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ABSTRACT: DNA immunization by gene gun against a variety of infectious diseases has yielded promising results in animal models. Skin-based DNA vaccination against these diseases is not only an attractive option for the clinic but can aid in the discovery and optimization of vaccine candidates. Vaccination against the protozoan parasite Plasmodium presents unique challenges: (a) most parasite-associated antigens are stage-specific; (b) antibodies capable of neutralizing the parasite during the probing of the mosquitoes have to be available at high titers in order to prevent infection of the liver; (c) immunity to liver-stage infection needs to be absolute in order to prevent subsequent blood-stage parasitemia. Gene gun vaccination has successfully been used to prevent the infection of mice with the rodent malaria strain P. berghei and has been employed in a macaque model of human P. falciparum. DNA plasmid delivery by gene gun offers the opportunity to economically and efficiently test novel malaria vaccine candidates and vaccination strategies, which include the evaluation of novel molecular adjuvant strategies. Here we describe the procedures involved in making and delivering a pre-clinical malaria DNA vaccine by gene gun as well as the correct approach for the in vivo evaluation of the vaccine. Furthermore, we discuss various approaches that either have already been tested or could be employed to improve DNA vaccines against malaria.
Methods in molecular biology (Clifton, N.J.) 01/2013; 940:269-84.
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Yanan Liu,
Xiaoli Tian, Wolfgang W Leitner,
Michael E Aldridge,
Junying Zheng,
Zhiya Yu,
Nicholas P Restifo,
Richard Weiss,
Sandra Scheiblhofer,
Chong Xie,
Ren Sun,
Genhong Cheng,
Gang Zeng
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ABSTRACT: In search of intrinsic factors that contribute to the distinctively strong immunogenicity of a non-mutated cancer/testis antigen, we found that NY-ESO-1 forms polymeric structures through disulfide bonds. NY-ESO-1 binding to immature dendritic cells was dependent on its polymeric structure and involved Toll-like receptor-4 (TLR4) on the surface of immature dendritic cells in mouse and human. Gene gun-delivered plasmid encoding the wild-type NY-ESO-1 readily induced T cell-dependent antibody (Ab) responses in wild-type C57BL/10 mice but not TLR4-knock-out C57BL/10ScNJ mice. Disrupting polymeric structures of NY-ESO-1 by cysteine-to-serine (Cys-to-Ser) substitutions lead to diminished immunogenicity and altered TLR4-dependence in the induced Ab response. To demonstrate its adjuvant effect, NY-ESO-1 was fused with a major mugwort pollen allergen Art v 1 and a tumor-associated antigen, carbonic anhydrase 9. Plasmid DNA vaccines encoding the fusion genes generated robust immune responses against otherwise non-immunogenic targets in mice. Polymeric structure and TLR4 may play important roles in rendering NY-ESO-1 immunogenic and thus serve as a potent molecular adjuvant. NY-ESO-1 thus represents the first example of a cancer/testis antigen that is a also damage-associated molecular pattern.
Journal of Biological Chemistry 09/2011; 286(43):37077-84. · 4.77 Impact Factor
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ABSTRACT: MFG-E8 (also called lactadherin and SED1) is a secreted glycoprotein that has been previously implicated in enhancement of vascular endothelial growth factor-dependent angiogenesis. Major sources of MFG-E8 in vivo and precise mechanisms of MFG-E8 action remain undetermined. The objective of this study was to identify important sources of MFG-E8 in vivo and further elucidate the role(s) of MFG-E8 in the regulation of angiogenesis.
We used knockout mice and anti-MFG-E8 antibodies to study MFG-E8 function in vivo. In melanomas and in retinas of mice with oxygen-induced retinopathy, MFG-E8 colocalized with pericytes rather than endothelial cells, and platelet-derived growth factor receptor β+ pericytes/pericyte precursors purified from tumors contained large amounts of MFG-E8 mRNA. Tumor- and retinopathy-associated angiogenesis was diminished in MFG-E8 knockout mice, and pericyte coverage of neovessels was reduced. Inhibition of MFG-E8 production by 10T1/2 cells (surrogate pericyte/pericyte precursors) using small interfering RNAs and short hairpin RNAs, or inhibition of MFG-E8 action with some anti-MFG-E8 antibodies, selectively attenuated migration in vitro. Significantly, the anti-MFG-E8 antibodies that inhibited 10T1/2 cell migration in vitro also inhibited pathological angiogenesis in vivo.
