RESUMEN
Noroviruses, a major cause of acute gastroenteritis worldwide, present antigenic diversity that must be considered for the development of an effective vaccine. In this study, we explored approaches to increase the broad reactivity of virus-like particle (VLP) norovirus vaccine candidates. The immunogenicity of a GII.4 "Consensus" VLP that was engineered from sequences of three genetically distinct naturally occurring GII.4 strains was examined for its ability to induce cross-reactive immune responses against different clusters of GII.4 noroviruses. Rabbits immunized with GII.4 Consensus VLPs developed high serum antibody titers against VLPs derived from a number of distinct wild-type GII.4 viruses, including some that had been circulating over 30 years ago. Because the sera exhibited low cross-reactivity with antigenically distinct GI norovirus strains, we investigated the serum antibody response to a bivalent vaccine formulation containing GI.1 (Norwalk virus) and GII.4 Consensus VLPs that was administered to animals under varying conditions. In these studies, the highest homologous and heterologous antibody titers to the bivalent vaccine were elicited following immunization of animals by the intramuscular route using Alhydrogel (Al(OH)(3)) as adjuvant. Our data indicate that the use of both genetically engineered norovirus VLPs that incorporate relevant epitopes from multiple strains and multivalent vaccine formulations increase the breadth of the immune response to diverse variants within a genotype and, thus, prove helpful in the rational design of VLP-based vaccines against human noroviruses.
Asunto(s)
Norovirus/inmunología , Vacunación/métodos , Vacunas Virales/administración & dosificación , Vacunas Virales/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Hidróxido de Aluminio/administración & dosificación , Animales , Anticuerpos Antivirales/sangre , Reacciones Cruzadas , Femenino , Genotipo , Inyecciones Intramusculares , Norovirus/clasificación , Norovirus/genética , Conejos , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Vacunas de Virosoma/administración & dosificación , Vacunas de Virosoma/genética , Vacunas de Virosoma/inmunología , Vacunas Virales/genéticaRESUMEN
Immunotherapies that induce durable immune control of chronic HIV infection may eliminate the need for life-long dependence on drugs. We investigated a DNA vaccine formulated with a novel genetic adjuvant that stimulates immune responses in the blood and gut for the ability to improve therapy in rhesus macaques chronically infected with SIV. Using the SIV-macaque model for AIDS, we show that epidermal co-delivery of plasmids expressing SIV Gag, RT, Nef and Env, and the mucosal adjuvant, heat-labile E. coli enterotoxin (LT), during antiretroviral therapy (ART) induced a substantial 2-4-log fold reduction in mean virus burden in both the gut and blood when compared to unvaccinated controls and provided durable protection from viral rebound and disease progression after the drug was discontinued. This effect was associated with significant increases in IFN-γ T cell responses in both the blood and gut and SIV-specific CD8+ T cells with dual TNF-α and cytolytic effector functions in the blood. Importantly, a broader specificity in the T cell response seen in the gut, but not the blood, significantly correlated with a reduction in virus production in mucosal tissues and a lower virus burden in plasma. We conclude that immunizing with vaccines that induce immune responses in mucosal gut tissue could reduce residual viral reservoirs during drug therapy and improve long-term treatment of HIV infection in humans.
Asunto(s)
Vacunas contra el SIDA/farmacología , Síndrome de Inmunodeficiencia Adquirida/prevención & control , Vacunas contra el SIDAS/farmacología , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Vacunas de ADN/microbiología , Síndrome de Inmunodeficiencia Adquirida/inmunología , Animales , Inmunización/métodos , Interferón gamma/inmunología , Macaca mulatta , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunologíaRESUMEN
A simple and rapid approach to vaccine stabilization has been applied to a novel virus-like particle (VLP) that contains the primary influenza antigens (hemagglutinin and neuraminidase surface proteins). A complement of spectroscopic and light scattering techniques was used to characterize the physical stability of influenza VLPs as a function of temperature and pH, two pharmaceutically relevant stress factors. The resulting data set was mathematically converted into a three-color empirical phase diagram (EPD) that illustrates changes in physical state as a function of these stress factors. Conditions of temperature and pH corresponding to apparent phase boundaries in the EPD were then used to screen for inhibitors of VLP aggregation from a library of generally recognized as safe compounds. Several potent inhibitors of VLP aggregation were identified; of these, trehalose, sorbitol, and glycine were all found to exert significant stabilizing effects on viral protein tertiary structure and/or membrane integrity.
