RESUMEN
The international spread of infectious diseases is a global problem of health security. Vaccination is one of the most successful and profitable health interventions. Oral immunization has significant advantages over the widely used parental vaccines. Intestinal and free-living protozoa express on their surface a dense layer of proteins that protect them from hostile environmental conditions. The use of variable surface proteins (VSPs), such as those of the intestinal protozoan Giardia lamblia, is a feasible mechanism for the generation of oral vaccines, since they are highly immunogenic as well as resistant to changes in pH and proteases. In a recently published article, we showed that these properties of VSPs can be exploited to protect and enhance the immunogenicity of vaccine antigens, thus enabling their oral administration. We recently generated an oral vaccine against influenza virus composed of virus-like particles (VLPs) containing VSPs of G. lamblia and the HA antigen (viral hemagglutinin) in its envelope. When administered orally to mice, these coated particles elicit HA-specific humoral (systemic and local) and cellular responses, without the need of any additional adjuvant. Treated mice are protected against viral challenge as well as against the development of tumors expressing the HA vaccine antigen.
La propagación internacional de enfermedades infecciosas constituye un problema global de seguridad sanitaria. La vacunación es una de las intervenciones en salud más exitosas y efectivas. La administración por vía oral presenta ventajas significativas sobre la vía parental utilizada comúnmente. Protozoarios intestinales y de vida libre expresan en su superficie una densa capa de proteínas que los protegen de condiciones ambientales hostiles. La utilización de proteínas de superficie variante-específicas o VSPs (del inglés "Variant-specific Surface Proteins") tales como las del protozoario intestinal Giardia lamblia constituye un enfoque eficiente para la generación de vacunas orales, dada su alta inmunogenicidad y su resistencia a cambios de pH y proteasas. En un trabajo reciente mostramos que estas propiedades pueden ser explotadas para proteger antígenos vacunales y potenciar su inmunogenicidad, facilitando así su administración oral. Como modelo inicial, generamos una vacuna oral contra el virus de la influenza compuesta por partículas similares a virus (VLPs, del inglés "virus-like particles") que contienen en su envoltorio VSPs de G. lamblia y el antígeno HA (hemaglutinina del virus de la influenza). La administración oral a ratones de estas partículas recubiertas con VSPs y HA induce una respuesta inmune humoral (sistémica y de mucosa) y celular específica para HA sin la necesidad de adyuvantes externos. La respuesta inmune generada protege frente al desafío con el virus y también frente al desarrollo de tumores que expresan el antígeno vacunal HA.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Proteínas de la Membrana/inmunología , Proteínas Protozoarias/inmunología , Vacunas de Partículas Similares a Virus/inmunología , Vacunas/inmunología , Administración Oral , Animales , Giardia lamblia/química , Humanos , Inmunidad Humoral/efectos de los fármacos , Proteínas de la Membrana/administración & dosificación , Proteínas Protozoarias/administración & dosificación , Vacunas/administración & dosificación , Vacunas de Partículas Similares a Virus/administración & dosificaciónRESUMEN
Breast cancer is a worldwide health problem, and the complexity of the disease, as well as the lack of treatment specificity, generates an urgent need for developing prophylactic and therapeutic measures. Searching for novel epitope-based approaches able to induce tumour immunity, we designed virus-like particles (VLPs) derived from Human parvovirus B19 assembled of chimeric VP2 proteins displaying two epitopes from the insulin-like growth factor-1 receptor (IGF-1R). Here, we present the generation of two chimeric VP2s that retain the stability, solubility and conditions of purification and assembly of the native VP2. We generated versatile chimeric multiepitope anti-cancer vaccine candidates, which prevented and delayed tumour growth when used in a prophylactic scheme of 4 weekly immunizations prior to 4T1 cell inoculation in female BALB/c mice. The presence of specific antibodies against the displayed epitopes suggests their participation in the protective effect; in contrast, no significant proliferative T-cell responses were recorded following stimulation by specific epitopes. The results comprise an approach whereby fusing desired epitopes from cancer to the N-terminus of B19 VP2 protein can generate a library of chimeric VP2-desired epitopes for further assembly in a designed and personalized epitope delivery system.
