Pirouz Daftarian

Circulating urokinase receptor as a cause of focal segmental glomerulosclerosis

Focal segmental glomerulosclerosis (FSGS) is a cause of proteinuric kidney disease, compromising both native and transplanted kidneys. Treatment is limited because of a complex pathogenesis, including unknown serum factors. Here we report that serum soluble urokinase receptor (suPAR) is elevated in two-thirds of subjects with primary FSGS, but not in people with other glomerular diseases. We further find that a higher concentration of suPAR before transplantation underlies an increased risk for recurrence of FSGS after transplantation. Using three mouse models, we explore the effects of suPAR on kidney function and morphology. We show that circulating suPAR activates podocyte β3 integrin in both native and grafted kidneys, causing foot process effacement, proteinuria and FSGS-like glomerulopathy. Our findings suggest that the renal disease only develops when suPAR sufficiently activates podocyte β3 integrin. Thus, the disease can be abrogated by lowering serum suPAR concentrations through plasmapheresis, or by interfering with the suPAR–β3 integrin interaction through antibodies and small molecules targeting either uPAR or β3 integrin. Our study identifies serum suPAR as a circulating factor that may cause FSGS.

Recombinant Modified Vaccinia Virus Ankara Expressing a Soluble Form of Glycoprotein B Causes Durable Immunity and Neutralizing Antibodies against Multiple Strains of Human Cytomegalovirus

ABSTRACT Human cytomegalovirus (CMV) is a viral pathogen that infects both genders, who remain asymptomatic unless they receive immunosuppressive drugs or acquire infections that cause reactivation of latent virus. CMV infection also causes serious birth defects following primary maternal infection during gestation. A safe and effective vaccine to limit disease in this population continues to be elusive. A well-studied antigen is glycoprotein B (gB), which is the principal target of neutralizing antibodies (NAb) towards CMV in humans and has been implicated as the viral partner in the receptor-mediated infection by CMV in a variety of cell types. Antibody-mediated virus neutralization has been proposed as a mechanism by which host immunity could modify primary infection. Towards this goal, an attenuated poxvirus, modified vaccinia virus Ankara (MVA), has been constructed to express soluble CMV gB (gB680-MVA) to induce CMV NAb. Very high levels of gB-specific CMV NAb were produced after two doses of the viral vaccine. NAb were durable within a twofold range for up to 6 months. Neutralization titers developed in immunized mice are equivalent to titers found clinically after natural infection. This viral vaccine, expressing gB derived from CMV strain AD169, induced antibodies that neutralized CMV strains of three different genotypes. Remarkably, preexisting MVA and vaccinia virus (poxvirus) immunity did not interfere with subsequent immunizations of gB680-MVA. The safety characteristics of MVA, combined with the robust immune response to CMV gB, suggest that this approach could be rapidly translated into the clinic.

Peptide-Conjugated PAMAM Dendrimer as a Universal DNA Vaccine Platform to Target Antigen-Presenting Cells

DNA-based vaccines hold promise to outperform conventional antigen-based vaccines by virtue of many unique features. However, DNA vaccines have thus far fallen short of expectations, due in part to poor targeting of professional antigen-presenting cells (APC) and low immunogenicity. In this study, we describe a new platform for effective and selective delivery of DNA to APCs in vivo that offers intrinsic immune-enhancing characteristics. This platform is based on conjugation of fifth generation polyamidoamine (G5-PAMAM) dendrimers, a DNA-loading surface, with MHC class II-targeting peptides that can selectively deliver these dendrimers to APCs under conditions that enhance their immune stimulatory potency. DNA conjugated with this platform efficiently transfected murine and human APCs in vitro. Subcutaneous administration of DNA-peptide-dendrimer complexes in vivo preferentially transfected dendritic cells (DC) in the draining lymph nodes, promoted generation of high affinity T cells, and elicited rejection of established tumors. Taken together, our findings show how PAMAM dendrimer complexes can be used for high transfection efficiency and effective targeting of APCs in vivo, conferring properties essential to generate effective DNA vaccines.

Therapy of established B16-F10 melanoma tumors by a single vaccination of CTL/T helper peptides in VacciMax®

BackgroundMelanoma tumors are known to express antigens that usually induce weak immune responses of short duration. Expression of both tumor-associated antigens p53 and TRP2 by melanoma cells raises the possibility of simultaneously targeting more than one antigen in a therapeutic vaccine. In this report, we show that VacciMax® (VM), a novel liposome-based vaccine delivery platform, can increase the immunogenicity of melanoma associated antigens, resulting in tumor elimination.MethodsC57BL/6 mice bearing B16-F10 melanoma tumors were vaccinated subcutaneously 6 days post tumor implantation with a mixture of synthetic peptides (modified p53: 232–240, TRP-2: 181–188 and PADRE) and CpG. Tumor growth was monitored and antigen-specific splenocyte responses were assayed by ELISPOT.ResultsVaccine formulated in VM increased the number of both TRP2- and p53-specific IFN-γ producing splenocytes following a single vaccination. Vaccine formulated without VM resulted only in enhanced IFN-γ producing splenocytes to one CTL epitopes (TRP2:180–188), suggesting that VM overcomes antigen dominance and enhances immunogenicity of multiple epitopes. Vaccination of mice bearing 6-day old B16-F10 tumors with both TRP2 and p53-peptides formulated in VM successfully eradicated tumors in all mice. A control vaccine which contained all ingredients except liposomes resulted in eradication of tumors in no more than 20% of mice.ConclusionA single administration of VM is capable of inducing an effective CTL response to multiple tumor-associated antigens. The responses generated were able to reject 6-day old B16-F10 tumors.

