RESUMEN
The development of latency reversing agents that potently reactivate HIV without inducing global T cell activation would benefit the field of HIV reservoir research and could pave the way to a functional cure. Here, we explore the reactivation capacity of a lipid nanoparticle containing Tat mRNA (Tat-LNP) in CD4 T cells from people living with HIV undergoing antiretroviral therapy (ART). When combined with panobinostat, Tat-LNP induces latency reversal in a significantly higher proportion of latently infected cells compared to PMA/ionomycin (≈ 4-fold higher). We demonstrate that Tat-LNP does not alter the transcriptome of CD4 T cells, enabling the characterization of latently infected cells in their near-native state. Upon latency reversal, we identify transcriptomic differences between infected cells carrying an inducible provirus and non-infected cells (e.g. LINC02964, GZMA, CCL5). We confirm the transcriptomic differences at the protein level and provide evidence that the long non-coding RNA LINC02964 plays a role in active HIV infection. Furthermore, p24+ cells exhibit heightened PI3K/Akt signaling, along with downregulation of protein translation, suggesting that HIV-infected cells display distinct signatures facilitating their long-term persistence. Tat-LNP represents a valuable research tool for in vitro reservoir studies as it greatly facilitates the in-depth characterization of HIV reservoir cells' transcriptome and proteome profiles.
Asunto(s)
Productos del Gen tat , VIH-1 , Nanopartículas , ARN Viral , Latencia del Virus , Latencia del Virus/efectos de los fármacos , Latencia del Virus/genética , Productos del Gen tat/genética , Productos del Gen tat/metabolismo , ARN Viral/administración & dosificación , ARN Viral/genética , ARN Viral/metabolismo , Nanopartículas/administración & dosificación , Nanopartículas/química , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/genética , Infecciones por VIH/virología , Panobinostat/farmacología , Terapia Antirretroviral Altamente Activa , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/virología , Antígenos CD4/genética , Antígenos CD4/metabolismo , VIH-1/efectos de los fármacos , VIH-1/genética , Provirus/efectos de los fármacos , Provirus/genética , Análisis de Expresión Génica de una Sola Célula , Proteína p24 del Núcleo del VIH/genética , Proteína p24 del Núcleo del VIH/metabolismo , ARN Largo no Codificante/metabolismo , Células Cultivadas , Humanos , Ionomicina/farmacologíaAsunto(s)
Anticuerpos Antivirales/sangre , Vacunas contra la COVID-19/administración & dosificación , COVID-19/prevención & control , Inmunoglobulina G/sangre , ARN Mensajero/inmunología , ARN Viral/inmunología , SARS-CoV-2/inmunología , Anciano , Anciano de 80 o más Años , Anticuerpos Antivirales/inmunología , Vacuna BNT162 , COVID-19/inmunología , Vacunas contra la COVID-19/genética , Vacunas contra la COVID-19/inmunología , Femenino , Humanos , Inmunoglobulina G/inmunología , Masculino , Persona de Mediana Edad , ARN Mensajero/administración & dosificación , ARN Mensajero/sangre , ARN Viral/administración & dosificación , ARN Viral/genética , SARS-CoV-2/genética , Resultado del TratamientoRESUMEN
Self-amplifying RNA replicons are promising platforms for vaccine generation. Their defects in one or more essential functions for viral replication, particle assembly, or dissemination make them highly safe as vaccines. We previously showed that the deletion of the envelope (E) gene from the Middle East respiratory syndrome coronavirus (MERS-CoV) produces a replication-competent propagation-defective RNA replicon (MERS-CoV-ΔE). Evaluation of this replicon in mice expressing human dipeptidyl peptidase 4, the virus receptor, showed that the single deletion of the E gene generated an attenuated mutant. The combined deletion of the E gene with accessory open reading frames (ORFs) 3, 4a, 4b, and 5 resulted in a highly attenuated propagation-defective RNA replicon (MERS-CoV-Δ[3,4a,4b,5,E]). This RNA replicon induced sterilizing immunity in mice after challenge with a lethal dose of a virulent MERS-CoV, as no histopathological damage or infectious virus was detected in the lungs of challenged mice. The four mutants lacking the E gene were genetically stable, did not recombine with the E gene provided in trans during their passage in cell culture, and showed a propagation-defective phenotype in vivo. In addition, immunization with MERS-CoV-Δ[3,4a,4b,5,E] induced significant levels of neutralizing antibodies, indicating that MERS-CoV RNA replicons are highly safe and promising vaccine candidates.