Asunto(s)
Neoplasias de la Mama/prevención & control , Epítopos/metabolismo , Parvovirus B19 Humano/metabolismo , Receptor IGF Tipo 1/inmunología , Vacunas de Partículas Similares a Virus/administración & dosificación , Animales , Apoptosis , Neoplasias de la Mama/inmunología , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/inmunología , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Epítopos/genética , Femenino , Humanos , Inmunización , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Parvovirus B19 Humano/genética , Resultado del Tratamiento , Vacunas de Partículas Similares a Virus/genética , Vacunas de Partículas Similares a Virus/inmunologíaRESUMEN
Intestinal and free-living protozoa, such as Giardia lamblia, express a dense coat of variant-specific surface proteins (VSPs) on trophozoites that protects the parasite inside the host's intestine. Here we show that VSPs not only are resistant to proteolytic digestion and extreme pH and temperatures but also stimulate host innate immune responses in a TLR-4 dependent manner. We show that these properties can be exploited to both protect and adjuvant vaccine antigens for oral administration. Chimeric Virus-like Particles (VLPs) decorated with VSPs and expressing model surface antigens, such as influenza virus hemagglutinin (HA) and neuraminidase (NA), are protected from degradation and activate antigen presenting cells in vitro. Orally administered VSP-pseudotyped VLPs, but not plain VLPs, generate robust immune responses that protect mice from influenza infection and HA-expressing tumors. This versatile vaccine platform has the attributes to meet the ultimate challenge of generating safe, stable and efficient oral vaccines.
Asunto(s)
Giardia lamblia/química , Vacunas contra la Influenza/inmunología , Proteínas de la Membrana/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Proteínas Protozoarias/inmunología , Vacunas de Partículas Similares a Virus/inmunología , Adyuvantes Inmunológicos , Administración Oral , Animales , Presentación de Antígeno/efectos de los fármacos , Bioingeniería/métodos , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Células Dendríticas/virología , Femenino , Expresión Génica , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Humanos , Inmunidad Innata/efectos de los fármacos , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/genética , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Transgénicos , Neuraminidasa/genética , Neuraminidasa/inmunología , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/virología , Estabilidad Proteica , Proteínas Protozoarias/genética , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunología , Trofozoítos/química , Vacunación , Vacunas de Partículas Similares a Virus/administración & dosificación , Vacunas de Partículas Similares a Virus/genéticaRESUMEN
Paracoccidioidomycosis (PCM) is an infectious disease endemic to South America, caused by the thermally dimorphic fungi Paracoccidioides. Currently, there is no effective human vaccine that can be used in prophylactic or therapeutic regimes. We tested the hypothesis that the immunogenicity of the immunodominant CD4+ T-cell epitope (P10) of Paracoccidioides brasiliensis gp43 antigen might be significantly enhanced by using a hepatitis B virus-derived particle (VLP) as an antigen carrier. This chimera was administered to mice as a (His)6-purified protein (rPbT) or a replication-deficient human type 5 adenoviral vector (rAdPbT) in an immunoprophylaxis assay. The highly virulent Pb18 yeast strain was used to challenge our vaccine candidates. Fungal challenge evoked robust P10-specific memory CD4+ T cells secreting protective Th-1 cytokines in most groups of immunized mice. Furthermore, the highest level of fungal burden control was achieved when rAdPbT was inoculated in a homologous prime-boost regimen, with 10-fold less CFU recovering than in non-vaccinated mice. Systemic Pb18 spreading was only prevented when rAdPbT was previously inoculated. In summary, we present here VLP/P10 formulations as vaccine candidates against PCM, some of which have demonstrated for the first time their ability to prevent progression of this pernicious fungal disease, which represents a significant social burden in developing countries.