Two Distinct Pathways of Immuno-Modulation Improve Potency of p53 Immunization in Rejecting Established Tumors

The p53 gene product is overexpressed by almost 50% of cancers, making it an ideal target for cancer immunotherapy. We previously demonstrated rejection of established p53-overexpressing tumors without stimulating autoimmunity by immunization with modified vaccinia Ankara-expressing murine p53 (MVAp53). Tumor rejection was enhanced through antibody-mediated CTL-associated antigen 4 (CTLA-4) blockade. We examined the role of synthetic oligodeoxynucleotides (ODN) containing unmethylated cytosine-phosphate-guanine (CpG) motifs (CpG ODN) in enhancing MVAp53-mediated tumor rejection. CpG ODN with MVAp53 resulted in tumor rejection in BALB/c mice bearing poorly immunogenic 11A-1 murine mammary carcinomas or Meth A sarcomas and C57Bl/6 mice bearing MC-38 colon carcinomas. The effect was similar to that seen in tumor-bearing mice treated with MVAp53 along with CTLA-4 blockade. Monoclonal antibody depletion experiments demonstrated that the adjuvant effects of CpG ODN and CTLA-4 blockades were CD8 dependent. CpG ODN were partially natural killer cell dependent and ineffective in Toll-like Receptor 9−/− and interleukin 6−/− mice, whereas CTLA-4 blockade was partially CD4 dependent and functional in Toll-like Receptor 9−/− and interleukin 6−/− mice. In addition, when administered with MVAp53, both adjuvants enhanced p53-specific cytotoxicity and demonstrated an additive effect when combined. The combination of CpG ODN and CTLA-4 blockade worked synergistically to reject palpable 11A-1 and MC-38 tumors. These experiments demonstrate the potential for augmenting MVAp53-mediated antitumor immunity using CpG ODN and CTLA-4 blockade. This cell-free immunotherapy approach is a candidate for evaluation in cancer patients.

Rejection of large HPV-16 expressing tumors in aged mice by a single immunization of VacciMax® encapsulated CTL/T helper peptides

The incidence of cancer increases significantly in later life, yet few pre-clinical studies of cancer immunotherapy use mice of advanced age. A novel vaccine delivery platform (VacciMax®,VM) is described that encapsulates antigens and adjuvants in multilamellar liposomes in a water-in-oil emulsion. The therapeutic potential of VM-based vaccines administered as a single dose was tested in HLA-A2 transgenic mice of advanced age (48–58 weeks old) bearing large palpable TC1/A2 tumors. The VM-based vaccines contained one or more peptides having human CTL epitopes derived from HPV 16 E6 and E7. VM formulations contained a single peptide, a mixture of four peptides or the same four peptides linked together in a single long peptide. All VM formulations contained PADRE and CpG as adjuvants and ISA51 as the hydrophobic component of the water-in-oil emulsion. VM-formulated vaccines containing the four peptides as a mixture or linked together in one long peptide eradicated 19-day old established tumors within 21 days of immunization. Peptide-specific cytotoxic cellular responses were confirmed by ELISPOT and intracellular staining for IFN-γ producing CD8+ T cells. Mice rendered tumor-free by vaccination were re-challenged in the opposite flank with 10 million HLF-16 tumor cells, another HLA-A2/E6/E7 expressing tumor cell line. None of these mice developed tumors following the re-challenge. In summary, this report describes a VM-formulated therapeutic vaccine with the following unprecedented outcome: a) eradication of large tumors (> 700 mm3) b) in mice of advanced age c) in less than three weeks post-immunization d) following a single vaccination.

Immunization with Th-CTL Fusion Peptide and Cytosine-Phosphate-Guanine DNA in Transgenic HLA-A2 Mice Induces Recognition of HIV-Infected T Cells and Clears Vaccinia Virus Challenge