These studies strongly implicate MFG-E8 in pericyte/pericyte precursor function and indicate that MFG-E8-directed therapeutics may merit further development.
Arteriosclerosis Thrombosis and Vascular Biology 09/2011; 31(9):2024-34. · 6.37 Impact Factor
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ABSTRACT: The immunological mechanisms responsible for protection against malaria infection vary among Plasmodium species, host species and the developmental stage of parasite, and are poorly understood. A challenge with live parasites is the most relevant approach to testing the efficacy of experimental malaria vaccines. Nevertheless, in the mouse models of Plasmodium berghei and Plasmodium yoelii, parasites are usually delivered by intravenous injection. This route is highly artificial and particularly in the P. berghei model produces inconsistent challenge results. The initial objective of this study was to compare an optimized intravenous (IV) delivery challenge model with an optimized single infectious mosquito bite challenge model. Finding shortcomings of both approaches, an alternative approach was explored, i.e., the subcutaneous challenge.
Mice were infected with P. berghei sporozoites by intravenous (tail vein) injection, single mosquito bite, or subcutaneous injection of isolated parasites into the subcutaneous pouch at the base of the hind leg. Infection was determined in blood smears 7 and 14 days later. To determine the usefulness of challenge models for vaccine testing, mice were immunized with circumsporozoite-based DNA vaccines by gene gun.
Despite modifications that allowed infection with a much smaller than reported number of parasites, the IV challenge remained insufficiently reliable and reproducible. Variations in the virulence of the inoculum, if not properly monitored by the rigorous inclusion of sporozoite titration curves in each experiment, can lead to unacceptable variations in reported vaccine efficacies. In contrast, mice with different genetic backgrounds were consistently infected by a single mosquito bite, without overwhelming vaccine-induced protective immune responses. Because of the logistical challenges associated with the mosquito bite model, the subcutaneous challenge route was optimized. This approach, too, yields reliable challenge results, albeit requiring a relatively large inoculum.
Although a single bite by P. berghei infected Anopheles mosquitoes was superior to the IV challenge route, it is laborious. However, any conclusive evaluation of a pre-erythrocytic malaria vaccine candidate should require challenge through the natural anatomic target site of the parasite, the skin. The subcutaneous injection of isolated parasites represents an attractive compromise. Similar to the mosquito bite model, it allows vaccine-induced antibodies to exert their effect and is, therefore not as prone to the artifacts of the IV challenge.
Malaria Journal 01/2010; 9:145. · 3.19 Impact Factor
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ABSTRACT: Carney complex, a multiple neoplasia syndrome, characterized primarily by spotty skin pigmentation and a variety of endocrine and other tumors, is caused by mutations in PRKAR1A, the gene that codes for the RIalpha subunit of protein kinase A (PKA). PKA controls cell proliferation in many cell types. The cAMP analogue 8-Cl-adenosine (8-Cl-ADO) is thought to inhibit cancer cell proliferation.
The objective of the study was to study the antiproliferative effects of 8-Cl-ADO on growth and proliferation in B-lymphocytes of Carney complex patients that have PKA defects and to determine whether 8-CL-ADO could be used as a therapeutic agent in the treatment of Carney complex-associated tumors.
We used a multiparametric approach (i.e. growth and proliferation assays, PKA, and PKA subunit assays, cAMP and (3)H-cAMP binding assays, and apoptosis assays) to understand the growth and proliferative effects of 8-Cl-ADO on human B-lymphocytes.
8-Cl-ADO inhibited proliferation, mainly through its intracellular transport and metabolism, which induced apoptosis. PKA activity, cAMP levels, and (3)H-cAMP binding were increased or decreased, respectively, by 8-Cl-ADO, whereas PKA subunit levels were differentially affected. 8-Cl-ADO also inhibited proliferation induced by G protein-coupled receptors for isoproterenol and adenosine, as well as proliferation induced by tyrosine kinase receptors.
8-Cl-ADO in addition to unambiguously inhibiting proliferation and inducing apoptosis in a PKA-independent manner also has PKA-dependent effects that are unmasked by a mutant PRKAR1A. Thus, 8-Cl-ADO could serve as a therapeutic agent in patients with Carney complex-related tumors.
