Search

We used optimized DNA expression vectors to compare two gene delivery methodologies in rhesus macaques, namely direct DNA injection and in vivo adaptive constant-current electroporation via the intramuscular route. The use of in vivo electroporation increased levels of gene expression and immune responses. We used an optimized HIV gag expression plasmid to show the development of new cellular immune responses in SIV-infected animals controlling viremia. Furthermore, after vaccination with SIV expression plasmids the recall responses to the SIV antigens were very high, indicating that DNA is a strong boost in the presence of antiretroviral treatment in SIV-infected animals. There was substantial animal-to-animal variability in DNA expression, revealed by plasma measurements of IL-15 produced by co-injected IL-15 DNA. IL-15 expression levels correlated with peak immune responses. Electroporation led to an expansion of antigen-specific CD4+ and CD8+ T cells of both central and effector memory phenotype. These results indicate that improved gene delivery and expression by electroporation dramatically increases immunogenicity of DNA vaccines. Electroporation is thus an important method to improve the effectiveness of DNA vaccination.

We show that co-expression of interleukin 15 (IL-15) and IL-15 receptor alpha (IL-15Ralpha) in the same cell allows for the intracellular interaction of the two proteins early after translation, resulting in increased stability and secretion of both molecules as a complex. In the absence of co-expressed IL-15Ralpha, a large portion of the produced IL-15 is rapidly degraded immediately after synthesis. Co-injection into mice of IL-15 and IL-15Ralpha expression plasmids led to significantly increased levels of the cytokine in serum as well as increased biological activity of IL-15. Examination of natural killer cells and T lymphocytes in mouse organs showed a great expansion of both cell types in the lung, liver, and spleen. The presence of IL-15Ralpha also increased the number of CD44(high) memory cells with effector phenotype (CD44(high)CD62L-). Thus, mutual stabilization of IL-15 and IL-15Ralpha leads to remarkable increases in production, stability, and tissue availability of bioactive IL-15 in vivo. The in vivo data show that the most potent form of IL-15 is as part of a complex with its receptor alpha either on the surface of the producing cells or as a soluble extracellular complex. These results explain the reason for coordinate expression of IL-15 and IL-15Ralpha in the same cell and suggest that the IL-15Ralpha is part of the active IL-15 cytokine rather than part of the receptor.

Rhesus macaques chronically infected with highly pathogenic simian immunodeficiency virus (SIV) SIVmac251 were treated with antiretroviral drugs and vaccinated with combinations of DNA vectors expressing SIV antigens. Vaccination during therapy increased cellular immune responses. After the animals were released from therapy, the virus levels of 12 immunized animals were significantly lower (P = 0.001) compared to those of 11 animals treated with only antiretroviral drugs. Vaccinated animals showed a persistent increase in immune responses, thus indicating both a virological and an immunological benefit following DNA therapeutic vaccination. Several animals show a long-lasting decrease in viremia, suggesting that therapeutic vaccination may provide an additional benefit to antiretroviral therapy.

A cohort of 22 rhesus macaques of Indian origin infected as neonates, juveniles, or adults by Rev-independent strains of SIV was monitored over several years. After the initial acute phase, virus replication was controlled and plasma virus loads were persistently below the threshold of the assay. The animals were monitored for up to 7.6 years after infection for viral loads, cellular and humoral immune responses, hematological changes, and overall health and no signs of immune dysfunction or AIDS were observed. This study represents several years of additional observation compared to the previously published results, and indicates that the Rev-independent SIV clones tested do not cause AIDS-like progressive disease within 7.6 years from infection. All the animals showed persistent humoral and cellular SIV-specific immune responses, consistent with chronic infection. Different Rev-independent SIV strains showed similar properties and lack of pathogenicity. Multicolor flow cytometric analysis demonstrated preservation of the Central Memory subset of T cells in the attenuated SIV-infected animals. This study demonstrates a potent, long-lasting control of the Rev-independent attenuated SIV in macaques independent of the age at virus exposure.

