RESUMO
This review focuses on a dengue virus (DENV) vaccine candidate based on a recombinant subunit approach which targets the DENV envelope glycoprotein (E). Truncated versions of E consisting of the N-terminal portion of E (DEN-80E) have been expressed recombinantly in the Drosophila S2 expression system and shown to have native-like conformation. Preclinical studies demonstrate that formulations containing tetravalent DEN-80E adjuvanted with ISCOMATRIX™ adjuvant induce high titer virus neutralizing antibodies and IFN-γ producing T cells in flavivirus-naïve non-human primates. The preclinical data further suggest that administration of such formulations on a 0, 1, 6 month schedule may result in higher maximum virus neutralizing antibody titers and better durability of those titers compared to administration on a 0, 1, 2 month schedule. In addition, the virus neutralizing antibody titers induced by adjuvanted tetravalent DEN-80E compare favorably to the titers induced by a tetravalent live virus comparator. Furthermore, DEN-80E was demonstrated to be able to boost virus neutralizing antibody titers in macaques that have had a prior DENV exposure. A monovalent version of the vaccine candidate, DEN1-80E, was formulated with Alhydrogel™ and studied in a proof-of-principle Phase I clinical trial by Hawaii Biotech, Inc. (NCT00936429). The clinical trial results demonstrate that both the 10 µg and 50 µg formulations of DEN1-80E with 1.25 mg of elemental aluminum were immunogenic when administered in a 3-injection series (0, 1, 2 months) to healthy, flavivirus-naïve adults. The vaccine formulations induced DENV-1 neutralizing antibodies in the majority of subjects, although the titers in most subjects were modest and waned over time. Both the 10 µg DEN1-80E and the 50 µg DEN1-80E formulations with Alhydrogel™ were generally well tolerated.
Assuntos
Ensaios Clínicos Fase I como Assunto , Vacinas contra Dengue/administração & dosagem , Vacinas contra Dengue/imunologia , Dengue/prevenção & controle , Avaliação Pré-Clínica de Medicamentos , Adjuvantes Imunológicos/administração & dosagem , Hidróxido de Alumínio/administração & dosagem , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Colesterol/administração & dosagem , Dengue/epidemiologia , Vacinas contra Dengue/genética , Vacinas contra Dengue/isolamento & purificação , Combinação de Medicamentos , Humanos , Esquemas de Imunização , Interferon gama/metabolismo , Macaca , Fosfolipídeos/administração & dosagem , Saponinas/administração & dosagem , Linfócitos T/imunologia , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/isolamento & purificação , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificaçãoRESUMO
We describe here the preclinical development of a dengue vaccine composed of recombinant subunit carboxy-truncated envelope (E) proteins (DEN-80E) for each of the four dengue serotypes. Immunogenicity and protective efficacy studies in Rhesus monkeys were conducted to evaluate monovalent and tetravalent DEN-80E vaccines formulated with ISCOMATRIX™ adjuvant. Three different doses and two dosing regimens (0, 1, 2 months and 0, 1, 2, and 6 months) were evaluated in these studies. We first evaluated monomeric (DEN4-80E) and dimeric (DEN4-80EZip) versions of DEN4-80E, the latter generated in an attempt to improve immunogenicity. The two antigens, evaluated at 6, 20 and 100 µg/dose formulated with ISCOMATRIX™ adjuvant, were equally immunogenic. A group immunized with 20 µg DEN4-80E and Alhydrogel™ induced much weaker responses. When challenged with wild-type dengue type 4 virus, all animals in the 6 and 20 µg groups and all but one in the DEN4-80EZip 100 µg group were protected from viremia. Two out of three monkeys in the Alhydrogel™ group had breakthrough viremia. A similar study was conducted to evaluate tetravalent formulations at low (3, 3, 3, 6 µg of DEN1-80E, DEN2-80E, DEN3-80E and DEN4-80E respectively), medium (10, 10, 10, 20 µg) and high (50, 50, 50, 100 µg) doses. All doses were comparably immunogenic and induced high titer, balanced neutralizing antibodies against all four DENV. Upon challenge with the four wild-type DENV, all animals in the low and medium dose groups were protected against viremia while two animals in the high-dose group exhibited breakthrough viremia. Our studies also indicated that a 0, 1, 2 and 6 month vaccination schedule is superior to the 0, 1, and 2 month schedule in terms of durability. Overall, the subunit vaccine was demonstrated to induce strong neutralization titers resulting in protection against viremia following challenge even 8-12 months after the last vaccine dose.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Colesterol/administração & dosagem , Vacinas contra Dengue/administração & dosagem , Vacinas contra Dengue/imunologia , Dengue/prevenção & controle , Fosfolipídeos/administração & dosagem , Saponinas/administração & dosagem , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Modelos Animais de Doenças , Combinação de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Feminino , Esquemas de Imunização , Macaca mulatta , Masculino , Vacinas Combinadas/administração & dosagem , Vacinas Combinadas/imunologia , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia , Viremia/prevenção & controleRESUMO
