Sagar Kudchodkar

Coimmunization with an Optimized IL-15 Plasmid Results in Enhanced Function and Longevity of CD8 T Cells That Are Partially Independent of CD4 T Cell Help

DNA vaccines are a promising technology for the induction of Ag-specific immune responses, and much recent attention has gone into improving their immune potency. In this study we test the feasibility of delivering a plasmid encoding IL-15 as a DNA vaccine adjuvant for the induction of improved Ag-specific CD8+ T cellular immune responses. Because native IL-15 is poorly expressed, we used PCR-based strategies to develop an optimized construct that expresses 80-fold higher than the native IL-15 construct. Using a DNA vaccination model, we determined that immunization with optimized IL-15 in combination with HIV-1gag DNA constructs resulted in a significant enhancement of Ag-specific CD8+ T cell proliferation and IFN-γ secretion, and strong induction of long-lived CD8+ T cell responses. In an influenza DNA vaccine model, coimmunization with plasmid expressing influenza A PR8/34 hemagglutinin with the optimized IL-15 plasmid generated improved long term CD8+ T cellular immunity and protected the mice against a lethal mucosal challenge with influenza virus. Because we observed that IL-15 appeared to mostly adjuvant CD8+ T cell function, we show that in the partial, but not total, absence of CD4+ T cell help, plasmid-delivered IL-15 could restore CD8 secondary immune responses to an antigenic DNA plasmid, supporting the idea that the effects of IL-15 on CD8+ T cell expansion require the presence of low levels of CD4 T cells. These data suggest a role for enhanced plasmid IL-15 as a candidate adjuvant for vaccine or immunotherapeutic studies.

Immunogenicity of a novel DNA vaccine cassette expressing multiple human immunodeficiency virus (HIV-1) accessory genes.

ObjectiveTo develop an HIV-1 accessory gene immunogen using a DNA vaccine approach. MethodsHIV-1 accessory genes vif, vpu and nef were modified to express under the control of a single promoter with cellular proteolytic cleavage sites between the coding sequences (VVN-P). Immune responses induced by these constructs were evaluated in mice. ResultsPDNA vaccine construct (pVVN-P) expressing Vif, Vpu and Nef was processed and the fusion protein was cleaved appropriately. Vif, Vpu and Nef as a fusion protein with proteolytic cleavage sites (VVN-P) is able to induce a significant level of cellular immune responses. We also observed that accessory genes Vif, Vpu and Nef (VVN-P) induced an effective T helper 1 proliferative response measured by cytokine production. Furthermore, expression cassette pVVN-P was able to induce cytotoxic T lymphocyte (CTL) responses against diverse HIV-1 viruses in infected target cells. ConclusionWe conclude that cell-mediated immune responses induced by accessory gene constructs from clade B may have a broader recognition of divergent HIV-1 viruses and should be further examined for both prophylactic and therapeutic vaccination schemes against HIV-1.

Induction of Potent Th1-Type Immune Responses from a Novel DNA Vaccine for West Nile Virus New York Isolate (WNV-NY1999)

West Nile virus (WNV) is a vectorborne pathogen that induces brain inflammation and death. Recently, confirmed cases of infection and deaths have occurred in the United States Mid-Atlantic region. In this study, a DNA vaccine encoding the WNV capsid protein was constructed, and the in vivo immune responses generated were investigated in DNA vaccine-immunized mice. Antigen-specific humoral and cellular immune responses were observed, including a potent induction of antigen-specific Th1 and cytotoxic T lymphocyte responses. Strong induction of Th1-type immune responses included high levels of antigen-specific elaboration of the Th1-type cytokines interferon-gamma and interleukin-2 and beta-chemokines RANTES (regulated upon activation, normal T cell-expressed and secreted) and macrophage inflammatory protein-1beta. Dramatic infiltration of CD4 and CD8 T cells and macrophages also was observed at the muscle injection site. These results support the potential utility of this method as a tool for developing immunization strategies for WNV and other emerging pathogens.