The Journal of clinical endocrinology and metabolism 09/2009; 94(10):4061-9. · 6.50 Impact Factor
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ABSTRACT: The US National Institute of Allergy and Infectious Diseases convened a workshop of malaria investigators and immunologists to foster collaborations and attract more immunologists into malaria research. Discussions highlighted research gaps and underscored the incomplete understanding of basic immune mechanisms that contribute to the pathogenesis of or protection against malaria.
Nature Immunology 08/2009; 10(7):673-8. · 26.01 Impact Factor
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ABSTRACT: Malaria represents a major global health problem but despite extensive efforts, no effective vaccine is available. Various vaccine candidates have been developed that provide protection in animal models, such as a gene gun-delivered DNA vaccine encoding the circumsporozoite protein (CSP) of Plasmodium berghei. A common shortcoming of most malaria vaccines is the requirement for multiple immunizations leaving room for improvement even for established vaccine candidates such as the CSP-DNA vaccine. In this study, we explored whether regulating apoptosis in DNA vaccine transfected host cells could accelerate the onset of protective immunity and provide significant protection after a single immunization. A pro-apoptotic gene (Bax) was used as a molecular adjuvant in an attempt to mimic the immunostimulatory apoptosis triggered by viral or virus-derived vaccines, while anti-apoptotic genes such as Bcl-XL may increase the life span of transfected cells thus prolonging antigen production. Surprisingly, co-delivery of either Bax or Bcl-XL greatly reduced CSP-DNA vaccine efficacy after a single immunization. Co-delivery of Bax for three immunizations still had a detrimental effect on protective immunity, while repeated co-delivery of Bcl-XL had no negative impact. The fine characterization of humoral and cellular immune response modulated by these two molecular adjuvants revealed a previously unknown effect, i.e., a shift in the Th-profile. These results demonstrate that pro- or anti-apoptotic molecules should not be used as molecular adjuvants without careful evaluation of the resulting immune response. This finding represents yet another example that strategies to enhance vaccine efficacy developed for other model systems such as viral diseases cannot easily be applied to any vaccine.
Vaccine 08/2009; 27(41):5700-8. · 3.77 Impact Factor
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ABSTRACT: A new langerin(+) DC subset has recently been identified in murine dermis (langerin(+) dDC), but the lineage and functional relationships between these cells and langerin(+) epidermal Langerhans cells (LC) are incompletely characterized. Selective expression of the cell adhesion molecule EpCAM by LC allowed viable LC to be easily distinguished from langerin(+) dDC in skin and lymphoid tissue and ex vivo as well. Differential expression of EpCAM and langerin revealed the presence of at least 3 distinct skin DC subsets. We determined that LC and langerin(+) dDC exhibit different migratory capabilities in vitro and repopulate distinct anatomic compartments in skin at different rates after conditional depletion in vivo. Langerin(+) dDC, in contrast to LC, did not require TGFbeta1 for development. Carefully timed gene gun immunization studies designed to take advantage of the distinct repopulation kinetics of langerin(+) dDC and LC revealed that langerin(+) dDC were required for optimal production of beta-galactosidase-specific IgG2a/c and IgG2b in the acute phase. In contrast, immunization via LC-deficient skin resulted in persistent and strikingly reduced IgG1 and enhanced IgG2a Ab production. Our data support the concepts that LC and langerin(+) dDC represent distinct DC subsets that have specialized functions and that LC are important immunoregulatory cells. The presence of at least 3 functionally distinct skin DC subsets may have particular relevance for vaccines that are administered epicutaneously.
Proceedings of the National Academy of Sciences 03/2009; 106(9):3312-7. · 9.68 Impact Factor
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ABSTRACT: Nucleic acid-based vaccines are effective in infectious disease models but have yielded disappointing results in tumor models when tumor-associated self-antigens are used. Incorporation of helper epitopes from foreign antigens into tumor vaccines might enhance the immunogenicity of DNA vaccines without increasing toxicity. However, generation of fusion constructs encoding both tumor and helper antigens may be difficult, and resulting proteins have unpredictable physical and immunologic properties. Furthermore, simultaneous production of equal amounts of highly immunogenic helper and weakly immunogenic tumor antigens in situ could favor development of responses against the helper antigen rather than the antigen of interest. We assessed the ability of 2 helper antigens (beta-galactosidase or fragment C of tetanus toxin) encoded by one plasmid to augment responses to a self-antigen (lymphoma-associated T-cell receptor) encoded by a separate plasmid after codelivery into skin by gene gun. This approach allowed adjustment of the relative ratios of helper and tumor antigen plasmids to optimize helper effects. Incorporation of threshold (minimally immunogenic) amounts of helper antigen plasmid into a DNA vaccine regimen dramatically increased T cell-dependent protective immunity initiated by plasmid-encoded tumor-associated T-cell receptor antigen. This simple strategy can easily be incorporated into future vaccine trials in experimental animals and possibly in humans.
