A combination of intradermal jet-injection and electroporation overcomes in vivo dose restriction of DNA vaccines.

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.

Intradermal HIV-1 DNA Immunization Using Needle-Free Zetajet Injection Followed by HIV-Modified Vaccinia Virus Ankara Vaccination Is Safe and Immunogenic in Mozambican Young Adults: A Phase I Randomized Controlled Trial.

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.

Intracellular targeting of CEA results in Th1-type antibody responses following intradermal genetic vaccination by a needle-free jet injection device.

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.

Evaluation of a needle-free delivery platform for prime-boost immunization with DNA and modified vaccinia virus ankara vectors expressing herpes simplex virus 2 glycoprotein D.

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.

Pharmacokinetic bioequivalence of enfuvirtide using a needle-free device versus standard needle administration.

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.

Toll-Like Receptor 9 Activation Rescues Impaired Antibody Response in Needle-free Intradermal DNA Vaccination.

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.

Studies on the use of needle-free injection device on proteins.

In the following communication we report the evaluation of 18 proteins that were processed by a specific needle free injection device. The processed protein samples were analyzed by two HPLC techniques, reversed-phase liquid chromatography (RPLC) and size-exclusion chromatography (SEC). These techniques are two of the most widely used analytical techniques in the biopharmaceutical industry for the characterization, integrity assessment and stability study of peptide and protein products. The results indicate that needle free injection, using the specific device of this study, is not damaging to the studied proteins and does not generate aggregates. We found no evidence of the predicted possible effects of needle free injections, and concluded that needle free delivery is in general not different than any other delivery system and that its use should be evaluated on a case by case basis. It has to be noted that there are various needle free device designs and our work was performed using an Iject from Bioject. Our conclusions therefore should be limited to the Iject design we used in this study. In the reported experiments we used commercially available (economical) model proteins, which facilitate the use of the results for future comparison and reference. The work reported here can serve as a reference to illustrate the benign nature of our needle free injection device. It also highlights an interesting analogy between a set of phobias that were seen to have plagued the early stages of biochemistry and HPLC, on the one hand, and some attitudes that appear to hinder the widespread acceptance of needle free injection at present time, on the other. These phobias were identified and named by Professor Csaba Horváth, the father of HPLC, as barophobia, siderophobia and lithophobia. Today a wealth of evidence is available to indicate that those phobias are ungrounded and that the negative observations can be explained in most cases by adsorption and prevented by proper formulations and solvent conditions.

HIV-DNA Given with or without Intradermal Electroporation Is Safe and Highly Immunogenic in Healthy Swedish HIV-1 DNA/MVA Vaccinees: A Phase I Randomized Trial.

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-1ß 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.

Priming with a simplified intradermal HIV-1 DNA vaccine regimen followed by boosting with recombinant HIV-1 MVA vaccine is safe and immunogenic: a phase IIa randomized clinical trial.

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.

Safety, tolerability, and lack of antibody responses after administration of a PfCSP DNA malaria vaccine via needle or needle-free jet injection, and comparison of intramuscular and combination intramuscular/intradermal routes.

Introduction of a new vaccine requires choosing a delivery system that provides safe administration and the desired level of immunogenicity. The safety, tolerability, and immunogenicity of three monthly 2.5-mg doses of a PfCSP DNA vaccine were evaluated in healthy volunteers as administered intramuscularly (IM) by needle, IM by jet injection (Biojector or IM/intradermally (ID) by jet injection. Vaccine administration was well-tolerated. Adverse events were primarily mild and limited to the site of injection (98%). Jet injections (either IM or ID) were associated with approximately twice as many adverse events per immunization as needle IM, but nevertheless were strongly and consistently preferred in opinion polls taken during the study. No volunteers had clinically significant biochemical or hematologic changes or detectable anti-dsDNA antibodies. In conclusion, the injection of Plasmodium falciparum circumsporozoite (PfCSP) DNA vaccine appeared to be safe and well-tolerated when administered by any of the three modes of delivery. However, despite improved antibody responses following both jet injection and ID delivery in animal models, no antibodies could be detected in volunteers by immunofluorescence antibody test (IFAT) or enzyme-linked immunosorbent assay (ELISA) after DNA vaccination.

Jet injection devices for the needle-free administration of compounds, vaccines, and other agents.