Ibrutinib enhances chimeric antigen receptor T-cell engraftment and efficacy in leukemia

Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy is highly promising but requires robust T-cell expansion and engraftment. A T-cell defect in chronic lymphocytic leukemia (CLL) due to disease and/or therapy impairs ex vivo expansion and response to CAR T cells. To evaluate the effect of ibrutinib treatment on the T-cell compartment in CLL as it relates to CAR T-cell generation, we examined the phenotype and function of T cells in a cohort of CLL patients during their course of treatment with ibrutinib. We found that ≥5 cycles of ibrutinib therapy improved the expansion of CD19-directed CAR T cells (CTL019), in association with decreased expression of the immunosuppressive molecule programmed cell death 1 on T cells and of CD200 on B-CLL cells. In support of these findings, we observed that 3 CLL patients who had been treated with ibrutinib for ≥1 year at the time of T-cell collection had improved ex vivo and in vivo CTL019 expansion, which correlated positively together and with clinical response. Lastly, we show that ibrutinib exposure does not impair CAR T-cell function in vitro but does improve CAR T-cell engraftment, tumor clearance, and survival in human xenograft models of resistant acute lymphocytic leukemia and CLL when administered concurrently. Our collective findings indicate that ibrutinib enhances CAR T-cell function and suggest that clinical trials with combination therapy are warranted. Our studies demonstrate that improved T-cell function may also contribute to the efficacy of ibrutinib in CLL. These trials were registered at www.clinicaltrials.gov as #NCT01747486, #NCT01105247, and #NCT01217749.

Synthetic DNA vaccines: improved vaccine potency by electroporation and co-delivered genetic adjuvants

In recent years, DNA vaccines have undergone a number of technological advancements that have incited renewed interest and heightened promise in the field. Two such improvements are the use of genetically engineered cytokine adjuvants and plasmid delivery via in vivo electroporation (EP), the latter of which has been shown to increase antigen delivery by nearly 1000-fold compared to naked DNA plasmid delivery alone. Both strategies, either separately or in combination, have been shown to augment cellular and humoral immune responses in not only mice, but also in large animal models. These promising results, coupled with recent clinical trials that have shown enhanced immune responses in humans, highlight the bright prospects for DNA vaccines to address many human diseases.

Duration and specificity of humoral immune responses in mice vaccinated with the Alzheimer's disease-associated β-amyloid 1-42 peptide

Alzheimers disease (AD) is a neurodegenerative disorder characterized by overproduction of beta-amyloid (Abeta), which is formed from amyloid precursor protein (APP), with the subsequent pathologic deposition of Abeta in regions of the brain important for memory and cognition. Recently, vaccination of murine models of AD that exhibit Abeta deposition has halted or delayed the usual progression of the pathology of AD. Our group has demonstrated that vaccination of a doubly transgenic mouse model (expressing mutant APP and presenilin-1) with the Abeta 1-42 peptide protects these mice from the memory deficits they would ordinarily develop. This report further characterizes the Abeta 1-42 peptide vaccine in mice. Anti-Abeta response time course analysis indicated that at least three vaccinations (each 100 microg) were necessary to elicit a significant anti-Abeta titer. Subsequent vaccinations resulted in half-maximal antibody titers of at least 10,000, and these titers were maintained for at least 5 months after the final boost. Peptide binding competition studies indicated that the highest humoral responses are generated against the N terminus of the Abeta peptide. Also, measurement of specific murine Ig isotypes in Abeta-vaccinated mice demonstrated a predominant IgG(1) and IgG(2b) response, suggesting a type 2 (Th2) T-helper cell immune response, which drives humoral immunity. Finally, lymphocyte proliferation assay experiments using Abeta peptides and splenocytes from vaccinated mice demonstrated that the vaccine specifically stimulates T-cell epitopes present within the Abeta peptide.

Preferential incorporation of glucosamine into the galactosamine moieties of chondroitin sulfates in articular cartilage explants

OBJECTIVE To determine the metabolic fate of glucosamine (GlcN) in intact articular cartilage tissue. METHODS Intact articular cartilage explants were cultured for up to 13 days in Dulbeccos modified Eagles medium supplemented with 1) 1-13C-labeled GlcN, 2) 1-13C-labeled glucose (Glc), or 3) no labeling. Every 3-4 days, samples were removed and frozen in liquid nitrogen for carbon-13 magnetic resonance spectroscopic (MRS) analysis. The metabolic products of the labeled precursors were determined from the MRS data based on resonance positions and comparison with known standards and published values. RESULTS GlcN was taken up by the chondrocytes and incorporated selectively into the hexosamine, but not the hexuronic acid, components of the glycosaminoglycan chains of articular cartilage proteoglycan. The data also demonstrated that GlcN is the substrate of choice for the galactosamine moieties of the chondroitin sulfates, incorporating at levels 300% higher than with an equivalent amount of labeled Glc. CONCLUSION The results indicate that GlcN facilitates the production of proteoglycan components that are synthesized through the hexosamine biochemical pathway.

Adenovirus encoding HIV-1 Vpr activates caspase 9 and induces apoptotic cell death in both p53 positive and negative human tumor cell lines.

