We assessed the safety and immunogenicity of HIV-DNA priming using Zetajet™, a needle-free device intradermally followed by intramuscular HIV-MVA boosts, in 24 healthy Mozambicans. Volunteers were randomized to receive three immunizations of 600 μg (n = 10; 2 × 0.1 ml) or 1,200 μg (n = 10; 2 × 0.2 ml) of HIV-DNA (3 mg/ml), followed by two boosts of 108 pfu HIV-MVA. Four subjects received placebo saline injections. Vaccines and injections were safe and well tolerated with no difference between the two priming groups. After three HIV-DNA immunizations, IFN-γ ELISpot responses to Gag were detected in 9/17 (53%) vaccinees, while none responded to Envelope (Env). After the first HIV-MVA, the overall response rate to Gag and/or Env increased to 14/15 (93%); 14/15 (93%) to Gag and 13/15 (87%) to Env. There were no significant differences between the immunization groups in frequency of response to Gag and Env or magnitude of Gag responses. Env responses were significantly higher in the higher dose group (median 420 vs. 157.5 SFC/million peripheral blood mononuclear cell, p = .014). HIV-specific antibodies to subtype C gp140 and subtype B gp160 were elicited in all vaccinees after the second HIV-MVA, without differences in titers between the groups. Neutralizing antibody responses were not detected. Two (13%) of 16 vaccinees, one in each of the priming groups, exhibited antibodies mediating antibody-dependent cellular cytotoxicity to CRF01_AE. In conclusion, HIV-DNA vaccine delivered intradermally in volumes of 0.1-0.2 ml using Zetajet was safe and well tolerated. Priming with the 1,200 μg dose of HIV-DNA generated higher magnitudes of ELISpot responses to Env.

The delivery of plasmid DNA to the skin can target distinct subsets of dermal dendritic cells to confer a superior immune response. The needle-free immunization technology offers a reliable, safe and efficient means to administer intradermal (ID) injections. We report here that the ID injection of DNA vectors using an NF device (NF-ID) elicits a superior cell-mediated immune response, at much lesser DNA dosage, comparable in magnitude to the traditional intramuscular immunization. However, the humoral response is significantly impaired, possibly at the stage of B cell isotype switching. We found that the NF-ID administration deposits the DNA primarily on the epidermis resulting in a rapid loss of the DNA as well as the synthesized antigen due to the faster regeneration rate of the skin layers. Therefore, despite the immune-rich nature of the skin, the NF-ID immunization of DNA vectors may be limited by the impaired humoral response. Additional booster injections are required to augment the antibody response. As an alternative and a viable solution, we rescued the IgG response by coadministration of a Toll-like receptor 9 agonist, among other adjuvants examined. Our work has important implication for the optimization of the emerging needle-free technology for ID immunization.

BACKGROUND:Intradermal priming with HIV-1 DNA plasmids followed by HIV-1MVA boosting induces strong and broad cellular and humoral immune responses. In our previous HIVIS-03 trial, we used 5 injections with 2 pools of HIV-DNA at separate sites for each priming immunization. The present study explores whether HIV-DNA priming can be simplified by reducing the number of DNA injections and administration of combined versus separated plasmid pools.METHODS:In this phase IIa, randomized trial, priming was performed using 5 injections of HIV-DNA, 1000 μg total dose, (3 Env and 2 Gag encoding plasmids) compared to two "simplified" regimens of 2 injections of HIV-DNA, 600 μg total dose, of Env- and Gag-encoding plasmid pools with each pool either administered separately or combined. HIV-DNA immunizations were given intradermally at weeks 0, 4, and 12. Boosting was performed intramuscularly with 108 pfu HIV-MVA at weeks 30 and 46.RESULTS:129 healthy Tanzanian participants were enrolled. There were no differences in adverse events between the groups. The proportion of IFN-γ ELISpot responders to Gag and/or Env peptides after the second HIV-MVA boost did not differ significantly between the groups primed with 2 injections of combined HIV-DNA pools, 2 injections with separated pools, and 5 injections with separated pools (90%, 97% and 97%). There were no significant differences in the magnitude of Gag and/or Env IFN-γ ELISpot responses, in CD4+ and CD8+ T cell responses measured as IFN-γ/IL-2 production by intracellular cytokine staining (ICS) or in response rates and median titers for binding antibodies to Env gp160 between study groups.CONCLUSIONS:A simplified intradermal vaccination regimen with 2 injections of a total of 600 μg with combined HIV-DNA plasmids primed cellular responses as efficiently as the standard regimen of 5 injections of a total of 1000 μg with separated plasmid pools after boosting twice with HIV-MVA.TRIAL REGISTRATION:World Health Organization International Clinical Trials Registry Platform PACTR2010050002122368.