We evaluated immunogenicity of a novel Th-CTL fusion peptide composed of the pan DR Th epitope and a CTL epitope derived from HIV-pol in two transgenic HLA-A*0201/Kb mouse models. The immunogenicity of peptides of this structure is highly dependent on coadministered cytosine-phosphate-guanine DNA. Initial evaluations of peptide-specific immunity are based on results of chromium release assay, intracellular cytokine, and tetramer staining. Significant cytotoxic T cell responses are found upon a single immunization with as low as 0.1 nmol both peptide and cytosine-phosphate-guanine DNA. Splenocytes from immunized mice recognize naturally processed HIV-pol expressed from vaccinia virus (pol-VV). Translation of immunologic criteria into more relevant assays was pursued using systemic challenge of immunized mice with pol-VV. Only mice receiving both peptide and DNA together successfully cleared upward of 6 logs of virus from ovaries, compared with controls. Challenge with pol-VV by intranasal route of intranasal immunized mice showed a significant reduction in the levels of VV in lung compared with naive mice. A convincing demonstration of the relevance of these vaccines is the robust lysis of HIV-infected Jurkat T cells (JA2/R7/Hyg) by immune splenocytes from peptide- and DNA-immunized mice. This surprisingly effective immunization merits consideration for clinical evaluation, because it succeeded in causing immune recognition and lysis of cells infected with its target virus and reduction in titer of highly pathogenic VV.

A Targeted and Adjuvanted Nanocarrier Lowers the Effective Dose of Liposomal Amphotericin B and Enhances Adaptive Immunity in Murine Cutaneous Leishmaniasis

BACKGROUND Amphotericin B (AmB), the most effective drug against leishmaniasis, has serious toxicity. As Leishmania species are obligate intracellular parasites of antigen presenting cells (APC), an immunopotentiating APC-specific AmB nanocarrier would be ideally suited to reduce the drug dosage and regimen requirements in leishmaniasis treatment. Here, we report a nanocarrier that results in effective treatment shortening of cutaneous leishmaniasis in a mouse model, while also enhancing L. major specific T-cell immune responses in the infected host. METHODS We used a Pan-DR-binding epitope (PADRE)-derivatized-dendrimer (PDD), complexed with liposomal amphotericin B (LAmB) in an L. major mouse model and analyzed the therapeutic efficacy of low-dose PDD/LAmB vs full dose LAmB. RESULTS PDD was shown to escort LAmB to APCs in vivo, enhanced the drug efficacy by 83% and drug APC targeting by 10-fold and significantly reduced parasite burden and toxicity. Fortuitously, the PDD immunopotentiating effect significantly enhanced parasite-specific T-cell responses in immunocompetent infected mice. CONCLUSIONS PDD reduced the effective dose and toxicity of LAmB and resulted in elicitation of strong parasite specific T-cell responses. A reduced effective therapeutic dose was achieved by selective LAmB delivery to APC, bypassing bystander cells, reducing toxicity and inducing antiparasite immunity.

Red-Shifted Aequorin Variants Incorporating Non-Canonical Amino Acids: Applications in In Vivo Imaging

The increased importance of in vivo diagnostics has posed new demands for imaging technologies. In that regard, there is a need for imaging molecules capable of expanding the applications of current state-of-the-art imaging in vivo diagnostics. To that end, there is a desire for new reporter molecules capable of providing strong signals, are non-toxic, and can be tailored to diagnose or monitor the progression of a number of diseases. Aequorin is a non-toxic photoprotein that can be used as a sensitive marker for bioluminescence in vivo imaging. The sensitivity of aequorin is due to the fact that bioluminescence is a rare phenomenon in nature and, therefore, it does not suffer from autofluorescence, which contributes to background emission. Emission of bioluminescence in the blue-region of the spectrum by aequorin only occurs when calcium, and its luciferin coelenterazine, are bound to the protein and trigger a biochemical reaction that results in light generation. It is this reaction that endows aequorin with unique characteristics, making it ideally suited for a number of applications in bioanalysis and imaging. Herein we report the site-specific incorporation of non-canonical or non-natural amino acids and several coelenterazine analogues, resulting in a catalog of 72 cysteine-free, aequorin variants which expand the potential applications of these photoproteins by providing several red-shifted mutants better suited to use in vivo. In vivo studies in mouse models using the transparent tissue of the eye confirmed the activity of the aequorin variants incorporating L-4-iodophehylalanine and L-4-methoxyphenylalanine after injection into the eye and topical addition of coelenterazine. The signal also remained localized within the eye. This is the first time that aequorin variants incorporating non-canonical amino acids have shown to be active in vivo and useful as reporters in bioluminescence imaging.

Serotonin Activates Bacterial Quorum Sensing and Enhances the Virulence of Pseudomonas aeruginosa in the Host.

Bacteria in humans play an important role in health and disease. Considerable emphasis has been placed in understanding the role of bacteria in host-microbiome interkingdom communication. Here we show that serotonin, responsible for mood in the brain and motility in the gut, can also act as a bacterial signaling molecule for pathogenic bacteria. Specifically, we found that serotonin acts as an interkingdom signaling molecule via quorum sensing and that it stimulates the production of bacterial virulence factors and increases biofilm formation in vitro and in vivo in a novel mouse infection model. This discovery points out at roles of serotonin both in bacteria and humans, and at phenotypic implications not only manifested in mood behavior but also in infection processes in the host. Thus, regulating serotonin concentrations in the gut may provide with paradigm shifting therapeutic approaches.