RESUMEN
Adenoviruses are used extensively as gene transfer agents, both experimentally and clinically. However, targeting of liver cells by adenoviruses compromises their potential efficacy. In cell culture, the adenovirus serotype 5 fiber protein engages the coxsackievirus and adenovirus receptor (CAR) to bind cells. Paradoxically, following intravascular delivery, CAR is not used for liver transduction, implicating alternate pathways. Recently, we demonstrated that coagulation factor (F)X directly binds adenovirus leading to liver infection. Here, we show that FX binds to the Ad5 hexon, not fiber, via an interaction between the FX Gla domain and hypervariable regions of the hexon surface. Binding occurs in multiple human adenovirus serotypes. Liver infection by the FX-Ad5 complex is mediated through a heparin-binding exosite in the FX serine protease domain. This study reveals an unanticipated function for hexon in mediating liver gene transfer in vivo.
Asunto(s)
Adenovirus Humanos/fisiología , Proteínas de la Cápside/metabolismo , Factor X/metabolismo , Hígado/virología , Transducción Genética , Internalización del Virus , Adenovirus Humanos/química , Adenovirus Humanos/clasificación , Animales , Proteínas de la Cápside/química , Proteínas Portadoras/metabolismo , Microscopía por Crioelectrón , Factor X/química , Hepatocitos/virología , Humanos , Imagenología Tridimensional , Ratones , Ratones Transgénicos , Modelos Moleculares , Filogenia , Unión Proteica/efectos de los fármacos , Dominios y Motivos de Interacción de Proteínas , Resonancia por Plasmón de Superficie , Warfarina/farmacologíaRESUMEN
A common viral immune evasion strategy involves mutating viral surface proteins in order to evade host neutralizing antibodies. Such immune evasion tactics have not previously been intentionally applied to the development of novel viral gene delivery vectors that overcome the critical problem of anti-vector immunity. Recombinant, replication-incompetent adenovirus serotype 5 (rAd5) vector-based vaccines for human immunodeficiency virus type 1 and other pathogens have proved highly immunogenic in preclinical studies but will probably be limited by the high prevalence of pre-existing anti-Ad5 immunity in human populations, particularly in the developing world. Here we show that rAd5 vectors can be engineered to circumvent anti-Ad5 immunity. We constructed novel chimaeric rAd5 vectors in which the seven short hypervariable regions (HVRs) on the surface of the Ad5 hexon protein were replaced with the corresponding HVRs from the rare adenovirus serotype Ad48. These HVR-chimaeric rAd5 vectors were produced at high titres and were stable through serial passages in vitro. HVR-chimaeric rAd5 vectors expressing simian immunodeficiency virus Gag proved comparably immunogenic to parental rAd5 vectors in naive mice and rhesus monkeys. In the presence of high levels of pre-existing anti-Ad5 immunity, the immunogenicity of HVR-chimaeric rAd5 vectors was not detectably suppressed, whereas the immunogenicity of parental rAd5 vectors was abrogated. These data demonstrate that functionally relevant Ad5-specific neutralizing antibodies are focused on epitopes located within the hexon HVRs. Moreover, these studies show that recombinant viral vectors can be engineered to circumvent pre-existing anti-vector immunity by removing key neutralizing epitopes on the surface of viral capsid proteins. Such chimaeric viral vectors may have important practical implications for vaccination and gene therapy.
Asunto(s)
Adenoviridae/genética , Adenoviridae/inmunología , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Ingeniería Genética , Vectores Genéticos/genética , Vectores Genéticos/inmunología , Adenoviridae/clasificación , Adenoviridae/fisiología , Animales , Linfocitos T CD8-positivos/inmunología , ADN Recombinante/genética , Terapia Genética , Macaca mulatta/inmunología , Ratones , Ratones Endogámicos C57BL , Pruebas de Neutralización , VacunasRESUMEN
The deployment of adenovirus serotype 5 (Ad5)-based vectors is hampered by preexisting immunity. When such vectors are delivered intravenously, hepatocyte transduction is mediated by the hexon-coagulation factor X (FX) interaction. Here, we demonstrate that human sera efficiently block FX-mediated cellular binding and transduction of Ad5-based vectors in vitro. Neutralizing activity correlated well with the ability to inhibit Ad5-mediated liver transduction, suggesting that prescreening patient sera in this manner accurately predicts the efficacy of Ad5-based gene therapies. Neutralization in vitro can be partially bypassed by pseudotyping with Ad45 fiber protein, indicating that a proportion of neutralizing antibodies are directed against the Ad5 fiber.