Asunto(s)
Infecciones por Coronavirus/prevención & control , Coronavirus del Síndrome Respiratorio de Oriente Medio/genética , Coronavirus del Síndrome Respiratorio de Oriente Medio/inmunología , ARN Viral/administración & dosificación , Replicón , Vacunas Virales/administración & dosificación , Animales , Anticuerpos Neutralizantes/biosíntesis , Anticuerpos Antivirales/biosíntesis , Infecciones por Coronavirus/genética , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/virología , Virus Defectuosos/genética , Virus Defectuosos/inmunología , Femenino , Eliminación de Gen , Genes env , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Coronavirus del Síndrome Respiratorio de Oriente Medio/patogenicidad , ARN Viral/genética , ARN Viral/inmunología , Vacunas de ADN , Vacunas de Partículas Similares a Virus/administración & dosificación , Vacunas de Partículas Similares a Virus/genética , Vacunas de Partículas Similares a Virus/inmunología , Vacunas Virales/genética , Vacunas Virales/inmunología , Virulencia/genética , Virulencia/inmunologíaRESUMEN
The pathogenesis of Perioperative neurocognitive disorders (PND) is a synergistic effect of many factors. Up to now, the exact mechanism remains unclear. The dopamine pathway in the brain is one of the paths involved in the means of cognitive function. Therefore, the purpose of this study was to investigate the relationship between changes in dopamine transporters in the ventral tegmental area (VTA) of the midbrain and postoperative cognitive dysfunction in elderly rats. In this study, a mental dysfunction model in elderly rats was established after splenectomy under general anesthesia. Eighty male SD rats, aged 18-20 months, with a body mass of 300-500 g. Randomly divided into eight groups: Normal group (Normal, N) and Sham group (sham, S), Model 3 day group(PND, P3), Model 7 day group(PND, P7), Virus 3 days AAV·DAT·RNAi (AAV3), Virus 7 days AAV·DAT·RNAi (AAV7), Virus control for three days AAV·NC(NC3), Virus control for seven days AAV·NC(NC7). The results show that knockdown of dopamine transporter in the VTA region can significantly improve the cognitive dysfunction of elderly rats after surgery. These results suggest that dopamine transporter in the VTA region is involved in cognitive dysfunction in elderly rats. The effect of DAT changes in the VTA region on postoperative cognitive function in elderly rats may be related to the regulation of α-syn and Aß1-42 protein aggregation in the hippocampus.
Asunto(s)
Envejecimiento/metabolismo , Cognición/fisiología , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Mesencéfalo/metabolismo , Área Tegmental Ventral/metabolismo , Péptidos beta-Amiloides/análisis , Péptidos beta-Amiloides/metabolismo , Animales , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/administración & dosificación , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/análisis , Mesencéfalo/química , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/metabolismo , ARN Viral/administración & dosificación , ARN Viral/análisis , ARN Viral/metabolismo , Ratas , Ratas Sprague-Dawley , Área Tegmental Ventral/química , alfa-Sinucleína/análisis , alfa-Sinucleína/metabolismoRESUMEN
Several vaccines have demonstrated efficacy against SARS-CoV-2 mediated disease, yet there is limited data on the immune response induced by heterologous vaccination regimens using alternate vaccine modalities. Here, we present a detailed description of the immune response, in mice, following vaccination with a self-amplifying RNA (saRNA) vaccine and an adenoviral vectored vaccine (ChAdOx1 nCoV-19/AZD1222) against SARS-CoV-2. We demonstrate that antibody responses are higher in two-dose heterologous vaccination regimens than single-dose regimens. Neutralising titres after heterologous prime-boost were at least comparable or higher than the titres measured after homologous prime boost vaccination with viral vectors. Importantly, the cellular immune response after a heterologous regimen is dominated by cytotoxic T cells and Th1+ CD4 T cells, which is superior to the response induced in homologous vaccination regimens in mice. These results underpin the need for clinical trials to investigate the immunogenicity of heterologous regimens with alternate vaccine technologies.