BACKGROUND:We compared safety and immunogenicity of intradermal (ID) vaccination with and without electroporation (EP) in a phase I randomized placebo-controlled trial of an HIV-DNA prime HIV-MVA boost vaccine in healthy Swedish volunteers.METHODS:HIV-DNA plasmids encoding HIV-1 genes gp160 subtypes A, B and C; Rev B; Gag A and B and RTmut B were given ID at weeks 0, 6 and 12 in a dose of 0.6 mg. Twenty-five volunteers received vaccine using a needle-free device (ZetaJet) with (n=16) or without (n=9) ID EP (Dermavax). Five volunteers were placebo recipients. Boosting with recombinant MVA-CMDR expressing HIV-1 Env, Gag, Pol of CRF01_AE (HIV-MVA) or placebo was performed at weeks 24 and 40. Nine of the vaccinees received a subtype C CN54 gp140 protein boost together with HIV-MVA.RESULTS:The ID/EP delivery was very well tolerated. After three HIV-DNA immunizations, no statistically significant difference was seen in the IFN-γ ELISpot response rate to Gag between HIV-DNA ID/EP recipients (5/15, 33%) and HIV-DNA ID recipients (1/7, 14%, p=0.6158). The first HIV-MVA or HIV-MVA+gp140 vaccination increased the IFN-γ ELISpot response rate to 18/19 (95%). CD4+ and/or CD8+ T cell responses to Gag or Env were demonstrable in 94% of vaccinees. A balanced CD4+ and CD8+ T cell response was noted, with 78% and 71% responders, respectively. IFN-γ and IL-2 dominated the CD4+ T cell response to Gag and Env. The CD8+ response to Gag was broader with expression of IFN-γ, IL-2, MIP-1β and/or CD107. No differences were seen between DNA vaccine groups. Binding antibodies were induced after the second HIV-MVA+/-gp140 in 93% of vaccinees to subtype C Env, with the highest titers among EP/gp140 recipients.CONCLUSION:Intradermal electroporation of HIV-DNA was well tolerated. Strong cell- and antibody-mediated immune responses were elicited by the HIV-DNA prime and HIV-MVA boosting regimen, with or without intradermal electroporation use.TRIAL REGISTRATION:International Standard Randomised Controlled Trial Number (ISRCTN) 60284968.

