677 Molecular Adjuvants as a Strategy To Augment Host Immune Responses Following rAAV Mediated Genetic Vaccination Molecular Therapy �������� ��� ���� ���������������� �������� ��� ������© �����������[.]
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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
non-immunogenic, and do 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
SV40-mediated antigen-specific responses so they both are stronger and
require fewer immunizations than do 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 1
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 <15% specific lysis, and those receiving SV(mIL-15) alone
showed <10% specific lysis In experiments where mice were
co-immunized with SV(mIL-12) and SV(gag239), strongest responses
were seen in the mice that received IL-12 and Gag simultaneously
(50% specific lysis) at effector:target (E:T) ratios as low as 10:1
Specific lysis (>=20%) was observed in all experimental groups Of
the mice receiving SV(gag239) + SV(mIL-15), all three groups that
received both cytokine and antigen in combination responded with
>=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
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
1 Depathment of Microbiology, Immunology & Pathology,
Colorado State University, Fort Collins, CO, United States;
2 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
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.
Augment Host Immune Responses Following rAAV Mediated Genetic Vaccination
K Reed Clark, Ruju Chen, Philip R Johnson
1 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
rAAV-2 vectors expressing either mGM-CSF or mIL-1rAAV-2 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 post-injection 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
co-administration 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 4 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 GM-CSF 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
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Copyright © The American Society of Gene Therapy
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INFECTIOUS DISEASES AND VACCINES
during antigen synthesis may represent a powerful strategy to
increase host immune responses following rAAV-2 mediated genetic
vaccination
HIV-1 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
1 Weill Medical College of Cornell University, New York, NY;
2 Duke University School of Medicine, Durham, NC; 3 Ottawa
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,
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 5 and
12 post-infection, p24 levels were: hdAdlacZ 470±42, AdE2b-lacZ
908±200, AdNull 0±0, no Ad 3770±1500 pg/ml (p<0.05, ANOVA)
This represents a greater than 3 log inhibition by AdNull, but a less
than one log inhibition by the E2b- or the fully deleted vectors To
assess the effects of these vectors on HIV-1 LTR transcription, we
infected THP-1 cells with increasing doses of Ad 1 hr prior to
infection with a VSV-G pseudotyped R5 HIV-1 construct with a
luciferase LTR reporter and HIV-1 LTR transcription was quantified
by assessing luciferase activity 72 hr following HIV-1 infection In
contrast to inhibition of HIV-1 LTR transcription by AdNull in a
dose-responsive fashion (6x10³-105 pu/cell, p<0.05 ANOVA), the
fully deleted and E2b deleted vectors had no significant inhibitory
effect on HIV-1 LTR transcription [for 2.5x104 pu/cell, % reduction
vs no Ad (mean ± SEM): hdAdlacZ 9±9%, AdE2b-lacZ 0±5%,
AdNull 54±3%] These data support the hypothesis that the E2b
region is responsible, in part, for the Ad-mediated inhibition of
HIV-1 replication in human monocytic cells, but does not exclude a
smaller contribution from other Ad genes
Dr Crystal has equity in, is a consultant to, and receives sponsored
research funds from, GenVec, Inc., Gaithersburg, Maryland, a
publicly-traded biotechnology company
Infection
Marc Egelhofer,1 Gunda Brandenburg,1 Holger Martinius,1 Patricia Schult-Dietrich,1 Christopher Baum,2 Ingrid Choi,1 Alexander Alexandrov,3 Dorothee von Laer.1
1 Georg-Speyer-Haus, Frankfurt a.M., Germany; 2 Hematology, Medizinische Hochschule, Hannover, Germany; 3 Fresenius ImmunTherapy, Munich, Germany.
As the limitation of antiretroviral drug therapy become evident, alternative therapeutic strategies for HIV infection are gaining interest Here, a novel gene therapeutic strategy involving the inhibition of HIV entry into gene-modified cells is described Entry inhibition was achieved by expressing a membrane-anchored peptide derived from the second heptad repeat of the HIV-1 gp41 transmembrane glycoprotein In solution, these peptides are known
to inhibit fusion between the viral and cellular membrane To achieve maximal expression and activity of the membrane-anchored peptide, the peptide itself, the scaffold for presentation of the peptide on the cell surface and the retroviral vector backbone were optimized This optimized construct effectively inhibited virus replication in cell lines and primary blood lymphocytes Inhibition was found to be
on the level of membrane fusion, most likely involving direct binding
of the membrane-anchored peptide to gp41 Preclinical development
of the antiviral vector has been completed and clinical trials are in preparation
DNA Vaccine Adjuvants
Jeng-Hwan Wang,1 Chih-Chien Hsu,1 Chin-Jin Li,1 Show-Jane Sun,1 Su-Jane Wang,1 Lie-Fen Shyur,1 Ning-Sun Yang.1
1 Institute of BioAgricultural Sciences, Academia Sinica, Taipei, Taiwan.
Various studies on vaccines have focused on the immuno-stimulatory activities of cytokines/chemokines as growth factors for immune cells or as cofactors or modifiers for modulation of specific immune responses Researchers have reported that some herbal remedies can increase and regulate the secretion of specific cytokines or chemokines from various immune cells We hypothesize here that toll-like receptors (TLRs) may be important for binding of various adjuvants and thus act as adjuvant receptors TLR signaling has been shown to induce the production of pro-inflammatory cytokines/chemokines and upregulate the expression of co-stimulatory molecules; thereby it may activate not only the innate but also the adaptive immunity In the present study, we show that,
upon treatment with a Dioscorea plant extract (Dx-I), the expression
level of three pro-inflammatory cytokines [Interleukin (IL)-1β,
chemokines [IL-8 and monocyte chemotactic protein-1 (MCP-1)] and cell surface receptors (CD14 and TLRs) are significantly affected
in tested human cells (monocytes, keratinocytes and Caco-2) in culture Being different from the known actions of lipopolysaccharide (LPS) on initiation of inflammation by release of a spectrum of pro-inflammatory cytokines and chemokines, only IL-8, but not
MCP-1, IL-1β, IL-12 or TNF-α protein were effectively expressed in the undifferentiated monocytes after treatment with Dx-I herbal extract,
in a dose-dependent manner Dx-I stimulates IL-8 production might through toll-like receptor 4-dependent pathways Dx-I also enhanced CD14, TLR2 and TLR4 protein expression levels in human cell cultures tested After Dx-I treatment, significant differences in expression levels of TLRs mRNA were observed among monocytes, keratinocytes and Caco-2 cells The results suggest that Dx-I apparently conferred a very different type of cellular response as compared with the well-known inflammatory agent, bacterial LPS, that is known to cause a neutrophil-dominated inflammatory