Asunto(s)
Vacunas contra la COVID-19/administración & dosificación , COVID-19/prevención & control , ARN Viral/administración & dosificación , SARS-CoV-2/inmunología , Vacunación/métodos , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , COVID-19/inmunología , Vacunas contra la COVID-19/genética , Vacunas contra la COVID-19/inmunología , ChAdOx1 nCoV-19 , Inmunización Secundaria , Inmunogenicidad Vacunal , Ratones , ARN Viral/genética , ARN Viral/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Linfocitos T Citotóxicos/inmunología , Células TH1/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
Although antibody levels progressively decrease following SARS-CoV-2 infection, the immune memory persists for months. Thus, individuals who naturally contracted SARS-CoV-2 are expected to develop a more rapid and sustained response to COVID-19 vaccines than naïve individuals. In this study, we analyzed the dynamics of the antibody response to the BNT162b2 mRNA COVID-19 vaccine in six healthcare workers who contracted SARS-CoV-2 in March 2020, in comparison to nine control subjects without a previous infection. The vaccine was well tolerated by both groups, with no significant difference in the frequency of vaccine-associated side effects, with the exception of local pain, which was more common in previously infected subjects. Overall, the titers of neutralizing antibodies were markedly higher in response to the vaccine than after natural infection. In all subjects with pre-existing immunity, a rapid increase in anti-spike receptor-binding domain (RBD) IgG antibodies and neutralizing antibody titers was observed one week after the first dose, which seemed to act as a booster. Notably, in previously infected individuals, neutralizing antibody titers 7 days after the first vaccine dose were not significantly different from those observed in naïve subjects 7 days after the second vaccine dose. These results suggest that, in previously infected people, a single dose of the vaccine might be sufficient to induce an effective response.
Asunto(s)
Anticuerpos Antivirales/inmunología , Vacunas contra la COVID-19/inmunología , COVID-19/inmunología , ARN Mensajero/inmunología , ARN Viral/inmunología , SARS-CoV-2/inmunología , Adulto , Formación de Anticuerpos , Vacuna BNT162 , COVID-19/prevención & control , COVID-19/virología , Vacunas contra la COVID-19/administración & dosificación , Vacunas contra la COVID-19/genética , Femenino , Humanos , Inmunoglobulina G/inmunología , Masculino , Persona de Mediana Edad , ARN Mensajero/administración & dosificación , ARN Mensajero/genética , ARN Viral/administración & dosificación , ARN Viral/genética , SARS-CoV-2/genéticaRESUMEN
Accumulative evidence of using double stranded (ds) RNA encapsulated into virus like particle (VLP) nanocarrier has open feasibility to fight against shrimp viral infection in aquaculture field. In this study, we co-encapsulated VP37 and VP28 dsRNA into hypodermal and hematopoietic necrosis virus (IHHNV) like particle and investigated its protection against white spot syndrome virus (WSSV). Five micrograms of each dsRNA were used as starting materials to load into VLP, while the loading efficiency was slightly different, i.e, VP37 dsRNA had somewhat a better load into VLP's cavity. It was apparent that co-encapsulation of dual dsRNA showed a superior WSSV silencing ability than the single dsRNA counterpart as evidence by the lower WSSV gene expression and its copy number in the gill tissues. Besides, we also demonstrated that co-encapsulated dual dsRNA into IHHNV-VLP stimulated the increased number of hemocytes and the corresponding PO activity as well as up-regulated proPO gene expression in hemocytes to resist viral invasion after an acute stage of WSSV infection. This synergistic action of dual dsRNA encapsulated into IHHNV-VLPs could thus act to delay time of shrimp death and reduced shrimp cumulative mortality greater than the single, naked dsRNA treatment and positive control groups. The obtaining results would encourage the feasibility to use it as a new weapon to fight WSSV infection in shrimp aquaculture.
