167 research outputs found
Inhibition of a vaccine-induced anti-tumor B cell response by soluble protein antigen in the absence of continuing T cell help
No full textDNA vaccination can elicit the production of anti-tumor antibodies, thus obviating the need to continuously administer passive antibody. This vaccination strategy is particularly important where antibodies have proven to be effective anti-tumor agents. To amplify antibody responses against weak tumor antigens, we previously designed DNA-fusion vaccines incorporating tumor sequences linked to microbial genes. By using a safe idiotypic (Id) antigen from a B cell tumor fused to a fragment C (FrC) sequence from tetanus toxin, we induced both anti-Id and anti-FrC antibodies. It was important to determine whether the antigen itself, either injected or released from residual tumor cells, would boost the antibody response. Id protein not only failed to boost the response, but permanently and rapidly inhibited it by ablating Id-specific memory B cells. In contrast, an Id protein-FrC conjugate boosted both Id-specific and FrC-specific responses. Strikingly, the depletion of CD4+ T cells converted the Id protein-FrC conjugate vaccine into an inhibitor. These findings support the hypothesis that the activation of memory B cells by a DNA vaccine encoding a protein antigen, in the presence of the protein itself, depends completely on T cell help. Furthermore, by using knockout mice, we have shown that inhibition of the Id-specific memory B cells by the Id protein is largely independent of the Fc{gamma}RIIB and, hence, independent of immune complexes. The principles revealed by using a DNA vaccine have implications for all cancer vaccines designed to induce and maintain antibody responses against weak autologous tumor antigens
Failure of vaccination with idiotypic protein or DNA, (+/-IL-2), the depletion of regulatory T cells, or the blockade of CTLA-4 to prolong dormancy in mice with BCL1 lymphoma
No full textImmunization of mice with the idiotype (Id) immunoglobulin from the murine B cell lymphoma, BCL1, before inoculating tumor cells can induce tumor dormancy. In this model, the tumor cells grow for a short period of time and then regress. The mice live for months or years with approximately 1 million tumor cells in their spleens. Some mice relapse due to decreases in the anti-Id antibody titers or the development of mutations in the residual tumor cells which render them refractory to negative signaling by the anti-Id antibody. In this study we determined whether we could eliminate the residual dormant cells by using a DNA vaccine against the Id or by immunomodulation of T-cell subsets in vivo. Our results demonstrate that dormancy can be maintained by further immunizations with either the BCL1 Id protein or DNA vaccine encoding its single-chain Fv fragment. We also found that a cytotoxic T-cell response was not induced by either in vivo administration of vaccine alone or by the vaccine plus interleukin-2. In addition the injection of anti-cytotoxic T-lymphocyte-associate antigen did not prolong dormancy. Finally, the in vivo administration of anti-CD25 to deplete regulatory T cells did not prolong dormancy. Dormancy in this model is dependent primarily upon anti-Id antibodies, our results suggest that other strategies to target residual dormant BCL1 cells are warranted. They also suggest that the elimination of dormant tumor may represent a greater challenge than the elimination of primary tumors
A Novel Platform to Generate Synthetic Vaccine Candidates
No full textVaccination remains the optimal means to prevent infectious disease by inducing antibodies that confer protective immunity against the pathogen in question [1-3]. However, there remain viruses against which no effective vaccines exists including human immunodeficiency virus (HIV), West Nile Virus (WNV) and hepatitis C virus (HCV). These viruses and others evade the immune response by undergoing rapid mutations in immunodominant epitopes [4-6]. In addition, although they usually express conserved epitopes that are important for inducing neutralizing antibodies, in many cases these are not immunodominant. Traditional techniques in vaccine development have not been able to overcome these barriers for these and other viruses. Subunit and peptide vaccines are very safe but it is often difficult to identify the key epitopes needed to make them effective.
New approaches to developing safe vaccines that induce broadly neutralizing antibodies are needed. Therefore, the long term goal of this project was to generate vaccine candidates for any virus for which a neutralizing antibody existed or could be made without prior knowledge of the protective epitope(s). Furthermore, we desired a way to administer these vaccine candidates safely and before exposure so as to induce neutralizing antibodies. To accomplish these goals, we began with the development of a platform to generate synthetic vaccine candidates. This platform consisted of 1) libraries of B cell epitopes or “shapes” prepared by displaying peptoid sequences on beads, 2) neutralizing monoclonal antibodies (MAbs) to select the peptoids that bound to the antibody’s antigen-combining site, and 3) protein G dynabeads (PGDs) and a magnet to bind and isolate antibody bound peptoid beads. Any sequences identified in the platform as potential B cell mimetics were further evaluated in two validation assays. The first consisted of a “color screening” assay to determine that the isolated on-bead peptoids were bound by antibody. The second confirmed that these peptoids would fail to be bound by antibody if an excess of the native antigen was added (i.e. that peptoid sequences were bound by the antibody’s binding sites).
