RESUMO
Synthetic oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs (CpG ODN) are potent adjuvants to protein antigens administered by parenteral or mucosal routes to BALB/c mice. To date, there have been no studies using combined parenteral/mucosal approaches with CpG DNA as adjuvant. In this study we evaluated different parenteral prime-mucosal boost and mucosal prime-parenteral boost strategies using hepatitis B surface antigen (HBsAg) alone or with different adjuvants: aluminum hydroxide (alum), cholera toxin (CT), CpG ODN. In addition, since CpG ODN has previously been shown to act synergistically with other adjuvants after parenteral or mucosal delivery, we also evaluated adjuvant combinations: alum+CpG ODN and CT+CpG ODN. The effects of adjuvant and administration strategy on systemic and mucosal humoral responses were measured, as well as cell-mediated immune responses (cytotoxic T lymphocyte activity). These results were compared to parenteral only or mucosal only strategies. Our findings demonstrate that parenteral immunization can prime for mucosal responses even when different lymph nodes were being targeted. HBsAg-specific immune responses (IgG in plasma, cytotoxic T lymphocytes) induced by parenteral prime could all be significantly enhanced by mucosal boosting and despite the fact that intramuscular immunization alone could not induce mucosal IgA, it could prime for a subsequent mucosal boost. In addition, the presence of adjuvant at time of boosting could influence the nature of subsequent immune responses (Th1 vs. Th2). Mice primed intranasally could have their systemic immune responses boosted with a parenteral administration and it was also possible to enhance mucosal responses induced by intranasal prime with an intramuscular boost.
Assuntos
Adjuvantes Imunológicos , Antígenos de Superfície da Hepatite B/imunologia , Vacinas contra Hepatite B/imunologia , Hepatite B/prevenção & controle , Oligodesoxirribonucleotídeos/imunologia , Vacinas Sintéticas/imunologia , Compostos de Alúmen , Animais , Toxina da Cólera/imunologia , Modelos Animais de Doenças , Feminino , Hepatite B/imunologia , Anticorpos Anti-Hepatite B/sangue , Imunidade nas Mucosas , Imunização Secundária , Camundongos , Camundongos Endogâmicos BALB C , Linfócitos T Citotóxicos/imunologia , Células Th1/imunologia , Células Th2/imunologiaRESUMO
Despite the existence for some time of effective prophylactic vaccines, hepatitis B virus (HBV) infection remains an important global concern. Improvements on existing vaccines could be beneficial, especially in situations where it is desirable or necessary to induce protective immunity more rapidly or with fewer doses. We have compared, in chimpanzees, a current HBV vaccine that contains recombinant hepatitis B surface antigen HBsAg) adsorbed to alum, with two novel vaccine strategies that have proven superior to the current vaccine in mice. The first approach was the use of oligodeoxynucleotides containing CpG motifs (CpG ODN) as an adjuvant to Engerix-B, a commercial HBV vaccine. The addition of CpG ODN to Engerix-B greatly improved the kinetics and magnitude of the humoral response, suggesting that CpG ODN might allow induction of protective immunity in humans more quickly and with fewer vaccine doses. All animals receiving either control or CpG-containing subunit vaccines at 0 and 4 weeks attained titers of HBsAg-specific antibody (anti-HBs) considered protective (> or =10 mIU/ml) and were indeed protected from challenge at 8 weeks with 10(3.5) 50% chimp infectious doses (CID(50)) of intravenous HBV. The second approach was a DNA vaccine with a plasmid vector optimized for content of immunostimulatory CpG motifs. Despite the fact that earlier studies had shown four doses of a similar DNA vaccine (except not optimized for CpG content) to induce strong humoral responses in 1 of 2 chimpanzees, in this study two doses of DNA vaccine (at 0 and 4 weeks) did not generate any detectable anti-HBs in either of 2 chimpanzees, although it did protect 1 that rapidly developed anti-HBs during the incubation period, suggesting priming of an antibody response. The poor results may be due to an inadequate number of doses or amount of plasmid DNA in these larger animals, but nevertheless point to the need to improve delivery methods for DNA vaccines for use in larger animals such as primates.
Assuntos
Adjuvantes Imunológicos/uso terapêutico , Vacinas contra Hepatite B , Hepatite B/prevenção & controle , Oligodesoxirribonucleotídeos/uso terapêutico , Vacinas de DNA , Vacinas Sintéticas , Compostos de Alúmen/uso terapêutico , Animais , Hepatite B/imunologia , Hepatite B/virologia , Anticorpos Anti-Hepatite B/biossíntese , Antígenos de Superfície da Hepatite B/genética , Antígenos de Superfície da Hepatite B/imunologia , Vacinas contra Hepatite B/imunologia , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/genética , Oligodesoxirribonucleotídeos/imunologia , Pan troglodytes , Vacinas de DNA/imunologia , Vacinas Sintéticas/imunologiaRESUMO
Oligodeoxynucleotides (ODN) with unmethylated deoxycytidyl-deoxyguanosine (CpG) dinucleotides (CpG ODN) mimic the immunostimulatory activity of bacterial DNA and are recognized by the Toll-like receptor 9 (TLR9). CpG ODN of the B-Class stimulate strong B cell and NK cell activation and cytokine production. The highest degrees of NK stimulation as well as IFN-alpha secretion by plasmacytoid DC were found to occur only with A-Class ODN. A third class of CpG ODN combines the immune effects of A- and B-Class CpG ODN. C-Class ODN strongly stimulate B cell or NK cell activation and IFN-alpha production. In contrast to the A-Class, the C-Class is wholly phosphorothioate, has no poly-G stretches, but has palindromic sequences combined with stimulatory CpG motifs. All classes stimulate TLR9-dependent signaling, but with strikingly different dose-response relationships that are quite in contrast to those observed for IFN-alpha. Effects similar to those on human cells were observed on mouse splenocytes. In contrast, splenocytes from TLR9-deficient mice did not show any response to the three CpG ODN classes. In vivo studies demonstrate that C-Class ODN are very potent Th1 adjuvants. C-Class ODN may represent new therapeutic drugs that combine the effects of A- and B-Class ODN for broad applications in infectious disease or cancer therapy.