Asunto(s)
Adenoviridae/inmunología , Factor X/metabolismo , Vectores Genéticos/inmunología , Hepatocitos/virología , Sueros Inmunes/metabolismo , Línea Celular Tumoral , Terapia Genética/métodos , Humanos , Pruebas de Neutralización , Transducción Genética , Acoplamiento ViralRESUMEN
A critical goal of vaccine development for a wide variety of pathogens is the induction of potent and durable mucosal immunity. However, it has been assumed that this goal would be difficult to achieve by systemic vaccination due to the anatomic and functional distinctness of the systemic and mucosal immune systems and the resultant compartmentalization of immune responses. In this study, we show that Ag-specific CD8(+) T lymphocytes traffic efficiently to mucosal surfaces following systemic vaccination. Intramuscular immunization with recombinant adenovirus (rAd) vector-based vaccines expressing SIV Gag resulted in potent, durable, and functional CD8(+) T lymphocyte responses at multiple mucosal effector sites in both mice and rhesus monkeys. In adoptive transfer studies in mice, vaccine-elicited systemic CD8(+) T lymphocytes exhibited phenotypic plasticity, up-regulated mucosal homing integrins and chemokine receptors, and trafficked rapidly to mucosal surfaces. Moreover, the migration of systemic CD8(+) T lymphocytes to mucosal compartments accounted for the vast majority of Ag-specific mucosal CD8(+) T lymphocytes induced by systemic vaccination. Thus, i.m. vaccination can overcome immune compartmentalization and generate robust mucosal CD8(+) T lymphocyte memory. These data demonstrate that the systemic and mucosal immune systems are highly coordinated following vaccination.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/trasplante , Movimiento Celular/inmunología , Epítopos de Linfocito T/inmunología , Inmunidad Mucosa , Adenovirus Humanos/genética , Adenovirus Humanos/inmunología , Animales , Linfocitos T CD8-positivos/virología , Movimiento Celular/genética , Epítopos de Linfocito T/biosíntesis , Vectores Genéticos/administración & dosificación , Vectores Genéticos/inmunología , Humanos , Inmunidad Celular/genética , Inmunidad Mucosa/genética , Memoria Inmunológica/genética , Inyecciones Intramusculares , Cinética , Macaca mulatta , Ratones , Ratones Endogámicos C57BL , Fase de Descanso del Ciclo Celular/genética , Fase de Descanso del Ciclo Celular/inmunología , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Replicación Viral/genética , Replicación Viral/inmunologíaRESUMEN
The development of a subunit vaccine for smallpox represents a potential strategy to avoid the safety concerns associated with replication-competent vaccinia virus. Preclinical studies to date with subunit smallpox vaccine candidates, however, have been limited by incomplete information regarding protective antigens and the requirement for multiple boost immunizations to afford protective immunity. Here we explore the protective efficacy of replication-incompetent, recombinant adenovirus serotype 35 (rAd35) vectors expressing the vaccinia virus intracellular mature virion (IMV) antigens A27L and L1R and extracellular enveloped virion (EEV) antigens A33R and B5R in a murine vaccinia virus challenge model. A single immunization with the rAd35-L1R vector effectively protected mice against a lethal systemic vaccinia virus challenge. The rAd35-L1R vector also proved more efficacious than the combination of four rAd35 vectors expressing A27L, L1R, A33R, and B5R. Moreover, serum containing L1R-specific neutralizing antibodies afforded postexposure prophylaxis after systemic vaccinia virus infection. In contrast, the combination of rAd35-L1R and rAd35-B5R vectors was required to protect mice against a lethal intranasal vaccinia virus challenge, suggesting that both IMV- and EEV-specific immune responses are important following intranasal infection. Taken together, these data demonstrate that different protective antigens are required based on the route of vaccinia virus challenge. These studies also suggest that rAd vectors warrant further assessment as candidate subunit smallpox vaccines.