The major accomplishments to emerge from this study were 1) the creation of an optimized magnetic screening platform for the isolation of peptide B cell epitopes from an on-bead library, 2) a magnetic screening platform optimized for the isolation of peptoid B cell epitopes from a peptoid library, and 3) the identification of potential peptoid B cell epitope mimetics of FLAG peptide from a peptoid library using a MAb. Taken together, a sensitive, specific, and reproducible platform to identify vaccine candidates from a peptoid library was created. This platform is particularly important for viruses like HIV, HCV, and WNV where mutation makes foreknowledge of conserved, neutralizing epitopes difficult.
Once sufficiently large and diverse libraries are created, the B cell epitope mimetics (vaccine candidates) identifiable by this platform will have several advantages over their peptide counterparts. These peptoid-based vaccines are “safe” as there is no potential for reversion, they are less expensive and faster to synthesize than peptides, they are not dependent on the twenty amino acids, and the B cell epitopes identified with this platform can be conjugated to carrier in such a way that the multivalency and immunodominance can be controlled making this platform advantageous both to the generation of new vaccine candidates and in reformulating current vaccines
Intradermal Administration of RiVax, a Ricin Vaccine
No full textRicin toxin is a CDC Level B Biothreat due to its extreme toxicity and ease of production. The most effective method for minimizing ricin toxicity in humans is prophylactic vaccination. We have previously described the efficacy and safety of RiVax, a recombinant mutant of ricin A chain (RTA). RiVax has no residual toxicity from either its ribotoxic site or its vascular leak-inducing site. When administered by intramuscular (IM) injection, it was safe and immunogenic in mice, rabbits, and humans. A three dose regimen of IM administered RiVax also protected mice from an LD50dose of ricin delivered by injection, gastric gavage or aerosol. In this study we have attempted to increase the utility and immunogenicity of RiVax. To this end, we have compared intradermal (ID) vs. IM administration of RiVax by evaluating the following parameters of vaccine efficacy: (1) short-term antibody responses and protection of mice from a 10X LD50of ricin following a three dose vaccine regimen; (2) long-term antibody responses and protection of mice from a 10X LD50of ricin following a three dose vaccine regimen; (3) protective effect of a single high dose of RiVax from a 10X LD50dose of ricin; (4) the minimum dose of ricin at which fully vaccinated animals are no longer protected; (5) the rate of antigen trafficking to draining lymph nodes (DLN) following administration of RiVax. In the short term, when RiVax was delivered with alum, very low doses of vaccine administered ID were superior to the same low doses administered IM, with regard to both antibody production and protection against ricin delivered by injection, gavage, or aerosol.
Low doses of ID vaccine were also superior in maintaining lung function in mice exposed to aerosolized ricin. Comparing the same parameters in the long term or after a single dose of RiVax, ID and IM vaccinations were equally effective. Both ID and IM vaccination were also similar in their ability to protect mice from a supra-lethal challenge with injected ricin. One possible explanation for the improved efficacy of low doses of RiVax administered ID was that the vaccine trafficked more effectively to the DLNs. This appeared to be the trend, albeit not a statistically significant one. Given the increased efficacy of low doses of ID vaccine in protecting mice against ricin delivered to the lung and gut, we suggest that it should be considered for testing in humans
Chimeric ANTI-CD19 Monoclonal Antibodies for the Treatment of Precursor B Cell Acute Lymphoblastic Leukemia
No full textThousands of people are diagnosed with B cell malignancies every year, yet the only FDA-approved immunotherapies for them are based on anti-CD20 monoclonal antibodies (MAbs). However, CD20 is not expressed on precursor B cell acute lymphoblastic leukemia (pre-B ALL), and CD20 expression is often lost following anti-CD20 immunotherapy. CD19 is a pan B cell membrane antigen that is restricted to the B cell lineage and expressed on B cell lymphomas and pre-B ALLs. Previous studies have shown that a murine anti-human CD19 MAb, HD37, has efficacy in SCID mice with human B cell tumors. Furthermore, homodimers consisting of two conjugated IgG molecules of HD37 are more effective than monomers at inducing tumor cell death. Yet, their large size prevents effective tumor penetration, and normal Fc effector funtions are often