Asunto(s)
Excipientes/química , Subtipo H1N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/química , Vacunas contra la Influenza/inmunología , Gripe Humana/prevención & control , Virión/inmunología , Dicroismo Circular , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Humanos , Concentración de Iones de Hidrógeno , Subtipo H1N1 del Virus de la Influenza A/química , Subtipo H1N1 del Virus de la Influenza A/enzimología , Luz , Neuraminidasa/química , Neuraminidasa/inmunología , Transición de Fase , Dispersión de Radiación , Espectrometría de Fluorescencia , Temperatura , Virión/químicaRESUMEN
Enveloped virus-like particle (VLP) vaccines containing influenza hemagglutinin (HA) and neuraminidase (NA) antigens are produced easily in insect or mammalian cells via the simultaneous expression of HA and NA along with a viral core protein, such as influenza matrix (M1) or a retroviral Gag protein. The size and shape of the resulting particles are dictated by the choice of the core component, but M1- and Gag-based VLPs are strongly immunogenic and protective in seasonal and highly pathogenic influenza challenge models. Current data are consistent with the hypothesis that influenza VLP vaccine efficacy is related to the particulate, multivalent composition coupled with the presence of correctly folded antigens with intact biological activities. This new influenza vaccine paradigm offers potential advantages over the conventional egg-based, split-vaccine platform in terms of enhanced immunogenicity and better breadth of protection.
Asunto(s)
Vacunas contra la Influenza/inmunología , Gripe Humana/prevención & control , Animales , Línea Celular , Productos del Gen gag/genética , Productos del Gen gag/inmunología , Hemaglutininas Virales/genética , Hemaglutininas Virales/inmunología , Humanos , Vacunas contra la Influenza/genética , Insectos , Neuraminidasa/genética , Neuraminidasa/inmunología , Vacunas de Virosoma/genética , Vacunas de Virosoma/inmunología , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/inmunología , Proteínas Virales/genética , Proteínas Virales/inmunologíaRESUMEN
Influenza-pseudotyped Gag virus-like particles (VLPs) were produced via the expression of influenza hemagglutinin (HA), neuraminidase (NA) and the murine leukemia virus Gag product in the baculovirus-insect cell expression system. Hemagglutination specific activities of sucrose gradient-purified VLPs were similar to those of egg-grown influenza viruses but particle morphologies were gamma retrovirus-like in the form of consistent 100nm spheres. Immunization of mice and ferrets demonstrated robust immunogenicity and protection from challenge with no measurable morbidity. Ferret data were striking in that immunization with H5N1 VLPs representing either A/Vietnam/1203/04 or A/Indonesia/5/05 resulted in solid protection against highly pathogenic A/Vietnam/1203/04 challenge with no detectable virus in the upper respiratory tract post-challenge in either group. H1N1 VLP immunization of ferrets resulted in partial protection against H5N1 challenge with markedly accelerated virus clearance from the upper respiratory tract relative to controls. The immunogenicity of influenza-pseudotyped VLPs was not dependent on the adjuvant properties of replication competent contaminating baculovirus. These data demonstrate robust vaccine protection of Gag-based, influenza-pseudotyped VLPs carrying a variety of influenza antigens and suggest applicability toward a number of additional respiratory viruses.
Asunto(s)
Productos del Gen gag/inmunología , Vacunas contra la Influenza/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Virión/inmunología , Animales , Baculoviridae/inmunología , Células Cultivadas , Femenino , Hurones/inmunología , Productos del Gen gag/administración & dosificación , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/aislamiento & purificación , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H5N1 del Virus de la Influenza A/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Neuraminidasa/inmunología , Infecciones por Orthomyxoviridae/inmunología , Factores de TiempoRESUMEN
A mouse model was employed to evaluate protective cellular immune responses induced by an immediate early antigen of HSV-2. Particle-mediated DNA vaccination of mice with a DNA plasmid-encoding ICP27 resulted in the induction of ICP27-specific IFN-gamma and TNF-alpha production in Balb/c mice, but little protection to intranasal challenge with wild type HSV-2. However, when the DNA vaccine was supplemented with as little as 50ng of a vector encoding the A and B subunits of the Escherichia coli heat labile enterotoxin (LT), animals were profoundly protected from morbidity and mortality. The ICP27+LT-mediated protection was correlated with a large increase in ICP27-specific IFN-gamma and TNF-alpha production but cytokine-specific monoclonal antibody treatment at the time of challenge showed that protection was mediated predominantly by IFN-gamma. Furthermore, depletion of T cell subsets prior to infectious challenge demonstrated that removal of either CD8+ or CD4+ T cells impaired protection with CD8+ T cells appearing to play a direct effector role. These data demonstrate that augmented cellular immune responses resulting from LT vector plus antigen vector administration to the skin are biologically significant, leading to enhanced protection against mucosal pathogenic challenge.