There is a need for novel rabies vaccines suitable for short course, pre- and post-exposure prophylactic regimens which require reduced doses of antigen to address the current worldwide supply issue. We evaluated in rhesus macaques the immunogenicity of a quarter-dose of a standard rabies vaccine formulated with Merck's amorphous aluminum hydroxylphosphate sulfate adjuvant, the saponin-based ISCOMATRIX™ adjuvant, or a synthetic TLR9 agonist. All adjuvants significantly increased the magnitude and durability of the humoral immune response as measured by rapid fluorescent focus inhibition test (RFFIT). Several three-dose vaccine regimens resulted in adequate neutralizing antibody of ≥ 0.5 IU/ml earlier than the critical day seven post the first dose. Rabies vaccine with ISCOMATRIX™ adjuvant given at days 0 and 3 resulted in neutralizing antibody titers which developed faster and were up to one log10 higher compared to WHO-recommended intramuscular and intradermal regimens and furthermore, passive administration of human rabies immunoglobulin did not interfere with immunogenicity of this reduced dose, short course vaccine regimen. Adjuvantation of whole-killed rabies vaccine for intramuscular injection may therefore be a viable alternative to intradermal application of non-adjuvanted vaccine for both pre- and post-exposure regimens.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Compostos de Alumínio/administração & dosagem , Colesterol/administração & dosagem , Fosfolipídeos/administração & dosagem , Vacina Antirrábica/administração & dosagem , Vacina Antirrábica/imunologia , Saponinas/administração & dosagem , Receptor Toll-Like 9/agonistas , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Combinação de Medicamentos , Feminino , Injeções Intramusculares , Macaca mulatta , Masculino , Testes de Neutralização , Fosfatos/administração & dosagem , Raiva/prevenção & controle , Sulfatos/administração & dosagem , Receptor Toll-Like 9/imunologia , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/imunologiaRESUMO
PURPOSE OF REVIEW: To consider how nonhuman primate (NHP) model systems can best contribute to HIV vaccine development. RECENT FINDINGS: We review the traditional roles of NHP model systems in vaccine development and compare this with how NHP models have been used in HIV vaccine research and development. Comparisons of the immune responses elicited by cellular immune response-inducing vaccines in macaques and humans illustrate the value of primate studies for the relative ranking of HIV vaccine concepts for their likely immunogenicity in humans. The unusual structures (e.g. long complementarity-determining regions) of known broadly neutralizing HIV antibodies (bNAbs) suggest that it is critical to test candidate env immunogens in NHPs, whose germline antibody repertoires resemble those of humans. Recent clinical efficacy trial results question the utility of existing NHP challenge models in predicting HIV vaccine efficacy in humans, and highlight the need to further develop models in which acquisition of infection can be reliably evaluated. When evaluated in models using low virus dose challenges that better approximate human sexual exposure to HIV - some vaccine and passive NAb interventions appear to protect against acquisition of infection. SUMMARY: NHP models have important roles in the preclinical evaluation, optimization, and ranking of novel HIV immunogens. The apparent vaccine efficacy observed using low virus dose challenge models provides an opportunity to investigate the correlates of protection.
Assuntos
Vacinas contra a AIDS/imunologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos/métodos , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , Animais , Indústria Farmacêutica/métodos , Humanos , PrimatasRESUMO
Safe and efficient methods for in vivo delivery of transgenes of interest must be developed so that the promise of these therapies can be practically used in the clinic. In this work, we describe the use of electrostimulation to enhance the in vivo efficiency of plasmid DNA delivery. The method was optimized to work over a range of moderate frequencies, utilizing low field strengths and simple symmetrical waveforms. After studying several parameters of delivery in mice, we demonstrate how this methodology can be employed to significantly improve both gene expression (over 16-fold) and the immunogenicity of HIV-1 vaccines (over 28-fold) compared to naked DNA in non-human primates. Compared to an efficient viral Ad5 vector system, the gene expression levels of DNA+electrostimulation were surprisingly within a factor of four of the viral delivery system.