not retained. Murine antibodies are also highly immunogenic. Therefore, the objective of this study was to construct, express, and test the in vitro and in vivo activities of chimeric divalent and tetravalent HD37 MAbs. Both chimeric HD37 MAbs and the murine HD37 MAb were equally effective at mediating antibody dependent cellular cytotoxicity (ADCC) with mouse effector cells. The anti-tumor activities of all three MAbs were identical in SCID mice xenografted with human B cell tumors. However, the chimeric tetravalent MAb has a higher binding affinity and a longer half-life of dissociation than either of the divalent MAbs. Moreover, the chimeric tetravalent MAb mediated ADCC and complement dependent cytotoxicty (CDC) more efficiently than the divalent MAbs when human effector cells and human complement were used. None of the MAbs were cytotoxic to target cells in the absence of effector cells or complement. These data suggest that 1) the HD37 MAbs effectively extend the mean survival time of SCID mice engrafted with human B cell tumors; 2) more than two of the tetravalent HD37 MAb's binding sites are active; and 3) because in vitro results show that the chimeric tetravalent MAb is more effective than the divalent MAbs at mediating ADCC and CDC with human effector cells and complement, the chimeric tetravalent HD37 MAb could be superior to the divalent MAbs in humans
The Anti-Tumor Activity of UV3, an Anti-CD54 Antibody, in SCID Mice Xenografted with a Variety of Human Tumor Cell Lines
No full textUV3, a monoclonal antibody that specifically recognizes human CD54, also known as intercellular adhesion molecule-1 (ICAM-1) was previously developed for the treatment of multiple myeloma. Even at low doses UV3 was highly effective at prolonging the survival of SCID mice with advanced multiple myeloma. Since CD54 is expressed on many different cancer types, we have now investigated the anti-tumor activity of UV3 in several other CD54+ tumors. A panel of 28 human non-Hodgkin's lymphoma, breast, prostate, non-small cell lung, pancreatic, and melanoma tumor cell lines was examined for reactivity with UV3, and 24 were strongly positive. A representative CD54+ cell line from each cancer type was then grown in SCID mice, and UV3 was administered using different dose regimens. UV3 prolonged survival and/or slowed tumor growth in all of the investigated tumor models, although it was not curative. When UV3 or gemcitabine were administered to SCID mice xenografted with non-small cell lung or pancreatic tumor cell lines, UV3 was as effective as the chemotherapy alone. However, the best anti-tumor responses were observed when gemcitabine and UV3 were administered together.
In order to better understand how UV3 mediates its anti-tumor activity, some mechanisms of action were also investigated. Previous studies in multiple myeloma cells indicated that UV3 did not directly inhibit tumor cell growth or cell adhesion and that the Fc portion of UV3 was required for activity in mice. Similarly, in this study, UV3 did not induce cell cycle arrest or apoptosis in any of the tumor cell lines evaluated, and UV3 did mediate Fc effector mechanisms. However, the involvement of both Fc-dependent and Fc-independent mechanisms is suggested by the results, although the specific Fc-independent mechanisms are unknown.
UV3 has already been chimerized (cUV3), and both toxicology studies and clinical trials are in the planning stage to assess the safety and activity of cUV3 in patients with one or more of these tumors
Understanding the Mechanism of Action of Rituximab in the Reversal of Multidrug Resistance in a Non-Hodgkins Lymphoma Cell Line
No full textIt has been previously demonstrated that an anti-CD20 monoclonal antibody (MAb) can reverse MDR in B lymphoma cells in vitro. However, the mechanisms underlying this effect are unknown. A recent study showed that anti-CD20 MAbs could induce rapid redistribution of CD20 into a detergent-insoluble embrane compartment (lipid rafts). By redistributing CD20 into rafts, Rituximab(tm) (RTX) modified their stability and organization. P-glycoprotein (P-gp) is the constituent protein of MDR tumor cells and is responsible for pumping chemotherapeutic agents out of cells. Because ~40% of P-gp is contained in lipid rafts, we hypothesized that when CD20 translocated into lipid rafts, it would displace P-gp. This displacement would lead to the reversal of MDR. To this end, we determined the function of the P-gp pump in the presence or absence of MAbs. In addition, the effects of dose and incubation time with MAbs and chemotherapeutic drugs were determined using Namalwa/MDR1 and three drug-sensitive cells. Finally, the iv distribution of P-gp and CD20 in membranes was monitored after treatment with MAbs by western blot analysis.