Blood 11/2008; 113(1):37-45. · 9.90 Impact Factor
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ABSTRACT: Protein kinase A (PKA) affects cell proliferation in many cell types and is a potential target for cancer treatment. PKA activity is stimulated by cAMP and cAMP analogs. One such substance, 8-Cl-cAMP, and its metabolite 8-Cl-adenosine (8-Cl-ADO) are known inhibitors of cancer cell proliferation; however, their mechanism of action is controversial. We have investigated the antiproliferative effects of 8-Cl-cAMP and 8-CL-ADO on human thyroid cancer cells and determined PKA's involvement.
We employed proliferation and apoptosis assays and PKA activity and cell cycle analysis to understand the effect of 8-Cl-ADO and 8-Cl-cAMP on human thyroid cancer and HeLa cell lines.
8-Cl-ADO inhibited proliferation of all cells, an effect that lasted for at least 4 d. Proliferation was also inhibited by 8-Cl-cAMP, but this inhibition was reduced by 3-isobutyl-1-methylxanthine; both drugs stimulated apoptosis, and 3-isobutyl-1-methylxanthine drastically reduced 8-Cl-cAMP-induced cell death. 8-Cl-ADO induced cell accumulation in G1/S or G2/M cell cycle phases and differentially altered PKA activity and subunit levels. PKA stimulation or inhibition and adenosine receptor agonists or antagonists did not significantly affect proliferation.
8-Cl-ADO and 8-Cl-cAMP inhibit proliferation, induce cell cycle phase accumulation, and stimulate apoptosis in thyroid cancer cells. The effect of 8-Cl-cAMP is likely due to its metabolite 8-Cl-ADO, and PKA does not appear to have direct involvement in the inhibition of proliferation by 8-Cl-ADO. 8-Cl-ADO may be a useful therapeutic agent to be explored in aggressive thyroid cancer.
Journal of Clinical Endocrinology & Metabolism 04/2008; 93(3):1020-9. · 6.50 Impact Factor
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ABSTRACT: Immune response against circumsporozoite protein (CSP) of Plasmodium berghei, a major surface protein on the sporozoite, confers protection in various murine malaria models. Engineered DNA vaccine encoding CSP and 3 copies of C3d caused an unexpected loss in protection attributed to the binding of C3d to the C-terminal region of CSP. Because the C3d region known as p28 represents the complement receptor (CR) 2-binding motif, we developed a CSP-3 copies of p28 DNA construct (CSP-3p28). CSP-3p28-immunized mice were better protected against P. berghei sporozoites than CSP-immunized mice 6 weeks after the 2nd boost, produced sufficient IgG1 anti-CSP and CSP C-terminus antibody and failed to produce IgG2a. CSP-3C3d-immunized mice were not protected, failed to produce IgG1 and produced high amounts of IgG2a. We conclude that use of the CR2-binding motif of C3d as molecular adjuvant to CSP results in anti-malaria protective immune response probably by targeting the chimeric protein to CR2.