Asunto(s)
Densovirinae/fisiología , Penaeidae/inmunología , ARN Bicatenario/administración & dosificación , ARN Viral/administración & dosificación , Vacunas de Partículas Similares a Virus/administración & dosificación , Proteínas del Envoltorio Viral/química , Virus del Síndrome de la Mancha Blanca 1/inmunología , Animales , Penaeidae/virología , Interferencia de ARNAsunto(s)
COVID-19/prevención & control , Inmunización Pasiva/métodos , Vacunación Masiva/métodos , ARN Mensajero/inmunología , ARN Viral/inmunología , SARS-CoV-2/genética , Aire/análisis , Convalecencia , Humanos , ARN Mensajero/administración & dosificación , ARN Mensajero/análisis , ARN Viral/administración & dosificación , ARN Viral/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , SARS-CoV-2/inmunología , SARS-CoV-2/aislamiento & purificaciónAsunto(s)
Betacoronavirus/fisiología , Infecciones por Coronavirus/virología , Modelos Animales de Enfermedad , Especificidad del Huésped , Pulmón/virología , Neumonía Viral/virología , Cornetes Nasales/virología , Adaptación Fisiológica , Adenosina Monofosfato/administración & dosificación , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/farmacología , Adenosina Monofosfato/uso terapéutico , Administración Intranasal , Alanina/administración & dosificación , Alanina/análogos & derivados , Alanina/farmacología , Alanina/uso terapéutico , Animales , Betacoronavirus/genética , COVID-19 , Chlorocebus aethiops , Infecciones por Coronavirus/tratamiento farmacológico , Femenino , Especificidad del Huésped/genética , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Mutación Missense , Mucosa Nasal/virología , Pandemias , Neumonía Viral/tratamiento farmacológico , ARN Viral/administración & dosificación , ARN Viral/genética , SARS-CoV-2 , Células Vero , Carga Viral , Replicación Viral , Tratamiento Farmacológico de COVID-19RESUMEN
Chikungunya virus (CHIKV) is the causative pathogen of chikungunya fever, a mosquito-borne viral disease causing highly debilitating arthralgia that can persist for months and progress to chronic arthritis. Our previous studies have identified the CHIKV live-attenuated vaccine candidate CHIKV-NoLS. Like most live-attenuated vaccines, attenuated replication of CHIKV-NoLS has the potential to limit scalable production. To overcome production limits, as well as other drawbacks of live-attenuated vaccines, we developed an in vivo liposome RNA delivery system to deliver the self-replicating RNA genome of CHIKV-NoLS directly into mice, allowing the recipients' body to produce the live-attenuated vaccine particles. CAF01 liposomes were able to deliver replication-competent CHIKV-NoLS RNA in vitro. Immunodeficient AG129 mice inoculated with liposome-delivered CHIKV-NoLS RNA developed viremia and disease signs representative of this lethal model of CHIKV infection, demonstrating de novo vaccine particle production in vivo. In immunocompetent C57BL/6 mice, liposome-delivered CHIKV-NoLS RNA inoculation was associated with reduced IgM and IgG levels with low antibody CHIKV-neutralizing capacity, compared to vaccination with the original live-attenuated vaccine CHIKV-NoLS. One dose of liposome-delivered CHIKV-NoLS RNA did not provide systemic protection from CHIKV wild-type (WT) challenge but was found to promote an early onset of severe CHIKV-induced footpad swelling. Liposome-delivered CHIKV-NoLS RNA inoculation did, however, provide local protection from CHIKV-WT challenge in the ipsilateral foot after one dose. Results suggest that in the presence of low CHIKV-specific neutralizing antibody levels, local inflammatory responses, likely brought on by liposome adjuvants, have a role in the protection of CHIKV-induced footpad swelling in the ipsilateral foot of mice inoculated with liposome-delivered CHIKV-NoLS RNA. Low IgG and CHIKV-specific neutralizing antibody levels may be responsible for early onset of severe swelling in the feet of CHIKV-WT-challenged mice. These results support previous studies that suggest CHIKV is vulnerable to antibody-mediated enhancement of disease. Further studies using booster regimes aim to demonstrate the potential for liposomes to deliver the self-replicating RNA genome of live-attenuated vaccines and offer a novel immunization strategy.