We found that RTX inhibited the function of the P-gp pump while other anti-CD20 MAbs had no effect. RTX-mediated growth-inhibition of Namalwa/MDR1 cells was both dose- and time-dependent. Namalwa/MDR1 cells were resistant to doxorubicin and vincristine, but RTX rendered the Namalwa/MDR1 cells as chemosensitive as the parental Namalwa cells. RTX induced the translocation of CD20 into lipid rafts and the translocation of P-gp out of rafts. Our results supported our hypothesis that the ability of RTX to reverse MDR was initiated when CD20 was translocated into lipid rafts. This coincided with the translocation of P-gp out of rafts. When P-gp was no longer present in rafts, it lost activity and MDR was reversed
Examining the Role of Regulatory Lymphocytes in a Mouse Model of BCL1 Tumor Dormancy
No full textCancer dormancy is a clinical state where residual tumor cells persist for long periods but do not cause detectable disease. However, tumor regrowth can occur and is accompanied by resistance to treatment and high mortality rates. The mechanisms that mediate tumor dormancy have been studied using the mouse B cell lymphoma (BCL1) model of tumor dormancy. However, the events that lead to cancer relapse are not known since the tumor microenvironment consists of many cell types that either facilitate or prevent tumor progression or have a dual role depending on the disease stage. Regulatory T (Treg) cells play a key role in maintaining systemic immune tolerance and have been described to promote cancer progression. The objective of this study was to determine the role of Treg cells in preventing BCL1 tumor dormancy by suppressing the anti-tumor immune responses. Surprisingly, we found that the total numbers of Treg cell were highest in mice bearing dormant tumor cells, whereas mice with the highest BCL1 tumor burden had the lowest number of Treg cells in their tumor microenvironment. Moreover, we compared the functional differences between Treg cells isolated from mice bearing dormant tumors and those bearing non-dormant tumors. We found that they equally suppressed T cell subsets from their respective tumor microenvironments. Treg cells have also been shown to suppress B cells. Since the BCL1 tumor cells are malignant B cells, we examined the effects of Treg cells on the tumor cells themselves. We found that Treg cells did not suppress the proliferation nor inhibit IgM secretion by the tumor cells. Interestingly, we then found that the BCL1 tumor cells shared features with regulatory B (Breg) cells. Like Treg cells, Breg cells also induce immune tolerance by suppressing effector T cells. The BCL1 tumor cells homogeneously expressed the characteristic phenotype (CD1dhiCD5+) and cytokine profile (secretion of high levels of IL-10) of the B10 subset of Breg cells. Moreover, the tumor cells directly induced T cell apoptosis through a cell-contact dependent, caspase-3-mediated pathway. Therefore, the adoption of Breg cell characteristics may be another approach that BCL1 tumor cells employ to evade immune responses
Understanding the Mechanism of Action of UV3, an Anti-CD54 Monoclonal Antibody, in the Therapy of Multiple Myeloma
No full textMultiple myeloma is a hematopoietic malignancy involving the uncontrolled proliferation of a single clone of plasma cells or plasma cell progenitors in the bone marrow. Previously, a monoclonal antibody called UV3, which recognizes human CD54/ICAM-1, was developed for the therapy of multiple myeloma. UV3 is highly effective at treating advanced multiple myeloma in SCID mice with human multiple myeloma xenografts. UV3 does not inhibit homotypic tumor cell adhesion or their adhesion to the bone marrow. UV3 does not induce apoptosis of tumor cells or block cell growth. Previous work evaluating F(ab)'2 fragments of UV3 demonstrated that they were effective in mediating anti-tumor activity, suggesting that other mechanisms also contributed to the anti-tumor activity of UV3.
One possibility to explain how UV3 exerts its anti-tumor activity could be that UV3 inhibits the secretion of pro-angiogenic cytokines and molecules, resulting in an inhibition of angiogenesis. To this end, our goal was to evaluate the angiogenic signals from human multiple myeloma cells and determine whether UV3 would interfere with such signals. In addition, we further examined the role of the Fc portion of UV3 in mediating anti-tumor activity. We found that multiple myeloma cell lines secrete some pro-angiogenic cytokines and molecules, and although UV3 may induce a minor anti-angiogenic effect, the Fc portion of UV3 was critical for its anti-tumor activity. In addition, we found that UV3 prolonged the survival of SCID mice with Daudi lymphoma, which suggests UV3 may be effective in treating a variety of hematological malignancies
On the application of support vector machines to the prediction of propagation losses at 169 MHz for smart metering applications
No full textRecently, the need of deploying new wireless networks for smart gas metering has raised the problem of radio
planning in the 169 MHz band. Unluckily, software tools commonly adopted for radio planning in cellular communication systems cannot be employed to solve this problem because of the substantially lower transmission frequencies characterising this application. In this study, a novel data-centric solution, based on the use of support vector machine techniques for classification and regression, is illustrated. The proposed method requires the availability of a limited set of received signal strength measurements and the knowledge of a three-dimensional map of the propagation environment of interest and generates both an estimate of the coverage area and a prediction of the field strength within it. Various numerical results show that the proposed method is able to achieve good accuracy at the price of an acceptable computational cost and of a limited effort for the acquisition of measurements in the considered environments
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