AIDS Research and Human RetrovirusesVol. 30, No. S1 Vaccine Development: Emerging InsightsIntradermal HIV-DNA Given with or without Intradermal Electroporation Is Safe and Highly Immunogenic in Healthy Swedish HIV-1 DNA/MVA VaccineesCharlotta Nilsson, Bo Hejdeman, Karina Godoy-Ramirez, Teghesti Tecleab, Gabriella Scarlatti, Andreas Bråve, Patricia L. Earl, Richard R. Stout, Merlin L. Robb, Robin Shattock, Gunnel Biberfeld, Eric Sandström, and Britta WahrenCharlotta NilssonThe Public Health Agency of Sweden, Solna, SwedenKarolinska Institutet, Department of Microbiology, Tumor and Cell Biology, Stockholm, SwedenKarolinska Institutet, Department of Laboratory Medicine, Huddinge, SwedenSearch for more papers by this author, Bo HejdemanSödersjukhuset and Karolinska Institutet, Venhälsan and Department of Education and Clinical Research, Stockholm, SwedenSearch for more papers by this author, Karina Godoy-RamirezThe Public Health Agency of Sweden, Solna, SwedenSearch for more papers by this author, Teghesti TecleabThe Public Health Agency of Sweden, Solna, SwedenSearch for more papers by this author, Gabriella ScarlattiIRCCS San Raffaele Scientific Institute, Milan, ItalySearch for more papers by this author, Andreas BråveThe Public Health Agency of Sweden, Solna, SwedenKarolinska Institutet, Department of Microbiology, Tumor and Cell Biology, Stockholm, SwedenSearch for more papers by this author, Patricia L. EarlNational Institutes of Health, National Institute of Allergy and Infectious Diseases, Bethesda, MD, United StatesSearch for more papers by this author, Richard R. StoutBioject Inc., Tualatin, OR, United StatesSearch for more papers by this author, Merlin L. RobbWalter Reed Army Institute of Research, Department of Retrovirology, Rockville, MD, United StatesSearch for more papers by this author, Robin ShattockImperial College, Department of Infectious Diseases, Division of Medicine, London, United KingdomSearch for more papers by this author, Gunnel BiberfeldThe Public Health Agency of Sweden, Solna, SwedenKarolinska Institutet, Department of Microbiology, Tumor and Cell Biology, Stockholm, SwedenSearch for more papers by this author, Eric SandströmSödersjukhuset and Karolinska Institutet, Venhälsan and Department of Education and Clinical Research, Stockholm, SwedenSearch for more papers by this author, and Britta WahrenKarolinska Institutet, Department of Microbiology, Tumor and Cell Biology, Stockholm, SwedenSearch for more papers by this authorPublished Online:30 Oct 2014https://doi.org/10.1089/aid.2014.5049.abstractAboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View article"Intradermal HIV-DNA Given with or without Intradermal Electroporation Is Safe and Highly Immunogenic in Healthy Swedish HIV-1 DNA/MVA Vaccinees." AIDS Research and Human Retroviruses, 30(S1), pp. A31–A32FiguresReferencesRelatedDetails Volume 30Issue S1Oct 2014 InformationCopyright 2014, Mary Ann Liebert, Inc.To cite this article:Charlotta Nilsson, Bo Hejdeman, Karina Godoy-Ramirez, Teghesti Tecleab, Gabriella Scarlatti, Andreas Bråve, Patricia L. Earl, Richard R. Stout, Merlin L. Robb, Robin Shattock, Gunnel Biberfeld, Eric Sandström, and Britta Wahren.Intradermal HIV-DNA Given with or without Intradermal Electroporation Is Safe and Highly Immunogenic in Healthy Swedish HIV-1 DNA/MVA Vaccinees.AIDS Research and Human Retroviruses.Oct 2014.A31-A32.http://doi.org/10.1089/aid.2014.5049.abstractPublished in Volume: 30 Issue S1: October 30, 2014PDF download

