RESUMO
DNA vaccines require a considerable enhancement of immunogenicity. Here, we optimized a prototype DNA vaccine against drug-resistant HIV-1 based on a weak Th2-immunogen, HIV-1 reverse transcriptase (RT). We designed expression-optimized genes encoding inactivated wild-type and drug-resistant RTs (RT-DNAs) and introduced them into mice by intradermal injections followed by electroporation. RT-DNAs were administered as single or double primes with or without cyclic-di-GMP, or as a prime followed by boost with RT-DNA mixed with a luciferase-encoding plasmid ("surrogate challenge"). Repeated primes improved cellular responses and broadened epitope specificity. Addition of cyclic-di-GMP induced a transient increase in IFN-γ production. The strongest anti-RT immune response was achieved in a prime-boost protocol with electroporation by short 100V pulses done using penetrating electrodes. The RT-specific response, dominated by CD4+ T-cells, targeted epitopes at aa 199-220 and aa 528-543. Drug-resistance mutations disrupted the epitope at aa 205-220, while the CTL epitope at aa 202-210 was not affected. Overall, multiparametric optimization of RT strengthened its Th2- performance. A rapid loss of RT/luciferase-expressing cells in the surrogate challenge experiment revealed a lytic potential of anti-RT response. Such lytic CD4+ response would be beneficial for an HIV vaccine due to its comparative insensitivity to immune escape.
Assuntos
Vacinas contra a AIDS , Farmacorresistência Viral , Infecções por HIV/terapia , Transcriptase Reversa do HIV/imunologia , Células Th2/imunologia , Vacinação/métodos , Vacinas de DNA , Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/genética , Animais , Calibragem , Células Cultivadas , Códon , Sistemas de Liberação de Medicamentos , Farmacorresistência Viral/genética , Farmacorresistência Viral/imunologia , Epitopos/genética , Epitopos/imunologia , Infecções por HIV/imunologia , Transcriptase Reversa do HIV/genética , HIV-1/genética , HIV-1/imunologia , Células HeLa , Humanos , Evasão da Resposta Imune/genética , Evasão da Resposta Imune/imunologia , Imunização Secundária/métodos , Imunização Secundária/normas , Imunogenicidade da Vacina/genética , Camundongos , Camundongos Endogâmicos BALB C , Melhoria de Qualidade , Células Th2/metabolismo , Vacinação/normas , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genéticaRESUMO
Implantation of reporter-labeled tumor cells in an immunocompetent host involves a risk of their immune elimination. We have studied this effect in a mouse model of breast cancer after the orthotopic implantation of mammary gland adenocarcinoma 4T1 cells genetically labelled with luciferase (Luc). Mice were implanted with 4T1 cells and two derivative Luc-expressing clones 4T1luc2 and 4T1luc2D6 exhibiting equal in vitro growth rates. In vivo, the daughter 4T1luc2 clone exhibited nearly the same, and 4T1luc2D6, a lower growth rate than the parental cells. The metastatic potential of 4T1 variants was assessed by magnetic resonance, bioluminescent imaging, micro-computed tomography, and densitometry which detected 100-µm metastases in multiple organs and bones at the early stage of their development. After 3-4 weeks, 4T1 generated 11.4 ± 2.1, 4T1luc2D6, 4.5 ± 0.6; and 4T1luc2, <1 metastases per mouse, locations restricted to lungs and regional lymph nodes. Mice bearing Luc-expressing tumors developed IFN-γ response to the dominant CTL epitope of Luc. Induced by intradermal DNA-immunization, such response protected mice from the establishment of 4T1luc2-tumors. Our data show that natural or induced cellular response against the reporter restricts growth and metastatic activity of the reporter-labelled tumor cells. Such cells represent a powerful instrument for improving immunization technique for cancer vaccine applications.