Assuntos
Linfócitos B/metabolismo , Interferon-alfa/biossíntese , Glicoproteínas de Membrana/imunologia , Oligodesoxirribonucleotídeos/classificação , Receptores de Superfície Celular/imunologia , Animais , Linfócitos B/imunologia , Humanos , Camundongos , NF-kappa B/metabolismo , Oligodesoxirribonucleotídeos/imunologia , Receptor Toll-Like 9 , Receptores Toll-LikeRESUMO
Oligodeoxynucleotides (ODN) with unmethylated CpG dinucleotides mimic the immune stimulatory activity of bacterial DNA in vertebrates and are recognized by Toll-like receptor 9 (TLR9). It is also possible to detect immune activation with certain phosphorothioate sequences that lack CpG motifs. These ODN are less potent than CpG ODN and the mechanism by which they stimulate mammalian leucocytes is not understood. We here provide several lines of evidence demonstrating that the effects induced by non-CpG ODN are mediated by TLR9. First, non-CpG ODN could not stimulate cytokine secretion from the splenocytes of TLR9-deficient (TLR9(-/-)) mice. Second, immunization of TLR9(+/+) but not TLR9(-/-) mice with non-CpG ODN enhanced antigen-specific antibody responses, although these were T helper type 2 (Th2)-biased. Third, reactivity to non-CpG ODN could be reconstituted by transfection of human TLR9 into non-responsive cells. In addition, we define a new efficient immune stimulatory motif aside from the CpG dinucleotide that consists of a 5'-TC dinucleotide in a thymidine-rich background. Non-CpG ODN containing this motif induced activation of human B cells, but lacked stimulation of Th1-like cytokines and chemokines. Our study indicates that TLR9 can mediate either efficient Th1- or Th2-dominated effects depending on whether it is stimulated by CpG or certain non-CpG ODN.
Assuntos
Citocinas/imunologia , Proteínas de Ligação a DNA/imunologia , Oligonucleotídeos/imunologia , Receptores de Superfície Celular/imunologia , Células Th2/imunologia , Animais , Formação de Anticorpos/imunologia , Linfócitos B/imunologia , Sequência de Bases , Divisão Celular/imunologia , Quimiocina CXCL10 , Quimiocinas CXC/imunologia , Células Dendríticas/imunologia , Feminino , Humanos , Interferons/imunologia , Interleucina-10/imunologia , Interleucina-6/imunologia , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/imunologia , Oligodesoxirribonucleotídeos/imunologia , Baço/imunologia , Timidina/imunologia , Receptor Toll-Like 9RESUMO
DNA-based immunization may be of prophylactic and therapeutic value for hepatitis C virus (HCV) infection. In efforts to improve the immunogenicity of a plasmid expressing the second envelope protein (E2) of HCV, we evaluated in mice the role of the antigen localization and demonstrated that membrane-bound and secreted forms induced higher titers of E2-specific antibodies, as well as earlier and higher seroconversion rates, than the intracellular form, but all three forms induced strong CTL. We also investigated whether E2-specific antibody responses could be enhanced by CpG optimization of the plasmid backbone and showed that removal of neutralizing CpG dinucleotides did not have a significant effect but addition of 64 immunostimulatory CpG motifs significantly enhanced anti-E2 titers. These results may have implications for the design and development of HCV DNA vaccines.
Assuntos
Hepacivirus/genética , Hepacivirus/imunologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Vacinas contra Hepatite Viral/genética , Animais , Sequência de Bases , Ilhas de CpG , DNA Viral/genética , Feminino , Vetores Genéticos , Hepatite C/imunologia , Hepatite C/prevenção & controle , Anticorpos Anti-Hepatite C/biossíntese , Antígenos da Hepatite C/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Linfócitos T Citotóxicos/imunologia , Vacinas de DNA/genéticaRESUMO
A series of benzotriazole phenyldifluoromethylphosphonic acids were found to be potent PTP-1B inhibitors. Molecular modeling on the X-ray crystal structure of the lead structure led to the design of potent PTP-1B inhibitors that show moderate selectivity against TC-PTP, a very closely related protein tyrosine phosphatase.