AIDS Research and Human RetrovirusesVol. 30, No. S1 Vaccine Clinical TrialsFree AccessPhase I HIV Vaccine Trial to Assess Safety and Immunogenicity of DNA Priming and MVA Boosting in Healthy Mozambican Young AdultsEdna Omar Viegas, Nelson Tembe, Charlotta Nilsson, Bindiya Meggi, Norma Mabota, Nádia Sitoe, Cremildo Maueia, Raquel Chissumba, Victória Cumbane, Eulália Macovela, Emília Gonçalves, Rick Stout, Gabriella Scarlatti, Britta Wahren, Soren Andersson, Mary Marovich, Merlin Robb, Nafissa Osman, Gunnel Biberfeld G, Ilesh Jani, and Eric SandströmEdna Omar ViegasInstituto Nacional de Saúde, Maputo, MozambiqueKarolinska Institutet, Stockholm, SwedenEduardo Mondlane University, Maputo, MozambiqueSearch for more papers by this author, Nelson TembeInstituto Nacional de Saúde, Maputo, MozambiqueKarolinska Institutet, Stockholm, SwedenEduardo Mondlane University, Maputo, MozambiqueSearch for more papers by this author, Charlotta NilssonKarolinska Institutet, Stockholm, SwedenPublic Health Agency of Sweden, Stockholm, SwedenSearch for more papers by this author, Bindiya MeggiInstituto Nacional de Saúde, Maputo, MozambiqueSearch for more papers by this author, Norma MabotaInstituto Nacional de Saúde, Maputo, MozambiqueSearch for more papers by this author, Nádia SitoeInstituto Nacional de Saúde, Maputo, MozambiqueSearch for more papers by this author, Cremildo MaueiaInstituto Nacional de Saúde, Maputo, MozambiqueSearch for more papers by this author, Raquel ChissumbaInstituto Nacional de Saúde, Maputo, MozambiqueSearch for more papers by this author, Victória CumbaneInstituto Nacional de Saúde, Maputo, MozambiqueSearch for more papers by this author, Eulália MacovelaEduardo Mondlane University, Maputo, MozambiqueHospital Central de Maputo, Maputo, MozambiqueSearch for more papers by this author, Emília GonçalvesEduardo Mondlane University, Maputo, MozambiqueHospital Central de Maputo, Maputo, MozambiqueSearch for more papers by this author, Rick StoutBioject Inc., Tualatin, OR, United StatesSearch for more papers by this author, Gabriella ScarlattiIRCCS San Raffaele Scientific Institute, Milan, ItalySearch for more papers by this author, Britta WahrenKarolinska Institutet, Stockholm, SwedenSearch for more papers by this author, Soren AnderssonÖrebro University Hospital, Örebro, SwedenSearch for more papers by this author, Mary MarovichWalter Reed Army Institute of Research, Rockville, MD, United StatesSearch for more papers by this author, Merlin RobbWalter Reed Army Institute of Research, Rockville, MD, United StatesSearch for more papers by this author, Nafissa OsmanEduardo Mondlane University, Maputo, MozambiqueHospital Central de Maputo, Maputo, MozambiqueSearch for more papers by this author, Gunnel Biberfeld GKarolinska Institutet, Stockholm, SwedenPublic Health Agency of Sweden, Stockholm, SwedenSearch for more papers by this author, Ilesh JaniInstituto Nacional de Saúde, Maputo, MozambiqueSearch for more papers by this author, and Eric SandströmKarolinska Institutet, Stockholm, SwedenSearch for more papers by this authorPublished Online:30 Oct 2014https://doi.org/10.1089/aid.2014.5412.abstractAboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail P26.16Background: Safety and immunogenicity of intradermal HIV-DNA priming at doses of 600μg or 1200μg followed by intramuscular HIV-MVA-CMDR boosts were evaluated in this study.Methods: Healthy volunteers (n=24), aged 18–26, recruited at a Youth Clinic in Maputo, Mozambique, were randomized into two groups: 10 volunteers received 600μg (0.1ml) or 1200μg (0.2ml), respectively, of DNA intradermally using a needle-free device, Zetajet (M0, M1, and M3), followed by MVA boosts (M6 and M9), and four received placebo. Safety endpoints were assessed clinically and by standard chemistry and haematology tests. Primary immunogenicity endpoint was determined by IFN- γ ELISpot.Results: The ratio of screening and enrollment was 3:1.The main reasons for screening failures were risk behaviour, abnormal CBC and positive syphilis tests. During the trial, 2 volunteers became pregnant and 1 was infected with HIV. None of them had completed their vaccination schedule. HIV infection was the only Serious Adverse Event registered. Two grade 3 and 2 grade 4 laboratory adverse events were registered without probable relationship to vaccination.After the first MVA, 14/15 (93%) of vaccinees were IFN-γ ELISpot reactive:13 (87%) to Env and 14 (93%) to Gag. A significantly higher median ELISpot response to Env was seen in high dose recipients (420 SFC/106 PBMCs) compared to low dose recipients (157 SFC/106 PBMCs), p=0.05. A non-significant difference in response to Gag was seen between groups. All 16 vaccinees (median titer 800, range 400–3200) exhibited binding antibodies (Ab) to subtype C gp140 Env but no difference in ELISpot or binding Ab responses was seen between groups after the second HIV-MVA. Neutralizing Ab responses were not detected against pseudovirus 93MW962.23 in TZM-bl assay or against SF162 in a PBMC-based assay.Conclusions: This DNA priming-MVA boosting strategy is safe and highly immunogenic in Mozambican vaccinees. High dose recipients exhibited higher magnitudes of IFN-γ ELISpot responses to Env than low dose recipients.FiguresReferencesRelatedDetails Volume 30Issue S1Oct 2014 InformationCopyright 2014, Mary Ann Liebert, Inc.To cite this article:Edna Omar Viegas, Nelson Tembe, Charlotta Nilsson, Bindiya Meggi, Norma Mabota, Nádia Sitoe, Cremildo Maueia, Raquel Chissumba, Victória Cumbane, Eulália Macovela, Emília Gonçalves, Rick Stout, Gabriella Scarlatti, Britta Wahren, Soren Andersson, Mary Marovich, Merlin Robb, Nafissa Osman, Gunnel Biberfeld G, Ilesh Jani, and Eric Sandström.Phase I HIV Vaccine Trial to Assess Safety and Immunogenicity of DNA Priming and MVA Boosting in Healthy Mozambican Young Adults.AIDS Research and Human Retroviruses.Oct 2014.A192-A192.http://doi.org/10.1089/aid.2014.5412.abstractPublished in Volume: 30 Issue S1: October 30, 2014PDF download

