RESUMO
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.
Assuntos
Vacinas contra a AIDS/imunologia , Vacinas contra a AIDS/farmacocinética , Infecções por HIV/prevenção & controle , Vacinas de DNA/imunologia , Vacinas de DNA/farmacocinética , Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/efeitos adversos , Animais , Eletroporação/métodos , Feminino , Infecções por HIV/imunologia , HIV-1/genética , HIV-1/imunologia , Humanos , Injeções Intradérmicas/métodos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos/administração & dosagem , Plasmídeos/metabolismo , Pele/química , Vacinas de DNA/administração & dosagem , Vacinas de DNA/efeitos adversos , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/efeitos adversos , Vacinas Sintéticas/imunologia , Integração ViralRESUMO
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.
Assuntos
Linfócitos B/imunologia , Vacinas Anticâncer/imunologia , Antígeno Carcinoembrionário/imunologia , Eletroporação/métodos , Linfócitos T/imunologia , Vacinação/métodos , Animais , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/genética , Antígeno Carcinoembrionário/genética , Imunização Secundária/métodos , Injeções Intradérmicas , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Vacinas de DNA/imunologiaRESUMO
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 .
Assuntos
Vacinas contra a AIDS/imunologia , Vacinas contra a AIDS/normas , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Vacinas de DNA/imunologia , Vacinas de DNA/normas , Vaccinia virus/imunologia , Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/efeitos adversos , Adulto , Formação de Anticorpos/imunologia , Feminino , Vetores Genéticos/genética , Vetores Genéticos/imunologia , HIV-1/genética , Humanos , Masculino , Pessoa de Meia-Idade , Linfócitos T/imunologia , Vacinas de DNA/efeitos adversos , Vacinas de DNA/genética , Vaccinia virus/genética , Adulto JovemRESUMO
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.
Assuntos
Especificidade de Anticorpos/imunologia , Antígeno Carcinoembrionário/imunologia , Injeções a Jato/instrumentação , Injeções Subcutâneas/instrumentação , Células Th1/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia , Animais , Sistemas de Liberação de Medicamentos/métodos , Células HeLa , Humanos , Imunidade Inata/imunologia , Injeções a Jato/métodos , Injeções Subcutâneas/métodos , Camundongos , Camundongos Endogâmicos BALB CRESUMO
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.
Assuntos
Anticorpos Antivirais/sangue , Biolística , Vetores Genéticos , Vacinas de DNA/administração & dosagem , Vaccinia virus/genética , Proteínas do Envelope Viral/imunologia , Animais , Citocinas/sangue , Herpesvirus Humano 2/imunologia , Vacinas contra Herpesvirus/administração & dosagem , Vacinas contra Herpesvirus/imunologia , Imunização/métodos , Imunização Secundária/métodos , Imunoterapia Ativa/métodos , Camundongos , Agulhas , Plasmídeos/administração & dosagem , Plasmídeos/genética , Vaccinia virus/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
A dengue-1 DNA vaccine containing sequences encoding premembrane and envelope proteins (DIME) was previously shown to elicit virus neutralizing antibodies in rhesus and Aotus monkeys, and the primates were partially protected from viremia upon challenge. To increase the neutralizing antibody levels and subsequent protection from virus challenge, four strategies were evaluated: (a) coimmunization with a plasmid expressing Aotus GM-CSF gene; (b) coimmunization with a plasmid containing human immunostimulatory sequences (ISS); (c) coimmunization with both the GM-CSF gene and ISS; and (d) delivery of vaccine using the needle-free Biojector system. Vaccination with the mixed formulation containing DIME, GM-CSF gene, and ISS, by either needle injection or Biojector, led to neutralizing antibody titers that were stable for up to 6 months after vaccination. Furthermore, 6 of 7 monkeys (85%), and 7 of 8 monkeys (87%) receiving this formulation were completely protected from viremia when challenged 1 and 6 months after vaccination, respectively. This is a significant improvement compared to our previous study in which one of three monkeys (33%) receiving just the DIME vaccine was completely protected from viremia at 6 months after immunization.