Asunto(s)
Toxinas Bacterianas/inmunología , Enterotoxinas/inmunología , Proteínas de Escherichia coli/inmunología , Herpes Simple/inmunología , Herpes Simple/prevención & control , Herpesvirus Humano 2/inmunología , Vacunas contra Herpesvirus/inmunología , Vacunas de ADN/inmunología , Proteínas Virales/inmunología , Secuencia de Aminoácidos , Animales , Toxinas Bacterianas/genética , Antígenos CD8/inmunología , Citocinas/metabolismo , Enterotoxinas/genética , Epítopos/inmunología , Proteínas de Escherichia coli/genética , Herpesvirus Humano 2/genética , Vacunas contra Herpesvirus/genética , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Vacunas de ADN/genética , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
DNA immunization in conjunction with antiretroviral therapy was evaluated in SIV-infected rhesus macaques treated with [R]-9-[2-phosphonylmethoxypropyl]adenine (PMPA). Macaques were immunized monthly with DNA vaccines expressing either SIV gag/tat or SIV gag/tat and 19 CD8+ T cell epitopes during 7 months of therapy. Half the animals from each group were additionally immunized before infection. Only 60% of the animals (4 controls, 20 vaccinated) responded to PMPA (ART responders). All 4 ART responder controls demonstrated viral rebound or CD4 decline after PMPA was withdrawn. In contrast, 17 of 20 vaccinated ART responders contained viral rebound for over 7 months after PMPA was withdrawn. Viral control correlated with stable CD4 counts, higher lymphoproliferation and an increase in the magnitude and breadth of the CD8+ T cell response. Immunizing before infection or with multi-epitopes enhanced these effects. These results demonstrate that DNA immunization during antiretroviral therapy may be an effective strategy to treat HIV infection.
Asunto(s)
Adenina/análogos & derivados , Organofosfonatos/administración & dosificación , Síndrome de Inmunodeficiencia Adquirida del Simio/tratamiento farmacológico , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Virus de la Inmunodeficiencia de los Simios/inmunología , Vacunas de ADN/administración & dosificación , Viremia/prevención & control , Adenina/administración & dosificación , Adenina/uso terapéutico , Animales , Fármacos Anti-VIH/administración & dosificación , Fármacos Anti-VIH/uso terapéutico , Recuento de Linfocito CD4 , Linfocitos T CD8-positivos/inmunología , Proliferación Celular , Progresión de la Enfermedad , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Productos del Gen gag/genética , Productos del Gen gag/inmunología , Productos del Gen tat/genética , Productos del Gen tat/inmunología , Inmunoterapia Activa/métodos , Macaca mulatta , Organofosfonatos/uso terapéutico , ARN Viral/sangre , Virus de la Inmunodeficiencia de los Simios/efectos de los fármacos , Estadística como Asunto , Tenofovir , Vacunas de ADN/inmunología , Privación de TratamientoRESUMEN
A phase I clinical trial was conducted to evaluate a monovalent influenza DNA vaccine containing the HA gene from A/Panama/2007/99 delivered by particle-mediated epidermal delivery (PMED). Three groups of 12 healthy adult subjects received a single dose on day 0 of either 1, 2 or 4 microg of DNA vaccine, delivered as 1, 2 or 4 PMED administrations. The PMED influenza DNA vaccine elicited serum hemagglutination-inhibition (HAI) antibody responses at all three dose levels, with the highest and most consistent responses in subjects vaccinated with the highest dose level. Antibody responses were greatest at the last time point tested, day 56. Treatment-related reactions were mild to moderate, and included skin reactions at the vaccine site. These results provide a preliminary indication of the safety and immunogenicity of a prototype epidermal DNA vaccine for influenza.
