>This data suggests that adaptive immune responses may play an important role in the progression of HR disease whereas innate immune responses may be active in LR patients.</p

Mycobacteria activate γδ T-cell anti-tumour responses via cytokines from type 1 myeloid dendritic cells: a mechanism of action for cancer immunotherapy

Attenuated and heat-killed mycobacteria display demonstrable activity against cancer in the clinic; however, the induced immune response is poorly characterised and potential biomarkers of response ill-defined. We investigated whether three mycobacterial preparations currently used in the clinic (BCG and heat-killed Mycobacterium vaccae and Mycobacterium obuense) can stimulate anti-tumour effector responses in human γδ T-cells. γδ T-cell responses were characterised by measuring cytokine production, expression of granzyme B and cytotoxicity against tumour target cells. Results show that γδ T-cells are activated by these mycobacterial preparations, as indicated by upregulation of activation marker expression and proliferation. Activated γδ T-cells display enhanced effector responses, as shown by upregulated granzyme B expression, production of the TH1 cytokines IFN-γ and TNF-α, and enhanced degranulation in response to susceptible and zoledronic acid-treated resistant tumour cells. Moreover, γδ T-cell activation is induced by IL-12, IL-1β and TNF-α from circulating type 1 myeloid dendritic cells (DCs), but not from type 2 myeloid DCs or plasmacytoid DCs. Taken together, we show that BCG, M. vaccae and M. obuense induce γδ T-cell anti-tumour effector responses indirectly via a specific subset of circulating DCs and suggest a mechanism for the potential immunotherapeutic effects of BCG, M. vaccae and M. obuense in cancer

Immunization of mice with the nef gene from Human Immunodeficiency Virus type 1: Study of immunological memory and long-term toxicology

<p>Abstract</p> <p>Background</p> <p>The human immunodeficiency virus type 1 (HIV-1) regulatory protein, Nef, is an attractive vaccine target because it is involved in viral pathogenesis, is expressed early in the viral life cycle and harbors many T and B cell epitopes. Several clinical trials include gene-based vaccines encoding this protein. However, Nef has been shown to transform certain cell types <it>in vitro</it>. Based on these findings we performed a long-term toxicity and immunogenicity study of Nef, encoded either by Modified Vaccinia virus Ankara or by plasmid DNA. BALB/c mice were primed twice with either DNA or MVA encoding Nef and received a homologous or heterologous boost ten months later. In the meantime, the Nef-specific immune responses were monitored and at the time of sacrifice an extensive toxicological evaluation was performed, where presence of tumors and other pathological changes were assessed.</p> <p>Results</p> <p>The toxicological evaluation showed that immunization with MVAnef is safe and does not cause cellular transformation or other toxicity in somatic organs.</p> <p>Both DNAnef and MVAnef immunized animals developed potent Nef-specific cellular responses that declined to undetectable levels over time, and could readily be boosted after almost one year. This is of particular interest since it shows that plasmid DNA vaccine can also be used as a potent late booster of primed immune responses. We observed qualitative differences between the T cell responses induced by the two different vectors: DNA-encoded nef induced long-lasting CD8⁺T cell memory responses, whereas MVA-encoded nef induced CD4⁺T cell memory responses. In terms of the humoral immune responses, we show that two injections of MVAnef induce significant anti-Nef titers, while repeated injections of DNAnef do not. A single boost with MVAnef could enhance the antibody response following DNAnef prime to the same level as that observed in animals immunized repeatedly with MVAnef. We also demonstrate the possibility to boost HIV-1 Nef-specific immune responses using the MVAnef construct despite the presence of potent anti-vector immunity.</p> <p>Conclusion</p> <p>This study shows that the nef gene vectored by MVA does not induce malignancies or other adverse effects in mice. Further, we show that when the nef gene is delivered by plasmid or by a viral vector, it elicits potent and long-lasting immune responses and that these responses can be directed towards a CD4⁺or a CD8⁺T cell response depending on the choice of vector.</p

Proteasomal protein degradation: adaptation of cellular proteolysis with impact on virus-and cytokine-mediated damage of heart tissue during myocarditis

Viral myocarditis is an inflammation of the heart muscle triggered by direct virus-induced cytolysis and immune response mechanisms with most severe consequences during early childhood. Acute and long-term manifestation of damaged heart tissue and disturbances of cardiac performance involve virus-triggered adverse activation of the immune response and both immunopathology, as well as, autoimmunity account for such immune-destructive processes. It is a matter of ongoing debate to what extent subclinical virus infection contributes to the debilitating sequela of the acute disease. In this review, we conceptualize the many functions of the proteasome in viral myocarditis and discuss the adaptation of this multi-catalytic protease complex together with its implications on the course of disease. Inhibition of proteasome function is already highly relevant as a strategy in treating various malignancies. However, cardiotoxicity and immune-related adverse effects have proven significant hurdles, representative of the target's wide-ranging functions. Thus, we further discuss the molecular details of proteasome-mediated activity of the immune response for virus-mediated inflammatory heart disease. We summarize how the spatiotemporal flexibility of the proteasome might be tackled for therapeutic purposes aiming to mitigate virus-mediated adverse activation of the immune response in the heart

HIV-1 Env associates with HLA-C free-chains at the cell membrane modulating viral infectivity