Asunto(s)
Vacuna contra Viruela/inmunología , Viruela/inmunología , Viruela/prevención & control , Virus Vaccinia/inmunología , Adenoviridae/genética , Animales , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/uso terapéutico , Peso Corporal , Quimioprevención/métodos , Virus de la Ectromelia/genética , Ensayo de Inmunoadsorción Enzimática , Vectores Genéticos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Pruebas de Neutralización , Vacuna contra Viruela/genética , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Proteínas Virales/genética , Proteínas Virales/inmunologíaRESUMEN
Prime-boost vaccination regimens with heterologous antigen delivery systems have indicated that redirection of the immune response is feasible. We showed earlier that T-cell responses to circumsporozoite (CS) protein improved significantly when the protein is primed with recombinant adenovirus serotype 35 coding for CS (rAd35.CS). The current study was designed to answer the question whether such an effect can be extended to liver-stage antigens (LSA) of Plasmodium falciparum such as LSA-1. Studies with mice have demonstrated that the LSA-1 protein induces strong antibody response but a weak T-cell immunity. We first identified T-cell epitopes in LSA-1 by use of intracellular gamma interferon (IFN-gamma) staining and confirmed these epitopes by means of enzyme-linked immunospot assay and pentamer staining. We show that a single immunization with rAd35.LSA-1 induced a strong antigen-specific IFN-gamma CD8(+) T-cell response but no measurable antibody response. In contrast, vaccinations with the adjuvanted recombinant LSA-1 protein induced remarkably low cellular responses but strong antibody responses. Finally, both priming and boosting of the adjuvanted protein by rAd35 resulted in enhanced T-cell responses without impairing the level of antibody responses induced by the protein immunizations alone. Furthermore, the incorporation of rAd35 in the vaccination schedule led to a skewing of LSA-1-specific antibody responses toward a Th1-type immune response. Our results show the ability of rAd35 to induce potent T-cell immunity in combination with protein in a prime-boost schedule without impairing the B-cell response.
Asunto(s)
Adenoviridae , Antígenos de Protozoos/inmunología , Linfocitos B/inmunología , Inmunización Secundaria , Vacunas contra la Malaria/inmunología , Plasmodium falciparum/metabolismo , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Antígenos de Protozoos/química , Relación Dosis-Respuesta Inmunológica , Mapeo Epitopo , Epítopos de Linfocito T , Femenino , Malaria Falciparum/inmunología , Ratones , Ratones Endogámicos BALB C , Plasmodium falciparum/química , Vacunas Sintéticas/inmunologíaRESUMEN
Recently, we described the molecular identification of dendritic cell-specific TrAnsMembrane protein (DC-STAMP), a multimembrane-spanning protein preferentially expressed by human DC (hDC). In this report, we describe the identification and expression profile of the murine homologue of DC-STAMP (mDC-STAMP) as well as the characterization of the DC-STAMP protein. The results demonstrate that mDC-STAMP is over 90% homologous to hDC-STAMP and is also preferentially expressed by DC in vitro and ex vivo. mDC-STAMP expression is enhanced by interleukin-4 and down-regulated upon DC maturation. Analysis of differently tagged DC-STAMP proteins further demonstrates that hDC-STAMP and mDC-STAMP are glycosylated and primarily localize to an intracellular compartment. Applying confocal microscopy and electron microscopy, we demonstrate that hDC-STAMP localizes to the endoplasmic reticulum (ER) in human embryonic kidney 293 cells as well as hDC transduced with an adenovirus encoding hDC-STAMP-green fluorescent protein fusion protein. These data imply that DC-STAMP may exert its effect in the ER.
Asunto(s)
Células Dendríticas/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Clonación Molecular , Perfilación de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Humanos , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Homología de Secuencia de AminoácidoRESUMEN
Delivery of the full-length tumor antigen might be more successful in immunotherapy than single peptides and has the advantage that patients no longer need to be selected for their HLA type. In this study, we tested the in vitro induction of CAMEL/NY-ESO-ORF2-specific T cells by dendritic cells infected with an adenovirus (Ad) type 5 vector containing the fiber shaft and knob of human serotype Ad35 (Ad5F35 vector). Our data show induction of CD8(+) T cells specific for the known HLA-A(*)0201-binding CAMEL/NY-ESO-ORF2(1-11) epitope by DC infected with Ad5F35-CAMEL, but not by DC pulsed with the recombinant CAMEL protein. In one healthy donor, even CD8(+) T cells specific for a new HLA-B7-binding CAMEL/NY-ESO-ORF2(46-54) epitope were raised. In conclusion, the in vitro induction of CAMEL/NY-ESO-ORF2-specific CD8(+) T cells in healthy donors by DC infected with Ad5F35-CAMEL strongly supports further investigation of the Ad5F35 vector as a vehicle for gene transfer into DC for the generation of tumor antigen-specific CD8(+) T cell responses in vivo.