Asunto(s)
Fiebre Chikungunya/prevención & control , Virus Chikungunya/inmunología , ARN Viral/administración & dosificación , Vacunas Virales/administración & dosificación , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Virus Chikungunya/genética , Femenino , Genoma Viral , Liposomas , Masculino , Ratones , Ratones Endogámicos C57BL , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/inmunología , Vacunas Virales/inmunologíaRESUMEN
Self-amplifying replicon RNA (RepRNA) promotes expansion of mRNA templates encoding genes of interest through their replicative nature, thus providing increased antigen payloads. RepRNA derived from the non-cytopathogenic classical swine fever virus (CSFV) targets monocytes and dendritic cells (DCs), potentially promoting prolonged antigen expression in the DCs, contrasting with cytopathogenic RepRNA. We engineered pestivirus RepRNA constructs encoding influenza virus H5N1 (A/chicken/Yamaguchi/7/2004) nucleoprotein (Rep-NP) or hemagglutinin (Rep-HA). The inherent RNase-sensitivity of RepRNA had to be circumvented to ensure efficient delivery to DCs for intracellular release and RepRNA translation; we have reported how only particular synthetic delivery vehicle formulations are appropriate. The question remained concerning RepRNA packaged in virus replicon particles (VRPs); we have now compared an efficient polyethylenimine (PEI)-based formulation (polyplex) with VRP-delivery as well as naked RepRNA co-administered with the potent bis-(3',5')-cyclic dimeric adenosine monophosphate (c-di-AMP) adjuvant. All formulations contained a Rep-HA/Rep-NP mix, to assess the breadth of both humoral and cell-mediated defences against the influenza virus antigens. Assessment employed pigs for their close immunological relationship to humans, and as natural hosts for influenza virus. Animals receiving the VRPs, as well as PEI-delivered RepRNA, displayed strong humoral and cellular responses against both HA and NP, but with VRPs proving to be more efficacious. In contrast, naked RepRNA plus c-di-AMP could induce only low-level immune responses, in one out of five pigs. In conclusion, RepRNA encoding different influenza virus antigens are efficacious for inducing both humoral and cellular immune defences in pigs. Comparisons showed that packaging within VRP remains the most efficacious for delivery leading to induction of immune defences; however, this technology necessitates employment of expensive complementing cell cultures, and VRPs do not target human cells. Therefore, choosing the appropriate synthetic delivery vehicle still offers potential for rapid vaccine design, particularly in the context of the current coronavirus pandemic.
Asunto(s)
Inmunidad Celular , Inmunidad Humoral , Vacunas contra la Influenza/inmunología , ARN Viral/inmunología , Replicón/inmunología , Animales , COVID-19 , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Subtipo H5N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/administración & dosificación , Pestivirus , ARN Viral/administración & dosificación , SARS-CoV-2 , Porcinos , Proteínas del Núcleo Viral/inmunologíaRESUMEN
We have developed an oncolytic Newcastle disease virus (NDV) that has potent in vitro and in vivo anti-tumor activities and attenuated pathogenicity in chickens. In this ex vivo study using the same recombinant NDV backbone with GFP transgene (NDV-GFP, designated as rNDV), we found that rNDV induces maturation of monocyte-derived immature dendritic cells (iDCs) by both direct and indirect mechanisms, which promote development of antigen-specific T cell responses. Addition of rNDV directly to iDCs culture induced DC maturation, as demonstrated by the increased expression of costimulatory and antigen-presenting molecules as well as the production of type I interferons (IFNs). rNDV infection of the HER-2 positive human breast cancer cell line (SKBR3) resulted in apoptotic cell death, release of proinflammatory cytokines, and danger-associated molecular pattern molecules (DAMPs) including high-mobility group protein B1 (HMGB1) and heat shock protein 70 (HSP70). Addition of rNDV-infected SKBR3 cells to iDC culture resulted in greatly enhanced upregulation of the maturation markers and release of type I IFNs by DCs than rNDV-infected DCs only. When co-cultured with autologous T cells, DCs pre-treated with rNDV-infected SKBR3 cells cross-primed T cells in an antigen-specific manner. Altogether, our data strongly support the potential of oncolytic NDV as efficient therapeutic agent for cancer treatment.