Asunto(s)
Epidermis , Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Gripe Humana/prevención & control , Vacunas de ADN/inmunología , Adulto , Anticuerpos Antivirales/biosíntesis , Relación Dosis-Respuesta Inmunológica , Femenino , Humanos , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/efectos adversos , Gripe Humana/inmunología , Masculino , Vacunas de ADN/administración & dosificación , Vacunas de ADN/efectos adversosRESUMEN
Particle-mediated DNA vaccines employ a physical, intracellular delivery device to achieve the deposition of plasmid DNA-based expression vectors directly into the interior of cells of the skin. The resultant bolus of transient antigen expression in keratinocytes and trafficking dendritic cells results in the induction of humoral and cellular immune responses in various animal models and humans, mimicking characteristics of live or live-vectored vaccines. Ultimately, DNA vaccine success in the clinic will depend on both the successful intracellular delivery of a plasmid vector and an immunostimulator or adjuvant to maximise humoral and cellular immune responses to the encoded antigen(s). To this end, recent DNA vaccine clinical trials are confirming the importance of an intracellular delivery system, while preclinical studies in animal models are demonstrating the feasibility of augmenting responses through the use of DNA-encoded immunostimulators. Particle-mediated DNA vaccines represent a promising tool for developing candidate vaccines against some of the more difficult infectious, parasitic and oncologic disease targets.
Asunto(s)
Adyuvantes Inmunológicos/uso terapéutico , Biolística/métodos , Piel/metabolismo , Vacunas de ADN , Animales , Antígenos/metabolismo , Ensayos Clínicos como Asunto , Islas de CpG , Células Dendríticas/citología , Epidermis/metabolismo , Vectores Genéticos , Humanos , Memoria Inmunológica , Queratinocitos/metabolismo , Plásmidos/metabolismo , Células TH1/inmunología , Células Th2/inmunologíaRESUMEN
Data generated in the early phases of experimentation in a new field of scientific exploration sometimes results in hasty conclusions about the generality of the data. To some degree, this has happened at least twice in the relatively new area of DNA immunization. Early data seemed to indicate firstly that particle-mediated epidermal DNA immunization induced predominantly Th2-type cellular immune responses, and secondly that DNA immunization was not very successful in humans. This review highlights the current body of data showing that particle-mediated DNA immunization is highly effective in the induction Th1-type responses and is an efficient method for inducing immune responses in humans.
Asunto(s)
Vacunas de ADN/administración & dosificación , Animales , ADN/administración & dosificación , Haplorrinos , Humanos , Ratones , Piel/inmunología , Vacunas de ADN/inmunologíaRESUMEN
Two plasmid vectors encoding the A and B subunits of cholera toxin (CT) and two additional vectors encoding the A and B subunits of the Escherichia coli heat-labile enterotoxin (LT) were evaluated for their ability to serve as genetic adjuvants for particle-mediated DNA vaccines administered to the epidermis of laboratory animals. Both the CT and the LT vectors strongly augmented Th1 cytokine responses (gamma interferon [IFN-gamma]) to multiple viral antigens when codelivered with DNA vaccines. In addition, Th2 cytokine responses (interleukin 4 [IL-4]) were also augmented by both sets of vectors, with the effects of the LT vectors on IL-4 responses being more antigen dependent. The activities of both sets of vectors on antibody responses were antigen dependent and ranged from no effect to sharp reductions in the immunoglobulin G1 (IgG1)-to-IgG2a ratios. Overall, the LT vectors exhibited stronger adjuvant effects in terms of T-cell responses than did the CT vectors, and this was correlated with the induction of greater levels of cyclic AMP by the LT vectors following vector transfection into cultured cells. The adjuvant effects observed in vivo were due to the biological effects of the encoded proteins and not due to CpG motifs in the bacterial genes. Interestingly, the individual LT A and B subunit vectors exhibited partial adjuvant activity that was strongly influenced by the presence or absence of signal peptide coding sequences directing the encoded subunit to either intracellular or extracellular locations. Particle-mediated delivery of either the CT or LT adjuvant vectors in rodents and domestic pigs was well tolerated, suggesting that bacterial toxin-based genetic adjuvants may be a safe and effective strategy to enhance the potency of both prophylactic and therapeutic DNA vaccines for the induction of strong cellular immunity.