Asunto(s)
Adenoviridae/genética , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Vectores Genéticos , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Animales , Antígenos de Superficie , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Cisteína Endopeptidasas/metabolismo , Mapeo Epitopo , Terapia Genética , Antígenos HLA-A/metabolismo , Antígeno HLA-B7/metabolismo , Humanos , Interleucinas/metabolismo , Complejos Multienzimáticos/metabolismo , Péptidos/inmunología , Péptidos/metabolismo , Complejo de la Endopetidasa Proteasomal , Proteínas Recombinantes de Fusión/genéticaRESUMEN
The coxsackievirus-adenovirus receptor (CAR) is the described primary receptor for adenovirus serotype 5 (Ad5), a common human pathogen that has been exploited as a viral vector for gene therapy and vaccination. This study showed that monocytes and dendritic cells (DCs), such as freshly isolated human blood myeloid DCs, plasmacytoid DCs and monocyte-derived DCs, are susceptible to recombinant Ad5 (rAd5) infection despite their lack of CAR expression. Langerhans cells and dermal DCs from skin expressed CAR, but blocking CAR only partly decreased rAd5 infection, together suggesting that other receptor pathways mediate viral entry of these cells. Lactoferrin (Lf), an abundant protein in many bodily fluids known for its antiviral and antibacterial properties, promoted rAd5 infection in all cell populations except plasmacytoid DCs using a CAR-independent process. Lf caused phenotypic differentiation of the DCs, but cell activation played only a minor role in the increase in infection frequencies. The C-type lectin receptor DC-SIGN facilitated viral entry of rAd5-Lf complexes and this was dependent on high-mannose-type N-linked glycans on Lf. These results suggest that Lf present at high levels at mucosal sites can facilitate rAd5 attachment and enhance infection of DCs. A better understanding of the tropism and receptor mechanisms of Ad5 may help explain Ad5 pathogenesis and guide the engineering of improved rAd vectors.
Asunto(s)
Adenovirus Humanos/fisiología , Proteínas Portadoras/fisiología , Moléculas de Adhesión Celular/fisiología , Células Dendríticas/virología , Lectinas Tipo C/fisiología , Receptores de Superficie Celular/fisiología , Receptores Virales/fisiología , Acoplamiento Viral , Células Cultivadas , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus , Humanos , Lactoferrina , Monocitos/virologíaRESUMEN
We recently characterized the domains of the human cofactor protein CD46 involved in binding species B2 adenovirus (Ad) serotype 35. Here, the CD46 binding determinants are mapped for the species B1 Ad serotypes 3 and 7 and for the species B2 Ad11. Ad3, 7 and 11 bound and transduced CD46-positive rodent BHK cells at levels similar to Ad35. By using antibody-blocking experiments, hybrid CD46-CD4 receptor constructs and CD46 single point mutants, it is shown that Ad3, 7 and 11 share many of the Ad35-binding features on CD46. Both CD46 short consensus repeat domains SCR I and SCR II were necessary and sufficient for optimal binding and transgene expression, provided that they were positioned at an appropriate distance from the cell membrane. Similar to Ad35, most of the putative binding residues of Ad3, 7 and 11 were located on the same glycan-free, solvent-exposed face of the SCR I or SCR II domains, largely overlapping with the binding surface of the recently solved fiber knob Ad11-SCR I-II three-dimensional structure. Differences between species B1 and B2 Ads were documented with competition experiments based on anti-CD46 antibodies directed against epitopes flanking the putative Ad-binding sites, and with competition experiments based on soluble CD46 protein. It is concluded that the B1 and B2 species of Ad engage CD46 through similar binding surfaces.