Vaccine 12/2007; 25(45):7732-6. · 3.77 Impact Factor
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ABSTRACT: The relevance of angiogenesis in tumor biology and as a therapeutic target is well established. MFG-E8 (also termed lactadherin) and developmental endothelial locus 1 (Del1) constitute a two-gene family of alpha(v)beta(3)/beta(5) ligands that regulate angiogenesis. After detecting MFG-E8 mRNA in murine tumor cell lines, we sought to determine if MFG-E8 influenced tumorigenesis in Rip1-Tag2 transgenic mice, a cancer model in which angiogenesis is critical. MFG-E8 mRNA and protein were increased in angiogenic islets and tumors in Rip1-Tag2 mice compared with normal pancreas. Frequencies of angiogenic islets and tumor burdens were decreased in MFG-E8-deficient Rip1-Tag2 mice compared with those in control Rip1-Tag2 mice. Invasive carcinomas were modestly underrepresented in MFG-E8-deficient mice, but tumor frequencies and survivals were comparable in these two strains. Absence of MFG-E8 also led to decreases in tumor vascular permeability without obvious changes in vascular morphology. Decreased proliferation was noted in angiogenic islets and increases in apoptotic cells were detected in islets and tumors. Compensatory increases in mRNA encoding proangiogenic proteins, including FGF2, in angiogenic islets, and Del1, in angiogenic islets and tumors, were also detected in MFG-E8-deficient mice. MFG-E8 and its homologue Del1 may represent relevant targets in cancer and other diseases in which angiogenesis is prominent.
Cancer Research 08/2007; 67(14):6777-85. · 7.86 Impact Factor
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ABSTRACT: C3d is a fragment of the complement factor C3 and is generated in the course of complement activation. When bound to antigen in single or multiple copies, the B cell receptor and complement receptor 2 become co-crosslinked resulting in decreased or increased B cell responses depending on the valence of the antigen-C3d construct. When antigen-C3d constructs are used for the purpose of generating a protective immune response (vaccines), they may either enhance the expected response or suppress it depending on the nature of the antigen. Various pathogens use C3d to evade the immune system by inhibiting complement activation, invading and homing in host cells or masking immunogenic areas of pathogen proteins. Therefore, future vaccination strategies for infectious diseases and cancer employing C3d as a molecular adjuvant need to be carefully evaluated before choosing a target antigen in order to take advantage of the adjuvant effect of the complement component while avoiding potential vaccine complications associated with immune escape mechanisms.
Clinical Immunology 12/2006; 121(2):177-85. · 4.05 Impact Factor
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ABSTRACT: The multiple neoplasia syndrome Carney complex (CNC) is caused by heterozygote mutations in the gene, which codes for the RIalpha regulatory subunit (PRKAR1A) of protein kinase A. Inactivation of PRKAR1A and the additional loss of the normal allele lead to tumors in CNC patients and increased cyclic AMP signaling in their cells, but the oncogenetic mechanisms in affected tissues remain unknown. Previous studies suggested that PRKAR1A down-regulation may lead to increased mitogen-activated protein kinase (MAPK) signaling. Here, we show that, in lymphocytes with PRKAR1A-inactivating mutations, there is increased extracellular signal-regulated kinase (ERK) 1/2 and B-raf phosphorylation and MAPK/ERK kinase 1/2 and c-Myc activation, whereas c-Raf-1 is inhibited. These changes are accompanied by increased cell cycle rates and decreased apoptosis that result in an overall net gain in proliferation and survival. In conclusion, inactivation of PRKAR1A leads to widespread changes in molecular pathways that control cell cycle and apoptosis. This is the first study to show that human cells with partially inactivated RIalpha levels have increased proliferation and survival, suggesting that loss of the normal allele in these cells is not necessary for these changes to occur.
Cancer Research 12/2006; 66(21):10603-12. · 7.86 Impact Factor
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Maximilian Gabler,
Sandra Scheiblhofer,
Kerstin Kern, Wolfgang W Leitner,
Angelika Stoecklinger,
Cornelia Hauser-Kronberger,
Beate Alinger,
Berta Lechner,
Monika Prinz,
Susanne Vrtala,
Rudolf Valenta,
Josef Thalhamer,
Richard Weiss
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ABSTRACT: Replicase-based DNA vaccines stimulate T(H)1-biased immune responses at ultralow doses and induce self-removal of transfected cells through apoptosis. Both aspects are important requirements for efficient and safe DNA-based immunotherapy of type I allergies.
A Sindbis virus replicon-based DNA vaccine encoding the major timothy grass pollen allergen Phl p 5 was evaluated for its antiallergic potential compared with a conventional DNA vaccine in a BALB/c mouse model of allergy.