Background The use of optimized delivery devices has been shown to enhance the potency of DNA vaccines. However, further optimization of DNA vaccine delivery is needed for this vaccine modality to ultimately be efficacious in humans. Methods Herein we evaluated antigen expression and immunogenicity after intradermal delivery of different doses of DNA vaccines by needle or by the Biojector jet-injection device, with or without the addition of electroporation (EP). Results Neither needle injection augmented by EP nor Biojector alone could induce higher magnitudes of immune responses after immunizations with a high dose of DNA. After division of a defined DNA dose into multiple skin sites, the humoral response was particularly enhanced by Biojector while cellular responses were particularly enhanced by EP. Furthermore, a close correlation between in vivo antigen expression and cell-mediated as well as humoral immune responses was observed. Conclusions These results show that two optimized DNA vaccine delivery devices can act together to overcome dose restrictions of plasmid DNA vaccines.

It is likely that gene-based vaccines will enter the human vaccine area soon. A few veterinary vaccines employing this concept have already been licensed, and a multitude of clinical trials against infectious diseases or different forms of cancer are ongoing. Highly important when developing novel vaccines are the safety aspects and also new adjuvants and delivery techniques needs to be carefully investigated so that they meet all short- and long-term safety requirements. One novel in vivo delivery method for plasmid vaccines is electroporation, which is the application of short pulses of electric current immediately after, and at the site of, an injection of a genetic vaccine. This method has been shown to significantly augment the transfection efficacy and the subsequent vaccine-specific immune responses. However, the dramatic increase in delivery efficacy offered by electroporation has raised concerns of potential increase in the risk of integration of plasmid DNA into the host genome. Here, we demonstrate the safety and lack of integration after immunization with a high dose of a multigene HIV-1 vaccine delivered intradermally using the needle free device Biojector 2000 together with electroporation using Derma Vax™ DNA Vaccine Skin Delivery System. We demonstrate that plasmids persist in the skin at the site of injection for at least four months after immunization. However, no association between plasmid DNA and genomic DNA could be detected as analyzed by qPCR following field inversion gel electrophoresis separating heavy and light DNA fractions. We will shortly initiate a phase I clinical trial in which healthy volunteers will be immunized with this multiplasmid HIV-1 vaccine using a combination of the delivery methods jet-injection and intradermal electroporation.

The traditional assay used to measure potency of inactivated influenza vaccines is a single-radial immunodiffusion (SRID) assay that utilizes an influenza strain-specific antibody to measure the content of virus hemagglutinin (HA) in the vaccine in comparison to a homologous HA reference antigen. Since timely preparation of potency reagents by regulatory authorities is challenging and always a potential bottleneck in influenza vaccine production, it is extremely important that additional approaches for reagent development be available, particularly in the event of an emerging pandemic influenza virus. An alternative method for preparation of strain-specific antibody that can be used for SRID potency assay is described. The approach does not require the presence or purification of influenza virus, and furthermore, is not limited by the success of the traditional technique of bromelain digestion and purification of virus HA. Multiple mammalian expression vectors, including plasmid and modified vaccinia virus Ankara (MVA) vectors expressing the HAs of two H5N1 influenza viruses and the HA of the recently emerging pandemic H1N1 (2009) virus, were developed. An immunization scheme was designed for the sequential immunization of animals by direct vector injection followed by protein booster immunization using influenza HA produced in vitro from MVA vector infection of cells in culture. Each HA antibody was highly specific as shown by hemagglutination inhibition assay and the ability to serve as a capture antibody in ELISA. Importantly, each H5N1 antibody and the pandemic H1N1 (2009) antibody preparation were suitable for use in SRID assays for determining the potency of pandemic influenza virus vaccines. The results demonstrate a feasible approach for addressing one of the potential bottlenecks in inactivated pandemic influenza vaccine production and are particularly important in light of the difficulties in preparation of potency reagent antibody for pandemic H1N1 (2009) virus vaccines.