The targeted delivery of genes whose products arrest the cell cycle and/or induce apoptosis represent an important tool for the understanding and controlling forms of unregulated cell growth. The vpr gene product of HIV-1 has been reported to interfere with cell growth and induce apoptosis, but the mechanism of its action is not clearly understood. In order to study these important properties of Vpr, we created a recombinant adenovirus H5.010CMV-vpr (adCMV-vpr) as a tool to deliver the vpr gene to various cell lines to examine its biology. Vpr protein expression was confirmed by Western blot analysis in adCMV-vpr infected cells. We tested the effects of adCMV-vpr on cell growth of several tumor cell lines. Infection of both p53 positive and p53 deficient tumor cell lines with adCMV-vpr resulted in dramatic induction of cell death in short-term assays. We observed that apoptosis was induced through the mitochondrial pathway as we observed changes in the cytochrome c content accompanied by caspase 9 activation. As Bcl-2 is reported to interfere with apoptosis through the mitochondrial pathway, we examined the effect of adCMV-vpr in Bcl-2 over expressing cell lines. We observed that Bcl-2 overexpression does not inhibit adCMV-vpr induced apoptosis. The properties of adCMV-vpr inducing apoptosis through caspase 9 in a p53 pathway independent manner suggest that this is an important reagent. Such a vector may give insight into approaches designed to limit the growth of pathogenic human cells.

HIV-1 viral protein R (Vpr) regulates viral replication and cellular proliferation in T cells and monocytoid cells in vitro

Among the putative accessory genes of HIV‐1, the 96‐amino‐acid virion‐associated vpr gene product has been described to have several novel biological activities. These include cytoplasmic‐to‐nuclear translocation, which empowers HIV to infect and replicate in non‐dividing cells and to increase viral replication, particularly in macrophages. Along with these viral effects, we found that HIV‐1 Vpr induces dramatic biological changes in the target cells of HIV infection, including induction of changes in transcriptional patterns, morphological changes, and complete inhibition of proliferation, which collectively was termed differentiation. These changes occur in the absence of other viral gene products, suggesting that Vpr mediates its proviral effects partially or perhaps solely through modulation of the state of the target cell rather than directly on the virus. The inhibition of proliferation in T cell lines has been extended by several groups to demonstrate that the inhibition of proliferation is through G2 cell cycle arrest, further supporting the idea that Vpr acts directly on cellular targets. We have recently described a role for Vpr in modulating the glucocorticoid pathway, which is involved in the regulation of the state of the cell, in cytoplasmic‐to‐nuclear translocation, and in modulation of host cell transcription. It is important to note that certain anti‐glucocorticoid compounds modulate Vpr activity in vitro. These results support the idea that the host cell contains specific receptor molecule(s) through which Vpr mediates its activity. Consequently, Vpr represents a unique target for anti‐HIV drug development and has significance for HIV‐1 disease progression. J. Leukoc. Biol. 62: 93–99; 1997.

In vivo protection against ZIKV infection and pathogenesis through passive antibody transfer and active immunisation with a prMEnv DNA vaccine

Significant concerns have been raised owing to the rapid global spread of infection and disease caused by the mosquito-borne Zika virus (ZIKV). Recent studies suggest that ZIKV can also be transmitted sexually, further increasing the exposure risk for this virus. Associated with this spread is a dramatic increase in cases of microcephaly and additional congenital abnormalities in infants of ZIKV-infected mothers, as well as a rise in the occurrence of Guillain Barre’ syndrome in infected adults. Importantly, there are no licensed therapies or vaccines against ZIKV infection. In this study, we generate and evaluate the in vivo efficacy of a novel, synthetic, DNA vaccine targeting the pre-membrane+envelope proteins (prME) of ZIKV. Following initial in vitro development and evaluation studies of the plasmid construct, mice and non-human primates were immunised with this prME DNA-based immunogen through electroporation-mediated enhanced DNA delivery. Vaccinated animals were found to generate antigen-specific cellular and humoral immunity and neutralisation activity. In mice lacking receptors for interferon (IFN)-α/β (designated IFNAR−/−) immunisation with this DNA vaccine induced, following in vivo viral challenge, 100% protection against infection-associated weight loss or death in addition to preventing viral pathology in brain tissue. In addition, passive transfer of non-human primate anti-ZIKV immune serum protected IFNAR−/− mice against subsequent viral challenge. This study in NHP and in a pathogenic mouse model supports the importance of immune responses targeting prME in ZIKV infection and suggests that additional research on this vaccine approach may have relevance for ZIKV control and disease prevention in humans.