Mice were intradermally prevaccinated with plasmid DNA, followed by sensitization and intranasal allergen provocation with recombinant Phl p 5. In vitro proliferation and cytokine secretion was measured in splenocyte cultures. Distribution of IgG1, IgG2a, and IgE antibody subclasses was determined by means of ELISA. IgE-mediated degranulation was measured with the basophil release assay. Bronchoalveolar lavage fluid was analyzed for eosinophils, IL-4, IL-5, IL-13, and IFN-gamma. Mucus production, inflammatory infiltrates, and epithelial damage were determined in lung sections.
Both vaccines induced T(H)1-biased immune responses, resulting in suppression of functional IgE, reduction of eosinophilia in bronchoalveolar lavage fluid, and alleviation of lung pathology. However, immunization with the replicon DNA vaccine elicited these effects at a 100-fold lower dose compared with the conventional DNA vaccine.
The increased immunogenicity of replicon-based DNA vaccines allows for application of extremely low doses, thereby eliminating the concerns associated with conventional DNA vaccines, which have to be administered at milligram amounts to induce immune reactions in human subjects.
Their high safety profile makes replicon-based DNA vaccines promising candidates for treatment of type I allergies in the clinic.
Journal of Allergy and Clinical Immunology 10/2006; 118(3):734-41. · 11.00 Impact Factor
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ABSTRACT: The immunogenicity and efficacy of nucleic acid vaccines can be greatly enhanced when antigen production is under the control of an alphaviral replicase enzyme. However, replicase-mediated mRNA overproduction does not necessarily result in enhanced antigen level. Instead, the strong adaptive immune response of alphavirus replicon-based vectors is due to their production of double-stranded RNA (dsRNA) intermediates, which trigger innate immunity. Because viral infections are known to trigger innate immune responses that lead to the rapid production of Type I Interferons (IFNs), namely IFN-alpha and IFN-beta, we investigated the role of Type I IFNs in the enhanced immunogenicity of replicase-based DNA vaccines. In vitro, cells transfected with replicase-based plasmids produce significantly more Type I IFNs than cells transfected with a conventional DNA plasmid. In vivo, replicase-based DNA vaccines yield stronger humoral responses in the absence of Type I IFN signaling but the lack of this signaling pathway in IFN-alphabeta receptor-/- (knockout) mice abolishes T cell mediated efficacy against tumors of both conventional and alphavirus replicase-based DNA vaccines. Moreover, the co-delivery of an IFNalpha-encoding plasmid significantly improved the efficacy of a weakly immunogenic conventional plasmid. These results suggest a central role for Type I IFNs in the mechanism of replicase-based DNA vaccines and indicate that vaccines can be enhanced by enabling their capacity to triggering innate anti-viral defense pathways.
Vaccine 07/2006; 24(24):5110-8. · 3.77 Impact Factor
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Douglas S Walsh,
Montip Gettayacamin, Wolfgang W Leitner,
Jeffrey A Lyon,
V Ann Stewart,
Gary Marit,
Sathit Pichyangkul,
Panita Gosi,
Pongsri Tongtawe,
Kent E Kester,
Carolyn A Holland,
Nelly Kolodny,
Joe Cohen,
Gerald Voss,
W Ripley Ballou,
D Gray Heppner
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ABSTRACT: RTS,S/AS02A, a recombinant Plasmodium falciparum vaccine based on the circumsporozoite protein (CSP) repeat and C-terminus regions, elicits strong humoral and Th1 cell-mediated immunity. In field studies, RTS,S/AS02A reduced malaria infection, clinical episodes, and disease severity. Heterologous prime-boost immunization regimens, optimally spaced, might improve the protective immunity of RTS,S/AS02A.
DNA plasmid encoding P. falciparum CSP (3D7) was administered to six experimental groups of rhesus monkeys (N = 5) by gene gun (coded as D), followed by a 1/5th human dose of RTS,S/AS02A (coded as R). Immunization regimens, including a numeral to denote weeks between immunizations, were D-4-R, D-16-R, D-4-D-4-R, D-4-D-16-R, D-16-D-4-R and D-16-D-16-R. A control group (N = 5) received a single 1/5th dose of RTS,S/AS02A. Endpoints were antibody (Ab) to homologous CSP repeat and C-terminus regions and delayed-type hypersensitivity (DTH) to CSP peptides.