Asunto(s)
Adenoviridae/fisiología , Sitios de Unión/genética , Proteína Cofactora de Membrana/metabolismo , Receptores Virales/metabolismo , Acoplamiento Viral , Adenoviridae/clasificación , Animales , Anticuerpos/metabolismo , Línea Celular , Cricetinae , Citometría de Flujo , Genes Reporteros , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Humanos , Proteína Cofactora de Membrana/química , Proteína Cofactora de Membrana/genética , Ratones , Modelos Moleculares , Mutación Puntual , Mapeo de Interacción de Proteínas , Receptores Virales/química , Receptores Virales/genética , Transducción GenéticaRESUMEN
Although replication-incompetent recombinant adenovirus (rAd) type 5 is a potent vaccine vector for stimulating T and B cell responses, high seroprevalence of adenovirus type 5 (Ad5) within human populations may limit its clinical utility. Therefore, alternative adenovirus serotypes have been studied as vaccine vectors. In this study, we characterized the ability of rAd5 and rAd35 to infect and induce maturation of human CD11c(+) myeloid dendritic cells (MDCs) and CD123(+) plasmacytoid dendritic cells (PDCs), and their ability to stimulate Ag-specific T cells. Both MDCs and PDCs were found to express the primary receptor for Ad35 (CD46) but not Ad5 (coxsackie-adenovirus receptor; CAR). Both dendritic cell (DC) subsets were also more susceptible to rAd35 than to rAd5. MDCs were more susceptible to both rAd35 and rAd5 than were PDCs. Whereas rAd35 used CD46 for entry into DCs, entry of rAd5 may be through a CAR-independent pathway. Exposure to rAd35 but not rAd5 induced high levels of IFN-alpha in PDCs and phenotypic differentiation in both DC subsets. MDCs and PDCs exposed to either rAd5 or rAd35 encoding for CMV pp65 were able to present pp65 and activate CMV-specific memory CD8(+) and CD4(+) T cells in a dose-dependent manner, but MDCs stimulated the highest frequencies of pp65-specific T cells. Responding T cells expressed multiple functions including degranulation (CD107a surface mobilization) and production of IFN-gamma, IL-2, TNF-alpha, and MIP-1beta. Thus, the ability of rAd35 to naturally target important DC subsets, induce their maturation, and appropriately present Ag to T cells may herald greater in vivo immunogenicity than has been observed with rAd5.
Asunto(s)
Adenovirus Humanos/genética , Adenovirus Humanos/inmunología , Células Dendríticas/inmunología , Células Dendríticas/virología , Memoria Inmunológica , Células Mieloides/inmunología , Células Mieloides/virología , Subgrupos de Linfocitos T/inmunología , Anticuerpos Bloqueadores/farmacología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Células Cultivadas , Técnicas de Cocultivo , Células Dendríticas/clasificación , Epítopos de Linfocito T/inmunología , Vectores Genéticos/inmunología , Células HeLa , Humanos , Memoria Inmunológica/genética , Inmunofenotipificación , Interferón-alfa/biosíntesis , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Proteína Cofactora de Membrana/inmunología , Proteína Cofactora de Membrana/metabolismo , Proteínas Recombinantes de Fusión/síntesis química , Proteínas Recombinantes de Fusión/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
Replication-incompetent adenovirus type 35 (rAd35) represents a potent vaccine carrier that elicits strong, antigen-specific T- and B-cell responses in diverse preclinical models. Moreover, Ad35 is rare in human populations, resulting in the absence of neutralizing antibodies against this carrier, in contrast to the commonly used rAd5. Therefore, rAd35 is being investigated as a vaccine carrier for a number of diseases for which an effective vaccine is needed, including malaria, AIDS and tuberculosis. However, it can be perceived that effective immunization will require insertion of multiple antigens into adenoviral vectors. We therefore wanted to create rAd35 vectors carrying double expression cassettes, to expand within one vector the number of insertion sites for foreign DNA encoding antigenic proteins. We show that it is possible to generate rAd35 vectors carrying two cytomegalovirus promoter-driven expression cassettes, provided that the polyadenylation signals in each expression cassette are not identical. We demonstrate excellent rAd35 vector stability and show that expression of a transgene is not influenced by the presence of a second expression cassette. Moreover, by using two model vaccine antigens, i.e. the human immunodeficiency virus-derived Env-gp120 protein and the Plasmodium falciparum-derived circumsporozoite protein, we demonstrate that potent T- and B-cell responses are induced to both antigens expressed from a single vector. Such rAd35 vectors thus expand the utility of rAd35 vaccine carriers for the development of vaccines against, for example, malaria, AIDS and tuberculosis.