Asunto(s)
Antígenos Fúngicos/inmunología , Linfocitos T CD4-Positivos/inmunología , Epítopos de Linfocito T/inmunología , Proteínas Fúngicas/inmunología , Vacunas Fúngicas/administración & dosificación , Glicoproteínas/inmunología , Paracoccidioides/crecimiento & desarrollo , Paracoccidioides/inmunología , Paracoccidioidomicosis/prevención & control , Animales , Citocinas/inmunología , Citocinas/metabolismo , Epítopos de Linfocito T/genética , Vacunas Fúngicas/inmunología , Virus de la Hepatitis B/genética , Inmunización , Epítopos Inmunodominantes/inmunología , Inmunogenicidad Vacunal , Memoria Inmunológica , Hígado/microbiología , Pulmón/microbiología , Ratones Endogámicos BALB C , Paracoccidioidomicosis/inmunología , Paracoccidioidomicosis/microbiología , Bazo/microbiología , Células TH1/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Vacunas de Partículas Similares a Virus/administración & dosificación , Vacunas de Partículas Similares a Virus/genética , Vacunas de Partículas Similares a Virus/inmunologíaRESUMEN
Two new vaccine candidates against dengue virus (DENV) infection were generated by fusing the coding sequences of the self-budding Z protein from Junin virus (Z-JUNV) to those of two cryptic peptides (Z/DENV-P1 and Z/DENV-P2) conserved on the envelope protein of all serotypes of DENV. The capacity of these chimeras to generate virus-like particles (VLPs) and to induce virus-neutralizing antibodies in mice was determined. First, recombinant proteins that displayed reactivity with a Z-JUNV-specific serum by immunofluorescence were detected in HEK-293 cells transfected with each of the two plasmids and VLP formation was also observed by transmission electron microscopy. Next, we determined the presence of antibodies against the envelope peptides of DENV in the sera of immunized C57BL/6 mice. Results showed that those animals that received Z/DENV-P2 DNA coding sequences followed by a boost with DENV-P2 synthetic peptides elicited significant specific antibody titers (≥6.400). Finally, DENV plaque-reduction neutralization tests (PRNT) were performed. Although no significant protective effect was observed when using sera of Z/DENV-P1-immunized animals, antibodies raised against vaccine candidate Z/DENV-P2 (diluted 1:320) were able to reduce in over 50 % the number of viral plaques generated by infectious DENV particles. This reduction was comparable to that of the 4G2 DENV-specific monoclonal cross-reactive (all serotypes) neutralizing antibody. We conclude that Z-JUNV-VLP is a valid carrier to induce antibody-mediated immune responses in mice and that Z/DENV-P2 is not only immunogenic but also protective in vitro against infection of cells with DENV, deserving further studies. On the other side, DENV's fusion peptide-derived chimera Z/DENV-P1 did not display similar protective properties.
Asunto(s)
Anticuerpos Neutralizantes/sangre , Vacunas contra el Dengue/inmunología , Virus del Dengue/genética , Portadores de Fármacos , Virus Junin/genética , Vacunas de Partículas Similares a Virus/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos Antivirales/sangre , Vacunas contra el Dengue/administración & dosificación , Vacunas contra el Dengue/genética , Ratones Endogámicos C57BL , Pruebas de Neutralización , Resultado del Tratamiento , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Vacunas de Partículas Similares a Virus/administración & dosificación , Vacunas de Partículas Similares a Virus/genética , Proteínas del Envoltorio Viral/genética , Ensayo de Placa ViralRESUMEN
Rabies is one of the most lethal infectious diseases in the world, with a mortality approaching 100%. There are between 60,000 and 70,000 reported annual deaths, but this is probably an underestimation. Despite the fact that there are vaccines available for rabies, there is a real need of developing more efficacious and cheaper vaccines. This is particularly true for veterinary vaccines because dogs are still the main vector for rabies transmission to human beings. In a previous work, we described the development and characterization of rabies virus-like particles (RV-VLPs) expressed in HEK293 cells. We showed that RV-VLPs are able to induce a specific antibodies response. In this work, we show that VLPs are able to protect mice against virus challenge. Furthermore, we developed a VLPs expressing HEK-293 clone (sP2E5) that grows in serum free medium (SFM) reaching high cell densities. sP2E5 was cultured in perfusion mode in a 5 L bioreactor for 20 days, and the RV-VLPs produced were capable of triggering a protective immune response without the need of concentration or adjuvant addition. Further, these VLPs are able to induce the production of rabies virus neutralizing antibodies. These results demonstrate that RV-VLPs are a promising rabies vaccine candidate.