Monkeys immunized twice with DNA, 16 weeks apart (D-16-D-4-R and D-16-D-16-R), developed higher levels of anti-C-terminus Abs than control monkeys (p<0.02). No CSP DNA priming regimen increased RTS,S/AS02A-induced Ab to CSP repeats. At 16 months after first immunization, D-R and D-D-R, but not control, monkeys had histologically confirmed DTH reactions against CSP C-terminus, which persisted at repeat testing 12 months later.
Two optimally spaced, particle-mediated epidermal deliveries of CSP DNA improved the humoral immunogenicity of a single dose of RTS,S/AS02A. Further, CSP DNA prime followed by one dose of RTS,S/AS02A gave biopsy proven DTH reactions against CSP C-terminus of up to 2 years duration, implying the induction of CD4+ memory T cells. Heterologous prime-boost strategies for malaria involving gene gun delivered DNA or more potent vectors, administered at optimal intervals, warrant further investigation.
Vaccine 06/2006; 24(19):4167-78. · 3.77 Impact Factor
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ABSTRACT: LYVE-1 is a surface bound hyaluronic acid (HA) receptor that is preferentially expressed by lymphatic endothelial cells (LEC). cDNA encoding full-length human LYVE-1 was coated onto gold particles that were then delivered via helium-assisted jet propulsion (gene gun) into the skin of Balb/C mice. LYVE-1 antisera, but not control pre-immune sera, recognized LYVE-1-transfected 293T cells by flow cytometry. While 40-70% of cultured human dermal microvascular endothelial cells (HMEC) were positive for LYVE-1 staining, human lung microvascular endothelial cells (LMEC) were negative. LYVE-1 antisera was used to effectively separate HMEC into LYVE-1 (hi) and LYVE-1(lo) populations that were enriched or depleted, respectively, for podoplanin, another LEC marker. By immunohistochemistry, LYVE-1 antisera detected CD31(lo) podoplanin(hi) lymphatic channels in normal and psoriatic human skin as well as in human tonsil. LYVE-1 antisera also blocked binding of FITC-labeled HA to HMEC (but not LMEC), demonstrating that these antibodies recognized regions of LYVE-1 required for HA binding. In summary, gene gun-assisted delivery of cDNA encoding LYVE-1 into skin resulted in reliable production of antisera that specifically and functionally recognized native LYVE-1 protein.
Microvascular Research 02/2006; 71(1):32-9. · 2.83 Impact Factor
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ABSTRACT: Replicase-based vaccines were introduced to overcome some of the deficiencies of conventional DNA- and RNA-based vaccines, including poor efficiency and low stability. At ultra-low doses, these alphavirus-derived vectors elicit cellular as well as humoral immune responses. Additionally, replicase-based vectors induce "self-removal" of the vaccine via apoptosis of transfected cells. This chapter describes the construction of a replicon-based DNA vaccine vector from commercially available plasmids. We present protocols for monitoring cellular immune responses following replicase-based immunization including measurement of allergen-specific proliferation of splenocytes, ELISPOT, a FACS-based cytokine secretion assay providing information about T-helper subsets, and a cytokine fluorescent bead immunoassay.
Methods in molecular medicine 02/2006; 127:221-35.
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ABSTRACT: Genetic immunization has proven a powerful method to induce antiallergic immune responses. The underlying functional principle has been described to be based on the recruitment of allergen-specific Th1 cells, CD8+ cells and the establishment of a Th1 cytokine milieu, which prevent the development of a Th2-biased response in a protective setup and can balance an ongoing Th2-type response in a therapeutic situation. Genetic immunization with plasmid DNA offers innovative solutions to the major problems associated with protein immunization, such as crosslinking of pre-existing immunoglobulin E on mast cells/basophils or induction of de novo synthesis of immunoglobulin E by the protein immunization itself. It easily enables the routine production of hypoallergenic vaccines, which do not translate native allergens, thus avoiding potential anaphylactic side effects. DNA vaccines can also be applied as mixtures of single vaccines, making them interesting candidates for treatment based on component-resolved diagnosis, followed by an individualized therapy with the relevant allergens. In addition to the description of up-to-date allergen gene vaccine approaches, this review gives an overview of animal studies dealing with the following topics: danger signals as the inherent adjuvant properties, methods to optimize the vaccine immunogenicity, modulation of the immune response, nonparenteral applications and low-dose vaccination strategies.
International Archives of Allergy and Immunology 02/2006; 139(4):332-45. · 2.40 Impact Factor