Asunto(s)
Adenoviridae/genética , Expresión Génica , Vectores Genéticos , Vacunas Virales/genética , Animales , Anticuerpos Antiprotozoarios/sangre , Anticuerpos Antivirales/sangre , Citomegalovirus/genética , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/inmunología , Interferón gamma/biosíntesis , Ratones , Ratones Endogámicos BALB C , Regiones Promotoras Genéticas , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Señales de Poliadenilación de ARN 3'/genética , Bazo/inmunología , Linfocitos T/inmunología , Vacunas Sintéticas/genética , Replicación Viral/genéticaRESUMEN
Previous studies have shown that the immunogenicity of rodent malaria parasite-derived circumsporozoite protein (CS) can be improved by deleting the glycosyl-phosphatidyl-inositol (GPI) signal sequence. To study whether GPI signal sequence deletion would also improve immunogenicity of CS derived from the major plasmodium species causing mortality in humans (P. falciparum), we tested different variants of the P. falciparum CS protein in the context of a live vector-based vaccine carrier (rAd35). We demonstrate that deletion of the GPI signal sequence from CS did not result in altered expression or secretion. In contrast, cellular localization was clearly altered, which perhaps helps to explain the significant improvement of anti-CS antibody and T-cell responses observed in mice using deletion variants in the context of the rAd35 carrier. Our results show that rational design of antigens is warranted for further development of malaria vaccines.
Asunto(s)
Glicosilfosfatidilinositoles/inmunología , Vacunas contra la Malaria/inmunología , Plasmodium falciparum/inmunología , Señales de Clasificación de Proteína/fisiología , Proteínas Protozoarias/inmunología , Adenoviridae/genética , Secuencia de Aminoácidos , Animales , Linfocitos B/inmunología , Línea Celular Tumoral , Femenino , Eliminación de Gen , Glicosilfosfatidilinositoles/genética , Glicosilfosfatidilinositoles/metabolismo , Humanos , Vacunas contra la Malaria/genética , Ratones , Ratones Endogámicos CBA , Datos de Secuencia Molecular , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Señales de Clasificación de Proteína/genética , Proteínas Protozoarias/biosíntesis , Proteínas Protozoarias/genética , Linfocitos T/inmunologíaRESUMEN
Recent evidence supports a role for vitamin K-dependent coagulation zymogens in adenovirus serotype 5 (Ad5, subgroup C) infection of hepatocytes. Here, we assessed the effect of virus-zymogen interaction on cellular transduction using a panel of fiber (f)-pseudotyped viruses derived from subgroup D (f47, f33, f24, f45, f17, f30). Each virus directly bound factor X (FX) as determined by surface plasmon resonance, resulting in enhanced cell surface binding. Infection of HepG2 cells was promoted by FX but not by FVII or FIX, while transduction of CHO cells was blocked in heparan sulfate proteoglycan-deficient cells. This suggests a broad role for FX in adenovirus infectivity.
Asunto(s)
Adenoviridae/clasificación , Adenoviridae/metabolismo , Factores de Coagulación Sanguínea/metabolismo , Precursores Enzimáticos/metabolismo , Adenoviridae/genética , Animales , Cricetinae , Humanos , Filogenia , Unión Proteica , Internalización del VirusRESUMEN
Recombinant adenovirus serotype 5 (rAd5) vector-based vaccines are currently being developed for both human immunodeficiency virus type 1 and other pathogens. The potential limitations associated with rAd5 vectors, however, have led to the construction of novel rAd vectors derived from rare Ad serotypes. Several rare serotype rAd vectors have already been described, but a detailed comparison of multiple rAd vectors from subgroups B and D has not previously been reported. Such a comparison is critical for selecting optimal rAd vectors for advancement into clinical trials. Here we describe the construction of three novel rAd vector systems from Ad26, Ad48, and Ad50. We report comparative seroprevalence and immunogenicity studies involving rAd11, rAd35, and rAd50 vectors from subgroup B; rAd26, rAd48, and rAd49 vectors from subgroup D; and rAd5 vectors from subgroup C. All six rAd vectors from subgroups B and D exhibited low seroprevalence in a cohort of 200 individuals from sub-Saharan Africa, and they elicited Gag-specific cellular immune responses in mice both with and without preexisting anti-Ad5 immunity. The rAd vectors from subgroup D were also evaluated using rhesus monkeys and were shown to be immunogenic after a single injection. The rAd26 vectors proved the most immunogenic among the rare serotype rAd vectors studied, although all rare serotype rAd vectors were still less potent than rAd5 vectors in the absence of anti-Ad5 immunity. These studies substantially expand the portfolio of rare serotype rAd vectors that may prove useful as vaccine vectors for the developing world.