HLA-C has been demonstrated to associate with HIV-1 envelope glycoprotein (Env). Virions lacking HLA-C have reduced infectivity and increased susceptibility to neutralizing antibodies. Like all others MHC-I molecules, HLA-C requires \u3b22-microglobulin (\u3b22m) for appropriate folding and expression on the cell membrane but this association is weaker, thus generating HLA-C free-chains on the cell surface. In this study, we deepen the understanding of HLA-C and Env association by showing that HIV-1 specifically increases the amount of HLA-C free chains, not bound to \u3b22m, on the membrane of infected cells. The association between Env and HLA-C takes place at the cell membrane requiring \u3b22m to occur. We report that the enhanced infectivity conferred to HIV-1 by HLA-C specifically involves HLA-C free chain molecules that have been correctly assembled with \u3b22m. HIV-1 Env-pseudotyped viruses produced in the absence of \u3b22m are less infectious than those produced in the presence of \u3b22m. We hypothesize that the conformation and surface expression of HLA-C molecules could be a discriminant for the association with Env. Binding stability to \u3b22m may confer to HLA-C the ability to preferentially act either as a conventional immune-competent molecule or as an accessory molecule involved in HIV-1 infectivity

Modulation of tumor sensitivity to effector mechanisms of cytotoxic lymphocytes

Today, ample evidence demonstrates a clear role for the immune system in the battle against cancer. However, the relatively high rate of mutation and proliferation of tumor cells, in combination with the selective pressure exerted by the immune system, can potentially lead to the generation of genetically altered tumor cells, which are able to evade recognition by the immune system and continue to grow and form tumors. Increased knowledge of the mechanisms allowing tumors to escape from the immune system is of great importance in facilitating the design of effective immunotherapeutic regimens against cancer. The work described in this thesis was aimed at identifying new mechanisms of tumor escape as well as possible ways to counteract them. We have identified TNF-alpha as a potent modulator of MHC class I antigen presentation in tumors. TNF-alpha-treatment led to enhanced expression of several molecules in the MHC class I antigen processing and presentation pathway, including the IFN-inducible subunits of the proteasome, LMP2, LMP7 and MECL-1, the transporters associated with antigen presentation (TAP) and MHC class I heavy chain. These changes resulted in increased stability of surface MHC class I complexes, presumably due to an increased supply of peptides suitable for binding to MHC class I molecules, and enhanced susceptibility of TNF-alpha-treated tumors to antigen-specific lysis by cytotoxic T-lymphocytes (CTLs). Our results suggest a role for TNF-alpha as a potent immunomodulator in IFN-gamma unresponsive tumors. Investigating the possible effects of cytokines on the sensitivity of tumor cells to different CTL effector mechanisms, we found that IFN-gamma protects uveal melanoma cells from CTL-mediated lysis. We also demonstrated that despite potent upregulation of antigen presentation in uveal melanoma cells, IFN-gamma-treated tumor cells were less sensitive to lysis by CTL. Granzyme B is an apoptosis-inducing effector molecule released by CTLs upon triggering of the T-cell receptor. IFNgamma-treated uveal melanoma cells bound less granzyme B than their untreated, or TNF-alpha-treated, counterparts. Cleavage of the granzyme B substrate Bid was reduced in uveal melanoma cells following treatment with IFN-gamma. This correlated with a reduced expression of the cationindependent mannose-6-phosphate receptor (CI-MPR), a receptor for granzyme B, and decreased CTL-lysis of IFN-treated uveal melanoma cells. In another study, we examined the regulatory role of IFN-gamma on the sensitivity of uveal melanoma cells to the lytic activity of perforin, another major constituent of cytolytic granules. We demonstrated that IFN-gamma induces resistance of uveal melanoma cells to plasma membrane lysis by perforin. This was not a result of proteolytic inactivation of perform by either cathepsin B, known to protect CTL from perforin-mediated suicide, or other proteases. Protection from perforin lysis correlated with IFN-gamma-induced growth arrest in the G1-phase of the cell cycle, and reduced binding of perform to IFN-gamma-treated OCM1 cells. In light of the current data, we propose a mechanism were IFN-gamma-induced growth arrest leading to structural changes in the plasma membrane results in decreased perforin binding capacity of the tumor cell and protection from perforin. Our results demonstrate that, in response to IFN-gamma, tumors can escape the immune system through the active acquisition of a CTL-resistant phenotype, characterized by impaired sensitivity to granule-mediated killing. The second major effector mechanism employed by CTL is the engagement of death receptors expressed on target cells. The production of soluble Fas ligand (sFasL) completely protected uveal melanoma cells from killing via Fas. Inhibition of metalloproteases on the surface of tumor cells prevented shedding of Fast, and rendered uveal melanoma cells sensitive to Fasmeditated lysis by CTL. The protective effect of Fast, was not due to tumor counter-attack or reduced lytic potential of CTL, but transfer of sFasL-containing culture supernatant protected normally Fas-sensitive cells from killing induced both by FasLexpressing lymphocytes and a agonistic antibody to Fas. We speculated that soluble Fast, bind to Fas receptors expressed on tumor cells, thereby preventing their activation by Fas-inducing effector molecules. Our findings demonstrate the existence of a novel mechanism of tumor escape from death receptor-mediated killing by cytotoxic lymphocytes, and point to a new rationale for the use of metalloprotease inhibitors as cancer therapeutic agents