Asunto(s)
Anticuerpos Neutralizantes/biosíntesis , Anticuerpos Antivirales/biosíntesis , Vacunas Antirrábicas/inmunología , Virus de la Rabia/inmunología , Vacunas de Partículas Similares a Virus/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Reactores Biológicos , Medio de Cultivo Libre de Suero , Perros , Células HEK293 , Humanos , Ratones , Rabia/prevención & control , Vacunas Antirrábicas/administración & dosificación , Virus de la Rabia/patogenicidad , Potencia de la Vacuna , Vacunas de Partículas Similares a Virus/administración & dosificaciónRESUMEN
Foot-and-mouth disease (FMD) has caused severe economic losses to millions of farmers worldwide. In this work, the coding genes of 141-160 epitope peptide (EP141-160) of VP1 were inserted into the coat protein (CP) genes of MS2 in prokaryotic expression vector, and the recombinant protein self-assembled into virus-like particles (VLP). Results showed that the CP-EP141-160 VLP had a strong immunoreaction with the FMD virus (FMDV) antigen in vitro, and also had an effective immune response in mice. Further virus challenge tests were carried out on guinea pigs and swine, high-titer neutralizing antibodies were produced and the CP-EP141-160 VLP vaccine could protect most of the animals against FMDV.
Asunto(s)
Anticuerpos Neutralizantes/biosíntesis , Anticuerpos Antivirales/biosíntesis , Proteínas de la Cápside/inmunología , Virus de la Fiebre Aftosa/genética , Virus de la Fiebre Aftosa/inmunología , Fiebre Aftosa/prevención & control , Vacunas de Partículas Similares a Virus/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Antivirales/inmunología , Proteínas de la Cápside/genética , Ensayo de Inmunoadsorción Enzimática , Epítopos , Fiebre Aftosa/inmunología , Adyuvante de Freund , Cobayas , Levivirus/genética , Ratones , Pruebas de Neutralización , Porcinos , Vacunas de Partículas Similares a Virus/administración & dosificación , Vacunas Virales/administración & dosificaciónRESUMEN
HPV L1-based virus-like particles vaccines (VLPs) efficiently induce temporary prophylactic activity through the induction of neutralizing antibodies; however, VLPs that can provide prophylactic as well as therapeutic properties for longer periods of time are needed. For this purpose, we generated a novel HPV 16 L1-based chimeric virus-like particle (cVLP) produced in plants that contains a string of T-cell epitopes from HPV 16 E6 and E7 fused to its C-terminus. In the present study, we analyzed the persistence of specific IgG antibodies with neutralizing activity induced by immunization with these cVLPs, as well as their therapeutic potential in a tumor model of C57BL/6 mice. We observed that these cVLPs induced persistent IgG antibodies for over 12 months, with reactivity and neutralizing activity for VLPs composed of only the HPV-16 L1 protein. Efficient protection for long periods of time and inhibition of tumor growth induced by TC-1 tumor cells expressing HPV-16 E6/E7 oncoproteins, as well as significant tumor reduction (57 %), were observed in mice immunized with these cVLPs. Finally, we discuss the possibility that chimeric particles of the type described in this work may be the basis for developing HPV prophylactic and therapeutic vaccines with high efficacy.