Asunto(s)
Infecciones por Adenoviridae/epidemiología , Adenoviridae/genética , Vectores Genéticos/genética , Vacunas Sintéticas/genética , Vacunas Virales/genética , Infecciones por Adenoviridae/sangre , África del Sur del Sahara/epidemiología , Animales , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , Ensayo de Inmunoadsorción Enzimática , Vectores Genéticos/inmunología , Humanos , Macaca mulatta , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Pruebas de Neutralización , Análisis de Secuencia de ADN , Estudios Seroepidemiológicos , SerotipificaciónRESUMEN
Previously, we have shown the potency of recombinant Adenovirus serotype 35 viral vaccines (rAd35) to induce strong immune response against the circumsporozoite protein (CS) of the plasmodium parasite. To further optimize immunogenicity of Ad35-based malaria vaccines we formulated rAd35.CS vaccine with aluminium phosphate adjuvant (AlPO(4)). In contrast to the conventional protein based vaccines no absorption to aluminium adjuvant was observed and rAd35 viral in vitro infectivity in mammalian cells was preserved. Immunization with Ad35.CS formulated with AlPO(4) resulted in significantly higher CS specific T and B cell responses in mice upon either single or prime-boost vaccination regimens as compared to rAd35.CS alone. With these results we report for the first time the feasibility of using an AlPO(4) adjuvant to increase the potency of a live adenovirus serotype 35-based vaccine.
Asunto(s)
Adenoviridae/inmunología , Adyuvantes Inmunológicos/farmacología , Compuestos de Aluminio/farmacología , Vacunas contra la Malaria/inmunología , Fosfatos/farmacología , Adyuvantes Inmunológicos/química , Compuestos de Aluminio/química , Animales , Formación de Anticuerpos/inmunología , Supervivencia Celular , Química Farmacéutica , Relación Dosis-Respuesta a Droga , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente Indirecta , Vectores Genéticos , Inmunidad Celular/inmunología , Inmunización Secundaria , Vacunas contra la Malaria/química , Ratones , Ratones Endogámicos BALB C , Fosfatos/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vacunas Sintéticas/química , Vacunas Sintéticas/inmunologíaRESUMEN
A workshop on viral vectors for malaria vaccine development, organized by the PATH Malaria Vaccine Initiative, was held in Bethesda, MD on October 20, 2005. Recent advancements in viral-vectored malaria vaccine development and emerging vector technologies were presented and discussed. Classic viral vectors such as poxvirus, adenovirus and alphavirus vectors have been successfully used to deliver malaria antigens. Some of the vaccine candidates have demonstrated their potential in inducing malaria-specific immunity in animal models and human trials. In addition, emerging viral-vector technologies, such as measles virus (MV), vesicular stomatitis virus (VSV) and yellow fever (YF) virus, may also be useful for malaria vaccine development. Studies in animal models suggest that each viral vector is unique in its ability to induce humoral and/or cellular immune responses. Those studies have also revealed that optimization of Plasmodium genes for mammalian expression is an important aspect of vaccine design. Codon-optimization, surface-trafficking, de-glycosylation and removal of toxic domains can lead to improved immunogenicity. Understanding the vector's ability to induce an immune response and the expression of malaria antigens in mammalian cells will be critical in designing the next generation of viral-vectored malaria vaccines.
Asunto(s)
Vectores Genéticos/genética , Vacunas contra la Malaria/biosíntesis , Vacunas Sintéticas/biosíntesis , Virus/genética , Adenoviridae/genética , Alphavirus/genética , Vacunas contra la Malaria/inmunología , Virus del Sarampión/genética , Poxviridae/genética , Vacunas Sintéticas/inmunología , Virus de la Estomatitis Vesicular Indiana/genética , Virus de la Fiebre Amarilla/genéticaRESUMEN
The high prevalence of preexisting immunity to adenovirus serotype 5 (Ad5) in human populations has led to the development of recombinant adenovirus (rAd) vectors derived from rare Ad serotypes as vaccine candidates for human immunodeficiency virus type 1 and other pathogens. Vaccine vectors have been constructed from Ad subgroup B, including rAd11 and rAd35, as well as from Ad subgroup D, including rAd49. However, the optimal combination of vectors for heterologous rAd prime-boost vaccine regimens and the extent of cross-reactive vector-specific neutralizing antibodies (NAbs) remain poorly defined. We have shown previously that the closely related vectors rAd11 and rAd35 elicited low levels of cross-reactive NAbs. Here we show that these cross-reactive NAbs correlated with substantial sequence homology in the hexon hypervariable regions (HVRs) and suppressed the immunogenicity of heterologous rAd prime-boost regimens. In contrast, vectors with lower hexon HVR homology, such as rAd35 and rAd49, did not elicit detectable cross-reactive vector-specific NAbs. Consistent with these findings, rAd35-rAd49 vaccine regimens proved more immunogenic than both rAd35-rAd5 and rAd35-rAd11 regimens in mice with anti-Ad5 immunity. These data suggest that optimal heterologous rAd prime-boost regimens should include two vectors that are both rare in human populations to circumvent preexisting antivector immunity as well as sufficiently immunologically distinct to avoid cross-reactive antivector immunity.