Asunto(s)
Adyuvantes Inmunológicos , Toxinas Bacterianas/genética , Toxina del Cólera/genética , Enterotoxinas/genética , Proteínas de Escherichia coli , Vectores Genéticos/administración & dosificación , Vacunas de ADN/inmunología , Vacunas contra el SIDA/genética , Vacunas contra el SIDA/inmunología , Animales , Anticuerpos Antivirales/sangre , Toxinas Bacterianas/administración & dosificación , Células CACO-2 , Toxina del Cólera/administración & dosificación , Enterotoxinas/administración & dosificación , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/inmunología , Humanos , Inmunización , Vacunas contra la Influenza/genética , Vacunas contra la Influenza/inmunología , Ratones , Plásmidos/genética , Porcinos , Linfocitos T/inmunología , Transfección , Vacunas de ADN/administración & dosificación , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/inmunologíaRESUMEN
An effective vaccine against human immunodeficiency virus (HIV) should protect against mucosal transmission of genetically divergent isolates. As a safe alternative to live attenuated vaccines, the immunogenicity and protective efficacy of a DNA vaccine containing simian immunodeficiency virus (SIV) strain 17E-Fr (SIV/17E-Fr) gag-pol-env was analyzed in rhesus macaques. Significant levels of cytotoxic T lymphocytes (CTL), but low to undetectable serum antibody responses, were observed following multiple immunizations. SIV-specific mucosal antibodies and CTL were also detected in rectal washes and gut-associated lymphoid tissues, respectively. Vaccinated and naive control monkeys were challenged intrarectally with SIV strain DeltaB670 (SIV/DeltaB670), a primary isolate whose env is 15% dissimilar to that of the vaccine strain. Four of seven vaccinees were protected from infection as determined by the inability to identify viral RNA or DNA sequences in the peripheral blood and the absence of anamnestic antibody responses postchallenge. This is the first report of mucosal protection against a primary pathogenic, heterologous isolate of SIV by using a commercially viable vaccine approach. These results support further development of a DNA vaccine for protection against HIV.
Asunto(s)
Inmunidad Mucosa , Glicoproteínas de Membrana , Vacunas contra el SIDAS/administración & dosificación , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Virus de la Inmunodeficiencia de los Simios/inmunología , Vacunación , Proteínas del Envoltorio Viral , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales/análisis , Productos del Gen env/genética , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/genética , Inmunoglobulina A/análisis , Inmunoglobulina G/análisis , Intestinos/inmunología , Tejido Linfoide/inmunología , Macaca mulatta , Datos de Secuencia Molecular , Recto/inmunología , Alineación de Secuencia , Virus de la Inmunodeficiencia de los Simios/genética , Linfocitos T Citotóxicos/inmunología , Vacunas de ADN/administración & dosificaciónRESUMEN
Due to the presence of a network of antigen-presenting cells and other cells with innate and adaptive immune functions, the skin is both a sensitive immune organ and a practical target site for vaccine administration. A handful of needle-free immunization technologies have emerged in recent years that aim to take advantage of these characteristics. Skin delivery technologies provide potentially safer alternatives to needle injection and promises increased efficacy in the prevention and/or therapy of infectious diseases, allergic disorders and cancer. In this review, we will cover advances in needle-free skin vaccination technologies and their potential applications to disease prevention and therapy. Emphasis will be placed on epidermal powder immunization and particle-mediated ('gene gun') DNA immunization, which use similar mechanical devices to deliver protein and DNA vaccines, respectively, into the viable epidermis.
Asunto(s)
Administración Cutánea , Vacunación/métodos , Vacunas/administración & dosificación , Vacunas/química , Adyuvantes Inmunológicos , Animales , Formación de Anticuerpos/inmunología , Química Farmacéutica , Sistemas de Liberación de Medicamentos , Humanos , Inmunidad Celular/inmunología , Polvos , Fenómenos Fisiológicos de la Piel , Vacunas de ADN/administración & dosificación , Vacunas de ADN/inmunologíaRESUMEN
A synthetic gene coding for the coat protein of tobacco mosaic virus (TMVCP) was expressed in E. coli under the direction of the lacUV5 promoter. Modification of the 3' end of the TMVCP gene by insertion of a region coding for an antigenic epitope from poliovirus type 3 resulted in the production of a hybrid TMVCP (TMVCP-polio 3). Both the E. coli-produced TMVCP and TMVCP-polio 3 were shown to assemble into virus-like rods under acidic conditions in E. coli extracts. Their purification was accomplished in a single step by chromatography on Sepharose 6B. TMVCP-polio 3 induced the formation of poliovirus neutralizing antibodies following injection into rats. The level of immune response was related to the degree of polymerization of the TMVCP-polio 3 preparations.