677 molecular adjuvants as a strategy to augment host immune responses following rAAV mediated genetic vaccination

677 Molecular Adjuvants as a Strategy To Augment Host Immune Responses Following rAAV Mediated Genetic Vaccination Molecular Therapy �������� ��� ���� ���������������� �������� ��� ������© �����������[.]

INFECTIOUS DISEASES AND VACCINES against HIV, such a strategy is complicated by very limited ability to boost immune responses, largely due to neutralizing antibody responses against the vector itself We have previously shown that large T antigen- (Tag)- deleted recombinant SV40 vectors are nonimmunogenic, and not elicit detectable neutralizing responses against themselves We have used these vectors to deliver lentivirus antigens in mice, successfully eliciting humoral and cell-mediated immune responses, particularly, strong cytotoxic lymphocyte responses We have further demonstrated that co-immunization with rSV40s encoding immunostimulatory cytokines boosts SV40mediated antigen-specific responses so they both are stronger and require fewer immunizations than responses elicited without immunostimulatory cytokines Therefore we investigated whether immunization protocols incorporating immunostimulatory cytokines, IL-12 or IL-15 delivered by rSV40 vectors, would augment Gag-specific immune responses, particularly cytotoxic T cell responses and cytolytic memory, in mice co-immunized with rSV40 encoding SIV gag Methods Mice received monthly injections of SV(gag239) ± SV(mIL-12) or SV(mIL-15) either alone or in combination Cloned Gag-expressing P815 cells were used as targets both in a cell-based ELISA to assay gag-binding antibody activity, and in a 51Cr-release assay, for measuring gag-specific cytolytic responses Direct 51Cr-release assays were performed 4d after final inoculation in SV(mIL-12) co-immunization studies, and after month (to test durability of cytotoxic responses) for SV(mIL-15) studies Results Co-administration of SV(cytokine) with SV(gag239) significantly affected Gag-specific cytolytic responses When immunizing with SV(gag239) alone, average CTL responses were 15% specific lysis Animals immunized with SV(mIL-12) alone showed =25% specific lysis at E:T ratios of 10:1, although responses were generally higher (30-50% lysis) at E:T ratios of 20:1 Conclusions Inoculation of mice with SV(mIL-12) or SV(mIL-15) in combination with SV(gag239), dramatically augmented Gag-specific cytolytic responses, compared with SV(gag239) alone While all IL-15 and Gag combinations gave good cytotoxic lymphocyte responses, mice receiving IL-12 and Gag simultaneously made the strongest responses Cytolytic responses elicited by SV(mIL-15) and SV(gag239) were durable in vivo These results indicate that rSV40s encoding immunostimulatory cytokines, such as IL-12 and IL-15, enhanced Gag-specific immune responses in mice co-immunized with SV(gag239), and so might be useful in HIV-1 vaccine development 676 Efficacy of Lentiviral Vector Transduced Anti-HIV-1 siRNAs in Progenitor Cell Derived T Cells and Macrophages Akhil Banerjea,1 Ming-Jie Li,2 Remling Leila,1 Nan-Sook Lee,2 John Rossi,2 Ramesh Akkina.1 Depathment of Microbiology, Immunology & Pathology, Colorado State University, Fort Collins, CO, United States; Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, CA, United States The potent sequence specific gene silencing mediated by small interfering RNAs (siRNAs) in a post transcriptional manner has drawn considerable attention recently, and can potentially be harnessed for gene therapy Using this approach several ground breaking studies showed remarkable suppression of HIV-1 gene Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy expression/replication To exploit this for possible in vivo therapeutic application, collective utilization of three main elements, namely, hematopoietic stem cells, retroviral vectors and in vivo animal models is necessary Based on this premise, we introduced anti-Rev-siRNAs into CD34+ hematopoietic progenitor cells using third generation HIV-1 based vectors Trangeneic CD34+ cells were allowed to differentiate into mature macrophages in vitro, and T cells in vivo in a SCID-hu mouse thymopoiesis model Expression of vector sequences or si-RNAs had no adverse effect on the lineage specific differentiation of these cells Reconstitution levels greater than 50% was achieved in mice injected with siRNA transduced CD34+ cells In vitro challenge of siRNA expressing macrophages and T lymphocytes showed remarkable inhibition of HIV-1 replication for a prolonged period These experiments highlight the promise of siRNAs for in vivo gene therapy against HIV-1 infection 677 Molecular Adjuvants as a Strategy To Augment Host Immune Responses Following rAAV Mediated Genetic Vaccination K Reed Clark, Ruju Chen, Philip R Johnson Center for Gene Therapy, Columbus Children’s Research Institute, Columbus Children’s Hospital, Columbus, OH