Assuntos
Vacinas contra a AIDS/imunologia , Eletroporação/métodos , Vetores Genéticos , Plasmídeos/genética , Transgenes , Vacinas de DNA/imunologia , Vacinas contra a AIDS/genética , Fosfatase Alcalina/genética , Animais , Linhagem Celular , Expressão Gênica , Técnicas de Transferência de Genes , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , Humanos , Macaca , Camundongos , Camundongos Endogâmicos BALB C , Vacinas de DNA/genéticaRESUMO
Current flu vaccines are based on killed or attenuated virus vaccines that must be altered each year to include the hemagglutinin and neuraminidase genes from a strain of virus predicted to predominate in the coming year. A vaccine that could protect against multiple strains of influenza A and B would be a major asset in the fight against flu-related mortality and morbidity. To support development of such a vaccine, we have developed a Flu Multiplex Assay based on a Luminex platform to assess serum antibody levels to two conserved peptides derived from influenza A (M2 protein) and influenza B (hemagglutinin protein). The peptides were synthesized with a biotin label and subsequently coupled to two different LumAvidin microspheres. We then tested various sera against both types of peptide in the multiplex assay format. The data show that sera from Rhesus macaques immunized with a single peptide react only with the homologous peptide while Rhesus macaques immunized with both peptides respond well to both peptides. Additionally, we were able to specifically compete reactivity to both peptides. We have tested serial bleeds from 100 pediatric patients at ages ranging from 16 to 56 weeks as well as single bleeds from over 100 healthy adults. No overall trend in titer relative to pediatric age was detected. Both demographics exhibited a minimal response to either the A/M2 or B/HA0 peptides. However, the average titer for the pediatric serum samples was significantly lower than that found in the adult population. The adult population exhibited a higher prevalence of low reactive samples. Assay reagents and parameters have been optimized and the assay is shown to be repeatable and robust. The assay will be used to support clinical vaccine trials of a bivalent peptide vaccine.
Assuntos
Anticorpos Antivirais/sangue , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Imunoensaio/métodos , Vírus da Influenza A/imunologia , Vírus da Influenza B/imunologia , Vacinas contra Influenza/imunologia , Proteínas da Matriz Viral/imunologia , Adolescente , Adulto , Animais , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Feminino , Humanos , Lactente , Macaca mulatta , Masculino , Peptídeos/imunologia , Padrões de Referência , Sensibilidade e EspecificidadeRESUMO
We describe the physiochemical characterization and immunological evaluation of plasmid DNA vaccine formulations containing a nonionic triblock copolymer adjuvant (CRL1005) in the presence and absence of a cationic surfactant, benzalkonium chloride (BAK). CRL1005 forms particles of 1-10 microns upon warming above its phase-transition temperature (approximately 6-8 degrees C) and the physical properties of the particles are altered by BAK. DNA/CRL1005 vaccines formulated with and without BAK were evaluated in rhesus macaques to determine the effect of CRL1005 and BAK on the ability of plasmid DNA to induce a cellular immune response. Immunogenicity results indicate that the addition of CRL1005 to human immunodeficiency virus-1 gag plasmid DNA formulated in phosphate-buffered saline leads to an enhancement in the gag-specific cellular immune response. Moreover, the addition of BAK to human immunodeficiency virus-1 gag plasmid DNA/CRL1005 formulations produces an additional enhancement in gag-specific cellular immunity. In vitro characterization studies of DNA/CRL1005 formulations indicate no detectable binding of DNA to CRL1005 particles in the absence of BAK, suggesting that the enhancement of cellular immunity induced by DNA/CRL1005 formulations is not due to enhanced DNA delivery. In the presence of BAK, however, results indicate that BAK binds to CRL1005 particles, producing cationic microparticles that bind DNA through electrostatic interactions. If BAK is present at the phase-transition temperature, it reduces the particle size from approximately 2 microns to approximately 300 nm, presumably by binding to hydrophobic surfaces during particle formation. Zeta potential measurements indicate that the surface charge of CRL1005-BAK particles changes from positive to negative upon DNA binding, and DNA bound to the surface of CRL1005-BAK particles was visualized by fluorescence microscopy. These results indicate that the addition of BAK to DNA/CRL1005 formulations leads to the formation of approximately 300 nm CRL1005-BAK-DNA particles that enhance the cellular immune response in rhesus monkeys.