Jet injection involves the use of a needle-free device that delivers a prescribed drug, vaccine, or compound intradermally, subcutaneously, or intramuscularly via high pressure produced by either a carbon-dioxide-filled or nitrogen-filled cartridge or a spring. During that procedure, the injector is held at an angle against the patient's skin, and a very fine stream of liquid medication is forced through a tiny orifice in the device, penetrates the skin in a selected volume ranging from 0.05 mL to 1.0 mL, and is deposited in the underlying tissue. When compared with methods of injection that require a needle, jet injection offers multiple benefits. It can be less painful for the patient, and it enhances compliance, reduces risks such as needlestick injuries and cross-contamination, eliminates the need for "sharps" disposal, and enables (with minimal training) the reliable, reproducible, and accurate delivery of medication. Patient convenience is also a factor: Jet injectors are designed for self-medication as well as professional use. It must be remembered, however, that treatment via jet injection is not always painless. Because of their formulations, some medications and vaccines produce a burning or stinging sensation, whether they are administered with a jet injector or a needle. Some compounded preparations, like the formulations included in this article, can be administered by jet injection, a practice that we suggest will increase in popularity as more drugs are prescribed for administration in the home setting. Because changes in drug concentration may be required to effect the transfer of an agent or ensure the accurate reconstitution of a lyophilized drug administered with a jet injector, the skill of a compounding pharmacist will be essential in preparing customized injectates. In this article, we address the use of needle-free technology in general; present examples of carbon-dioxide, spring-powered, and novel jet injection systems; and answer questions of interest to compounders about the use of jet injectors.

An alternative method for preparation of pandemic influenza strain-specific antibody for vaccine potency determination.

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.

Biodistribution, persistence and lack of integration of a multigene HIV vaccine delivered by needle-free intradermal injection and electroporation.

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.

Late administration of plasmid DNA by intradermal electroporation efficiently boosts DNA-primed T and B cell responses to carcinoembryonic antigen.

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.

Preclinical safety and biodistribution of Sindbis virus measles DNA vaccines administered as a single dose or followed by live attenuated measles vaccine in a heterologous prime-boost regimen.

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.

Broad immunogenicity of a multigene, multiclade HIV-1 DNA vaccine boosted with heterologous HIV-1 recombinant modified vaccinia virus Ankara.

BACKGROUND: A 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. METHODS: Forty 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). RESULTS: Vaccine-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. CONCLUSION: This HIV-DNA priming-MVA boosting approach is safe and highly immunogenic. TRIALS REGISTRATION: International Standard Randomised Controlled Trial number: ISRCTN32604572 .

Protection of mice against rotavirus challenge following intradermal DNA immunization by Biojector needle-free injection.

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 10 microg 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%).

A new multi-clade DNA prime/recombinant MVA boost vaccine induces broad and high levels of HIV-1-specific CD8(+) T-cell and humoral responses in mice.

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.

Heat-tolerant flowering plants of active geothermal areas in Yellowstone National Park.

A broad survey of most of the major geyser basins within Yellowstone National Park (Wyoming, USA) was conducted to identify the flowering plants which tolerate high rhizosphere temperatures (> or = 40 degrees C) in geothermally heated environments. Under such conditions, five species of monocots and four species of dicots were repeatedly found. The predominant flowering plants in hot soils (>40 degrees C at 2-5 cm depth) were grasses, primarily Dichanthelium lanuginosum. Long-term (weeks to months) rhizosphere temperatures of individual D. lanuginosum above 40 degrees C were recorded at several different locations, both in the summer and winter. The potential role of heat shock proteins (HSPs) in the apparent adaptation of these plants to chronically high rhizosphere temperatures was examined. Antibodies to cytoplasmic class I small heat shock proteins (sHSPs) and to HSP101 were used in Western immunoblot analyses of protein extracts from plants collected from geothermally heated soils. Relatively high levels of proteins reacting with anti-sHSP antibodies were consistently detected in root extracts from plants experiencing rhizosphere temperatures above 40 degrees C, though these proteins were usually not highly expressed in leaf extracts from the same plants. Proteins reacting with antibodies to HSP101 were also present both in leaf and root extracts from plants collected from geothermal soils, but their levels of expression were not as closely related to the degree of heat exposure as those of sHSPs.

Microplate quantification of plant leaf superoxide dismutases.

Superoxide dismutases (SODs) catalyze the dismutation of superoxide radicals in a broad range of organisms, including plants. Quantification of SOD activity in crude plant extracts has been problematic due to the presence of compounds that interfere with the dose-response of the assay. Although strategies exist to partially purify SODs from plant extracts, the requirement for purification limits the rapidity and practical number of assays that can be conducted. In this article, we describe modification of a procedure using o-dianisidine as substrate that permits relatively rapid quantification of SOD activity in crude leaf extracts in a microplate format. The method employs the use of a commercial apparatus that permits lysis of 12 tissue samples at once and the use of Pipes buffer to reduce interference from compounds present in crude leaf extracts. The assay provided a linear response from 1 to 50 units of SOD. The utility of the assay was demonstrated using tissue extracts prepared from a group of taxonomically diverse plants. Reaction rates with tissue extracts from two grasses were linear for at least 60 min. Tissues of certain species contained interfering compounds, most of which could be removed by ultrafiltration. The presence of plant catalases, peroxidases, and ascorbate in physiological quantities did not interfere with the assay. This approach provides a means to quantify SOD activity in relatively large numbers of plant samples provided that the possibility for the presence of interfering compounds is considered. The presence of interfering compounds in certain plant tissues necessitates caution in interpreting the effects of plant stresses on SOD.