Asunto(s)
Proteínas de la Cápside/inmunología , Carcinoma/prevención & control , Proteínas Oncogénicas Virales/inmunología , Proteínas E7 de Papillomavirus/inmunología , Infecciones por Papillomavirus/prevención & control , Vacunas contra Papillomavirus/inmunología , Proteínas Represoras/inmunología , Vacunas de Partículas Similares a Virus/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Proteínas de la Cápside/genética , Carcinoma/inmunología , Carcinoma/terapia , Modelos Animales de Enfermedad , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Femenino , Ratones , Ratones Endogámicos C57BL , Proteínas Oncogénicas Virales/genética , Proteínas E7 de Papillomavirus/genética , Infecciones por Papillomavirus/complicaciones , Infecciones por Papillomavirus/inmunología , Vacunas contra Papillomavirus/administración & dosificación , Vacunas contra Papillomavirus/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Represoras/genética , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Vacunas de Partículas Similares a Virus/administración & dosificación , Vacunas de Partículas Similares a Virus/genéticaRESUMEN
In previous studies we reported the cloning, expression and purification of the capsid protein from Dengue-2 virus. Subsequently, we described an in vitro-assembly process for the capsid protein, which resulted in nucleocapsid-like particles (recNLPs) that induced functional cell-mediated immunity and protection in mice. Moreover, our group reported the evaluation in non-human primates of the fusion protein P64k-domain III from Dengue-1 (PD10). This protein proved to be immunogenic and protective when Freund's adjuvant, but not alum, was used. Based on the previously demonstrated capacity of recNLPs to potentiate the immunogenicity of heterologous proteins, in this study we assess the immune response elicited by the formulation PD10-recNLPs-alum and its protective capacity against Dengue-1 and Dengue-2 virus. As expected, the humoral immune response was mainly directed against Dengue-1, while high levels of IFN-γ secretion were detected after stimulation with Dengue-1 and Dengue-2. Consistently, animals immunized with the bivalent formulation were significantly protected against challenge with either Dengue serotype. In conclusion, this report describes a novel formulation based on recombinant proteins and alum, which is protective against Dengue-1 and Dengue-2 in mice.
Asunto(s)
Vacunas contra el Dengue/administración & dosificación , Vacunas contra el Dengue/inmunología , Dengue/prevención & control , Vacunas de Partículas Similares a Virus/administración & dosificación , Vacunas de Partículas Similares a Virus/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Compuestos de Alumbre/administración & dosificación , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Proteínas de la Cápside/inmunología , Dengue/virología , Virus del Dengue/inmunología , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Interferón gamma/metabolismo , Leucocitos Mononucleares/inmunología , Ratones , Ratones Endogámicos BALB C , Pruebas de Neutralización , Análisis de Supervivencia , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunología , Proteínas Virales de Fusión/inmunologíaRESUMEN
The influenza pandemic of 2009 demonstrated the inability of the established global capacity for egg-based vaccine production technology to provide sufficient vaccine for the population in a timely fashion. Several alternative technologies for developing influenza vaccines have been proposed, among which non-replicating virus-like particles (VLPs) represent an attractive option because of their safety and immunogenic characteristics. VLP vaccines against pandemic influenza have been developed in tobacco plant cells and in Sf9 insect cells infected with baculovirus that expresses protein genes from pandemic influenza strains. These technologies allow rapid and large-scale production of vaccines (3-12 weeks). The 2009 influenza outbreak provided an opportunity for clinical testing of a pandemic influenza VLP vaccine in the midst of the outbreak at its epicenter in Mexico. An influenza A(H1N1)2009 VLP pandemic vaccine (produced in insect cells) was tested in a phase II clinical trial involving 4,563 healthy adults. Results showed that the vaccine is safe and immunogenic despite high preexisting anti-A(H1N1)2009 antibody titers present in the population. The safety and immunogenicity profile presented by this pandemic VLP vaccine during the outbreak in Mexico suggests that VLP technology is a suitable alternative to current influenza vaccine technologies for producing pandemic and seasonal vaccines.