Heterologous boost immunisation is considered the most efficient way to enhance DNA-primed immune responses. We have previously shown that administration of recombinant carcinoembryonic antigen (CEA) efficiently boosts humoral responses in mice primed with CEA DNA. However, clinical grade recombinant proteins are far more intriguing to produce than plasmid DNA. Therefore, the possibility to use plasmid DNA for both priming and boosting would be beneficial. With the prospect of future use in a clinical trial, we investigated if electroporation-mediated delivery of DNA could be used to boost DNA-primed immune responses to CEA. The Biojector was used to prime BALB/c mice intradermally three times with CEA66 DNA, encoding an intracellular modified form of CEA. Twelve weeks after the last prime, the animals received either one injection of recombinant CEA or one intradermal injection of twtCEA DNA, encoding the wild type CEA fused to a tetanus T helper epitope, in combination with electroporation. Boosting with rCEA protein did not enhance T cell responses to CEA but induced CEA-specific IgG in 4 of 8 mice. In contrast, intradermal delivery of twtCEA DNA by electroporation led to a tenfold increase in IFN-gamma-producing CD8+ T cells, compared to the levels obtained after the third priming immunisation. The DNA boost also induced high CEA-specific IgG titers in all immunised animals (8/8). The data suggests that a late DNA boost, in combination with enhanced DNA delivery by electroporation, could be used to enhance the efficiency of DNA vaccination and substitute for a heterologous protein boost vaccination.

BACKGROUNDA human immunodeficiency virus (HIV) vaccine that limits disease and transmission is urgently needed. This clinical trial evaluated the safety and immunogenicity of an HIV vaccine that combines a plasmid-DNA priming vaccine and a modified vaccinia virus Ankara (MVA) boosting vaccine.METHODSForty healthy volunteers were injected with DNA plasmids containing gp160 of HIV-1 subtypes A, B, and C; rev B; p17/p24 gag A and B, and RTmut B by use of a needle-free injection system. The vaccine was administered intradermally or intramuscularly, with or without recombinant granulocyte macrophage colony-stimulating factor, and boosted with a heterologous MVA containing env, gag, and pol of CRF01A_E. Immune responses were monitored with HIV-specific interferon (IFN)-gamma and interleukin (IL)-2 ELISpot and lymphoproliferative assays (LPAs).RESULTSVaccine-related adverse events were mild and tolerable. After receipt of the DNA priming vaccine, 11 (30%) of 37 vaccinees had HIV-specific IFN-gamma responses. After receipt of the MVA boosting vaccine, ELISpot assays showed that 34 (92%) of 37 vaccinees had HIV-specific IFN-gamma responses, 32 (86%) to Gag and 24 (65%) to Env. IFN-gamma production was detected in both the CD8(+) T cell compartment (5 of 9 selected vaccinees) and the CD4(+) T cell compartment (9 of 9). ELISpot results showed that 25 (68%) of 37 vaccinees had a positive IL-2 response and 35 (92%) of 38 had a positive LPA response. Of 38 subjects, a total of 37 (97%) were responders. One milligram of HIV-1 DNA administered intradermally was as effective as 4 mg administered intramuscularly in priming for the MVA boosting vaccine.CONCLUSIONThis HIV-DNA priming-MVA boosting approach is safe and highly immunogenic.TRIALS REGISTRATIONInternational Standard Randomised Controlled Trial number: ISRCTN32604572 .

Although growth and productivity of most crops are significantly reduced by exposure to temperatures in excess of 35°C, some plants thrive in geothermally heated soils characterized by chronic temperatures in excess of 40°C. The morphological adaptations that enable these plants to colonize thermal environments are poorly understood. We characterized trichome development, silica accumulation, stomatal density, and waxy cuticle development in populations of Dichanthelium lanuginosum var sericeum (Schmoll) isolated from thermal and non-thermal environments to determine whether morphological changes existed in populations adapted to thermal environments. Plants isolated from thermal environments of Yellowstone National Park developed an extensive series of trichomes when exposed to chronic temperatures of 45/35°C (day/night). In contrast, isolates from non-thermal environments of western Oregon showed minimal trichome development during exposure to the elevated temperatures. Leaves that developed during exposure to elevated temperatures had reduced thickness, though the reduction was less marked in plants from populations isolated from thermal environments. Plants isolated from thermal environments also had greater numbers of stomata on adaxial leaf surfaces relative to biotypes isolated from non-thermal environments. These results suggest phenotypic characteristics that may be useful in selection of new varieties of crop species with improved tolerance to supraoptimal temperatures.