Asunto(s)
Adenoviridae/inmunología , Reacciones Cruzadas/inmunología , Vectores Genéticos/inmunología , Vacunas Sintéticas/inmunología , Adenoviridae/genética , Animales , Anticuerpos/inmunología , Antígenos Virales/genética , Proteínas de la Cápside/genética , Técnicas para Inmunoenzimas , Ratones , Ratones Endogámicos C57BL , Pruebas de NeutralizaciónRESUMEN
Recombinant adenoviral vectors based on type 5 (rAd5) show great promise as a vaccine carrier. However, neutralizing activity against Ad5 is prevalent and high-titred among human populations, and significantly dampens Ad5-based vaccine modalities. The generation of alternative adenoviral vectors with low seroprevalence thus receives much research attention. Here, it is shown that a member from human adenovirus subgroup D, i.e. Ad49, does not cross-react with Ad5 neutralizing activity, making it a candidate serotype for vector development. Therefore, a plasmid system that allows formation of replication-incompetent adenovirus serotype 49 vaccine vectors (rAd49) was constructed and it was demonstrated that rAd49 can be successfully propagated to high titres on existing Ad5.E1-complementing cell lines such as PER.C6. Using an rAd49 vector carrying the luciferase marker gene, detailed seroprevalence studies were performed, demonstrating that rAd49 has low seroprevalence and neutralizing antibody titres worldwide. Also, we have initiated rAd49 vector receptor usage suggesting that rAd49 utilizes hCD46 as a cellular receptor. Finally, the immunogenicity of the rAd49 vector was assessed and it was shown that an rAd49.SIVGag vaccine induces strong anti-SIVGag CD8+ T-lymphocytes in naïve mice, albeit less than an rAd5.SIVGag vaccine. However, in mice with high anti-Ad5 immunity the rAd5.SIVGag vaccine was severely blunted, whereas the anti-SIVGag response was not significantly suppressed using the rAd49.SIVGag vaccine. These data demonstrate the potential of a replication deficient human group D adenoviral vector for vaccination purposes.
Asunto(s)
Adenovirus Humanos/crecimiento & desarrollo , Adenovirus Humanos/inmunología , Replicación Viral , Adenovirus Humanos/genética , Adenovirus Humanos/fisiología , Animales , Anticuerpos Antivirales , Línea Celular , Ingeniería Genética , Vectores Genéticos/genética , Vectores Genéticos/inmunología , Humanos , Proteína Cofactora de Membrana , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Especificidad de Órganos , Vacunas Sintéticas , Vacunas Virales/inmunologíaRESUMEN
Recombinant adenovirus (Ad) type 35 (rAd35) shows great promise as vaccine carrier with the advantage of low pre-existing immunity in human populations, in contrast to the more commonly used rAd5 vector. The rAd35 vector uses CD46 as a high-affinity receptor, which, unlike the rAd5 receptor, is expressed on human dendritic cells (DC), the most powerful APCs identified to date. In this study, we show that in contrast to rAd5, rAd35 infects migrated and mature CD83+ cutaneous DC with high efficiency (up to 80%), when delivered intradermally in an established human skin explant model. The high transduction efficiency is in line with high expression levels of CD46 detected on migratory cutaneous DC, which proved to be further increased upon intradermal administration of GM-CSF and IL-4. As compared with Ad5, these Ad35 infection characteristics translate into higher absolute numbers of skin-emigrated DC per explant that both express the transgene and are phenotypically mature. Finally, we demonstrate that upon intracutaneous delivery of a rAd35 vaccine encoding the circumsporozoite (CS) protein of Plasmodium falciparum, emigrated DC functionally express and process CS-derived epitopes and are capable of activating specific CD8+ effector T cells, as evidenced by activation of an HLA-A2-restricted CS-specific CD8+ T cell clone. Collectively, these data demonstrate the utility of rAd35 vectors for efficient in vivo human DC transduction.