Background We are developing HIV-1 genetic vaccines based on recombinant adeno-associated virus vectors (rAAV) We have previously demonstrated that rAAV-2 mediated delivery of SIV envelope and gag genes elicited antigen specific T-cell and antibody responses in mice and rhesus macaques when given via intramuscular injection To augment this novel vaccine approach, we have explored the use of molecular adjuvants as a means to increase host cellular and humoral immune responses following rAAV gene transfer We reasoned that since rAAV-2 vectors generally induce a mild inflammatory response at the site of injection, host immune responses might be increased by alteration of the local tissue milieu towards a more pro-inflammatory environment To this end, we have analyzed murine immune responses to enhanced green fluorescent protein (eGFP) antigen following co-delivery of rAAV2 vectors expressing either mGM-CSF or mIL-12 cytokines Additionally, CpG oligonucleotides were also evaluated for immune enhancement potential in the context of rAAV mediated gene transfer Methods A rAAV-2 eGFP expression vector (rAAV-2/CMV/eGFP) was directly injected into Balb/c mouse muscle (n=4) alone or as an admixture with each of the three molecular adjuvants (CpG ODN, rAAV-2/mIL-12, or rAAV-2/mGM-CSF) Twenty-one days postinjection animals were sacrificed and eGFP specific cellular immune responses were measured in total splenocytes against the H2-Kd restricted epitope HYLSTQSAL using an IFN-gamma ELISPOT assay Serum antibody titers were measured using an eGFP ELISA Results Analysis of eGFP ELISPOT levels revealed that coadministration of rAAV-2/mGM-CSF or CpG ODN (50 ug/dose) increased eGFP antigen specific cellular responses by an average of 1.8 and 3.2 -fold compared to animals receiving the rAAV/eGFP vector alone rAAV-2/mGM-CSF co-delivery significantly increased (>50-fold) anti-eGFP antibody levels in all animals assayed Moreover, histological analysis of rAAV-2/mGM-CSF muscle tissue revealed an extensive lymphocytic infiltrate and abundant myoytes possessing centralized nuclei that were not evident in the other vector inoculated animal tissues Conclusions We observed that host immune responses against a model antigen (eGFP) were augmented in the context of rAAV-2 mediated antigen gene transfer using either CpG ODN or an rAAV-2 vector encoding murine GMCSF Co-delivery of a rAAV/mIL-12 vector at this dosage did not appear to augment cellular or humoral immune responses These data suggest that modulation of the localized immune environment S263 INFECTIOUS DISEASES AND VACCINES during antigen synthesis may represent a powerful strategy to increase host immune responses following rAAV-2 mediated genetic vaccination 678 Ad E2b, in Part, Mediates Inhibition of HIV1 Replication in Human Monocytic Cells Robert J Kaner,1 Franck Rahaghi,1 Dmitri Igonkin,1 Andrea Amalfitano,2 Robin J Parks,3 John P Moore,1 Ronald G Crystal.1 Weill Medical College of Cornell University, New York, NY; Duke University School of Medicine, Durham, NC; 3Ottawa Health Research Institute, Ottawa, ON, Canada Previous studies have indicated that 1st generation adenovirus (Ad) gene transfer vectors (E1-, E3-) inhibit HIV-1 replication in human alveolar macrophages (AM; Am J Respir Cell Mol Biol 2002; 27: 214-219) This inhibition occurs at a step in the HIV-1 life cycle after reverse transcription of the HIV-1 RNA and at or prior to HIV-1 long terminal repeat (LTR) transcription The inhibition of HIV-1 replication by Ad is independent of the transgene, the Ad E4 region and the Ad capsid Since cellular nuclear factors are involved in both Ad and HIV-1 DNA replication, we hypothesized that Ad genes controlling DNA replication might play a role The Ad E2b region includes the Ad DNA polymerase and the preterminal protein genes that are needed for efficient initiation of Ad DNA replication and transcription of late proteins in wild type Ad Accordingly, we examined the effects of two Ad gene transfer vectors expressing the β-galactosidase gene, one a fully deleted helper-dependent Ad vector (hdAdlacZ) and the other a 1st generation vector additionally deleted in the E2b region (AdE2b-lacZ) Both vectors mediated expression of the β-galactosidase gene in the human monocytic cell line THP-1 at a dose of 2.5x104 particle units (pu)/cell To test the hypothesis that Ad gene(s) in the E2b region affect HIV-1 replication in human AM, cells (2x105/well), were infected with 1st generation AdNull, AdE2b-lacZ or hdAdlacZ 6x10³-105 pu/cell or no Ad for hr Following Ad infection, the AM were infected with the R5 HIV-1 strain JRFL, 10³ TCID50/ well To evaluate the effects on subsequent HIV-1 replication, HIV-1 p24 in the conditioned media was quantified by ELISA For the Ad dose of 2.5x104 pu/cell between days and 12 post-infection, p24 levels were: hdAdlacZ 470±42, AdE2b-lacZ 908±200, AdNull 0±0, no Ad 3770±1500 pg/ml (p