Measles still causes considerable morbidity and mortality among infants and young children in developing countries. To develop a new public health tool to reduce this burden, we designed two Sindbis virus replicon vaccines encoding measles virus (MV) hemagglutinin (H) and fusion (F) proteins (pMSIN-H and pMSINHFdU). Our goal is to administer the vaccines to young infants at 6 and 10 weeks of age to prime the immune system to safely and effectively respond to subsequent immunization at age approximately 14 weeks with the licensed attenuated measles vaccine. In preparation for a phase 1 clinical trial, studies of plasmid distribution, integration, and toxicology were performed in rabbits. Biodistribution was assessed after a single DNA immunization delivered intradermally by needle-free injection. Toxicity was assessed using a heterologous prime-boost regimen consisting of a repeat-dose DNA prime followed by a live-attenuated measles vaccine boost. The only vaccine-related adverse effects observed were minimal transient erythema, edema, and inflammation confined to the injection site. Plasmids were detected in the subcutis and muscle at the site of inoculation. A small proportion of animals exhibited plasmids in the regional lymph nodes. There was no evidence of plasmid integration into the host genome. Both Sindbis-based vaccine plasmids were immunogenic in rabbits; pMSIN-H elicited higher virus-neutralizing antibody levels. Both vaccines were shown to be well tolerated and suitable for clinical trials and they are currently being tested in phase 1 studies in young adults.

Hot springs panic grass, Dichanthelium lanuginosum var sericeum (Schmoll), successfully colonizes geo-thermally heated soils that are subject to chronic temperatures ranging from 40 to 50°C, occurs at altitudes in excess of 2500m, and tolerates low soil moisture content for extended periods of time. We utilized superoxide dismutase (SOD; EC 1.15.1.1) as an indicator of oxidative stress response to compare the impacts of temperature on wheat (Triticum aestivum L.), a cool-season grass, with the temperature responses of three isolates of D. lanuginosum. One of the D. lanuginosum isolates was collected from a non-thermal site and had reduced capacity to adapt to growth at elevated temperature. Wheat SOD activity in crude leaf extracts was significantly reduced by incubation at 45°C. In contrast, SOD activity in crude leaf extracts from a thermophilic D. lanuginosum isolate increased after incubation at 45°C. Significant increases in cellular chelating capacity occurred in wheat and the D. lanuginosum isolated from a non-thermal environment after plants were exposed to elevated temperatures. Ultra-filtration of leaf extracts through 10kDa molecular weight cutoff membranes removed much of the chelating activity and restored apparent SOD activity. The D. lanuginosum isolates with the greatest thermotolerance retained SOD activity when exposed to elevated temperatures and did not show statistically significant increases in cellular chelation potential. Wheat SOD activity increased in response to heat shock. The chelation activity was associated with low molecular weight components (<10kDa) that did not bind C18 media. Ultra-filtration removal of these low molecular weight chelating components increased SOD activity and replacement of low molecular weight chelating components with an equivalent amount of EDTA suppressed SOD activity.

The results presented here are from the preclinical evaluation in BALB/c mice of a DNA prime/modified vaccinia virus Ankara (MVA) boost multi-gene multi-subtype human immunodeficiency virus-1 (HIV-1) vaccine intended for use in humans. The plasmid DNA vaccine was delivered intradermally using a Biojector, and the MVA was delivered intramuscularly by needle. This combination of recombinant DNA and MVA proved to induce extraordinarily strong cellular responses, with more than 80% of the CD8(+) T cells specific for HIV-1 antigens. Furthermore, we show that the DNA priming increases the number of T-cell epitopes recognized after the MVA boost. In the prime/boost-immunized animals, a significant proportion of CD8(+) T cells were stained positive for both interferon-gamma (IFN-gamma) and interleukin-2 (IL-2), a feature that has been associated with control of HIV-1 infection in long-term non-progressors. The HIV-1-specific antibody levels were moderate after the plasmid DNA immunizations but increased dramatically after the MVA boost. Although the initial injection of MVA induced significant levels of vaccinia-neutralizing antibodies, the HIV-specific responses were still significantly boosted by the second MVA immunization. The results from this study demonstrate the potency of this combination of DNA plasmids and MVA construct to induce broad and high levels of immune responses against several HIV-1 proteins of different subtypes.

Mucosal administration (intranasal or oral) of a VP6 rotavirus vaccine to mice consistently elicits high levels of protection after rotavirus challenge (93–>99% reductions in fecal rotavirus shedding) but only when co-administered with an effective adjuvant such as LT(R192G). Here, we showed that Biojector needle-free injection of VP6-encoded plasmids also induced protection (85–93%) when they were co-administrated with LT(R192G)-encoded plasmids. A reduction in the amount of VP6 plasmid from 50 to 10μg reduced protection from 93 to 70%, but the immunized mice remained significantly (P<0.05) protected. Intramuscular needle injection of VP6/LT(R192G)-plasmids also induced significant protection (66%).