Studies of retroviral mRNA export identified two distinct RNA export elements utilizing conserved eukaryotic mRNA export mechanism(s), namely the Constitutive Transport Element (CTE) and the RNA Transport Element (RTE). Although RTE and CTE are potent in nucleocytoplasmic mRNA transport and expression, neither element is as powerful as the Rev-RRE posttranscriptional control. Here, we found that whereas CTE and the up-regulatory mutant RTEm26 alone increase expression from a subgenomic gag and env clones, the combination of these elements led to a several hundred-fold, synergistic increase. The use of the RTEm26-CTE combination is a simple way to increase expression of poorly expressed retroviral genes to levels otherwise only achieved via more cumbersome RNA optimization. The potent RTEm26-CTE element could be useful in lentiviral gene therapy vectors, DNA-based vaccine vectors, and gene transfer studies of other poorly expressed genes.

We have tested the efficacy of DNA immunization as a single vaccination modality for rhesus macaques followed by highly pathogenic SIVmac251 challenge. To further improve immunogenicity of the native proteins, we generated expression vectors producing fusion of the proteins Gag and Env to the secreted chemokine MCP3, targeting the viral proteins to the secretory pathway and to a beta-catenin (CATE) peptide, targeting the viral proteins to the intracellular degradation pathway. Macaques immunized with vectors expressing the MCP3-tagged fusion proteins developed stronger antibody responses. Following mucosal challenge with pathogenic SIVmac251, the vaccinated animals showed a statistically significant decrease in viral load (P = 0.010). Interestingly, macaques immunized with a combination of vectors expressing three forms of antigens (native protein and MCP3 and CATE fusion proteins) showed the strongest decrease in viral load (P = 0.0059). Postchallenge enzyme-linked immunospot values for Gag and Env as well as gag-specific T-helper responses correlated with control of viremia. Our data show that the combinations of DNA vaccines producing native and modified forms of antigens elicit more balanced immune responses able to significantly reduce viremia for a long period (8 months) following pathogenic challenge with SIVmac251.

Objective: We define characteristics of a dendritic cell (DC) precursor generated from murine lineage-negative (Lin(-)) Sca1(+) hematopoietic progenitor cells (HPC)., Materials and Methods: Lin(-)Sca1(+) HPC cultured 9 days in 100 ng/mL stem cell factor (SCF), 20 ng/mL interleukin-3 (IL-3), 50 ng/mL monocyte colony-stimulating factor (M-CSF), 5 ng/mL granulocyte-monocyte colony-stimulating factor (GM-CSF), and 25 ng/mL FLT3-ligand (FLT3-L) proliferate 387-fold and differentiate into DC precursors. Switch to > or =100 ng/mL GM-CSF + 1500 U/mL IL-4 or 500 U/mL tumor necrosis factor-alpha (TNF-alpha) for 3 days induces development into immature DC that are responsive to bacterial lipopolysaccharide (LPS)-induced maturation., Results: Lin(-)Sca1(+) HPC in the first 9 days of culture differentiate into DC precursors expressing surface CD11b(bright), CD11c(mod), CD86(low-mod), major histocompatibility class II antigen (MHC) II(low), DEC 205(low), but are surface CD40(-) and contain high levels of intracellular MHC II. Unlike immature DC described by others, these DC precursors are refractory to maturation with LPS and minimally stimulate allogeneic T lymphocytes in mixed leukocyte reactions (MLR). Switch to high-dose GM-CSF alone with IL-4 or TNF-alpha differentiates these DC precursors into immature DC. LPS treatment of the immature DC results in mature DC that express surface CD40(high) and CD86(high), secrete IL-1beta and IL-12, and strongly stimulate MLR., Conclusions: These studies define a distinct DC precursor derived from murine HPC that precedes development of immature and mature DC.