The route and method of immunization, as well as the cellular localization of the antigen, can influence the generation of an immune response. In general, intramuscular immunization results in Th1 responses, whereas intradermal delivery of DNA by gene gun immunization often results in more Th2 responses. Here we investigate how altering the cellular localization of the tumor antigen CEA (carcinoembryonic antigen) affects the quality and amplitude of DNA vaccine-induced antibody responses in mice following intradermal delivery of DNA by a needle-free jet injection device (Biojector). CEA was expressed either in a membrane-bound form (wild-type CEA) or in two truncated forms (CEA6 and CEA66) with cytoplasmic localization, where CEA66 was fused to a promiscuous T-helper epitope from tetanus toxin. Repeated intradermal immunization of BALB/c mice with DNA encoding wild-type CEA produced high antibody titers of a mixed IgG1/IgG2a ratio. In contrast, utilizing the DNA construct that resulted in intracellular targeting of CEA led to a reduced capacity to induce CEA-specific antibodies, but instead induced a Th1-biased immune response.

A previous report described a prime-boost immunization strategy using plasmid and modified vaccinia virus Ankara (MVA) vectors expressing herpes simplex: virus 2 glycoprotein D (gD). Enhanced humoral and cellular immune responses were elicited by the prime-boost combination compared to plasmid DNA immunization alone. Surprisingly, a more diverse antibody isotype response, and a greater antibody and cellular immune response, was obtained if the gD MVA vector was used as the priming immunization rather than the gD plasmid vector. The present report evaluates the use of a needle-free delivery platform (Biojector) for delivery of plasmid and MVA gD-expressing vectors in a prime-boost immunization strategy. Needle-free delivery of both plasmid and MVA gD expression vectors was efficient, reproducible, and elicited a strong immune response in immunized mice. Biojector delivery of plasmid DNA was able to evoke a broader isotype response and cellular immune response than that obtained by gene gun delivered plasmid DNA. Further, DNA priming by Biojector delivery as part of a prime-boost procedure with MVA-gD2 resulted in a diverse antibody isotype distribution and enhanced cellular immune responses, similar to the responses obtained when MVA-gD2 was used as the priming immunization. Thus, needle-free delivery of plasmid DNA may provide additional flexibility and options for effective prime-boost vaccination.

Study Objectives. To compare the relative bioavailability of enfuvirtide, a human immunodeficiency virus type 1 (HIV-1) fusion inhibitor, injected with the Biojector 2000 (B2000) needle-free device versus a 27-gauge half-inch needle-syringe; and to assess safety, tolerability, and patient preference for the two devices.Design. Open-label, randomized, two-period crossover bioequivalence evaluation.Setting. Clinical research center.Patients. Twenty-seven adults with HIV-1 viral loads below 1000 copies/ml.Intervention. Each patient received enfuvirtide 90 mg subcutaneously with the B2000 and with the needle-syringe, with a 1-week washout between treatments.Measurements and Main Results. Twenty-six and 27 patients were included in the bioequivalence and safety analyses, respectively. Plasma enfuvirtide concentrations were measured at baseline and at several intervals after each injection. The B2000:needle-syringe ratios of maximum concentration (C-max), area under the concentration-time curve from time zero extrapolated to infinity (AUC(0-infinity)), and AUC from time zero to tau (dosing interval) (AUC(0-tau)) served as criteria for bioequivalence determination. The two drug delivery systems were considered bioequivalent if the 90% confidence intervals (CIs) for the ratios were within 0.8-1.25. Safety and tolerability were evaluated based on documentation of adverse events, graded laboratory toxicities, and local injection-site reactions. Patient surveys provided feedback on device preference. Ratios of C-max, AUC(0-infinity), and AUC(0-tau) were 0.95 (90% CI 0.84-1.09), 0.99 (90% CI 0.93-1.05), and 0.99 (90% CI 0.93-1.05), respectively. The frequency of injection-site reactions was low, and severity was generally mild for both devices. Survey results showed 18 patients (69%) had a positive overall impression of the B2000 and 14 (54%) felt safer injecting with this device. Overall, 17 patients (65%) preferred the B2000 over the needle-syringe.Conclusion. Bioavailability of enfuvirtide with the B2000 and needle-syringe was equivalent based on C-max, AUC(0-tau), and AUC(0-infinity). Safety profiles and injection-site reactions were comparable between the devices, but patients preferred the B2000. Delivery of enfuvirtide with the B2000 is a feasible alternative to standard needle administration and warrants further evaluation.