Asunto(s)
Reactividad Cruzada , Células Dendríticas/inmunología , Inmunoterapia/métodos , Neoplasias/terapia , Viroterapia Oncolítica/métodos , Animales , Chlorocebus aethiops , Técnicas de Cocultivo , Células Dendríticas/metabolismo , Femenino , Células HeLa , Humanos , Interferón Tipo I/inmunología , Interferón Tipo I/metabolismo , Neoplasias/inmunología , Virus de la Enfermedad de Newcastle/genética , Virus de la Enfermedad de Newcastle/inmunología , Virus Oncolíticos/genética , Virus Oncolíticos/inmunología , ARN/administración & dosificación , ARN/genética , ARN Viral/administración & dosificación , ARN Viral/genética , Linfocitos T/inmunología , Células VeroRESUMEN
BACKGROUND: We evaluated safety and immunogenicity of the first mRNA vaccines against potentially pandemic avian H10N8 and H7N9 influenza viruses. METHODS: Two randomized, placebo-controlled, double-blind, phase 1 clinical trials enrolled participants between December 2015 and August 2017 at single centers in Germany (H10N8) and USA (H7N9). Healthy adults (ages 18-64â¯years for H10N8 study; 18-49â¯years for H7N9 study) participated. Participants received vaccine or placebo in a 2-dose vaccination series 3â¯weeks apart. H10N8 intramuscular (IM) dose levels of 25, 50, 75, 100, and 400⯵g and intradermal dose levels of 25 and 50⯵g were evaluated. H7N9 IM 10-, 25-, and 50-µg dose levels were evaluated; 2-dose series 6â¯months apart was also evaluated. Primary endpoints were safety (adverse events) and tolerability. Secondary immunogenicity outcomes included humoral (hemagglutination inhibition [HAI], microneutralization [MN] assays) and cell-mediated responses (ELISPOT assay). RESULTS: H10N8 and H7N9 mRNA IM vaccines demonstrated favorable safety and reactogenicity profiles. No vaccine-related serious adverse event was reported. For H10N8 (Nâ¯=â¯201), 100-µg IM dose induced HAI titersâ¯≥â¯1:40 in 100% and MN titersâ¯≥â¯1:20 in 87.0% of participants. The 25-µg intradermal dose induced HAI titersâ¯>â¯1:40 in 64.7% of participants compared to 34.5% of participants receiving the IM dose. For H7N9 (Nâ¯=â¯156), IM doses of 10, 25, and 50⯵g achieved HAI titersâ¯≥â¯1:40 in 36.0%, 96.3%, and 89.7% of participants, respectively. MN titersâ¯≥â¯1:20 were achieved by 100% in the 10- and 25-µg groups and 96.6% in the 50-µg group. Seroconversion rates were 78.3% (HAI) and 87.0% (MN) for H10N8 (100⯵g IM) and 96.3% (HAI) and 100% (MN) in H7N9 (50⯵g). Significant cell-mediated responses were not detected in either study. CONCLUSIONS: The first mRNA vaccines against H10N8 and H7N9 influenza viruses were well tolerated and elicited robust humoral immune responses. ClinicalTrials.gov NCT03076385 and NCT03345043.
Asunto(s)
Inmunogenicidad Vacunal , Vacunas contra la Influenza/genética , Vacunas contra la Influenza/inmunología , Gripe Humana/prevención & control , ARN Viral/inmunología , Adolescente , Adulto , Anticuerpos Antivirales/sangre , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Femenino , Voluntarios Sanos , Humanos , Subtipo H10N8 del Virus de la Influenza A , Subtipo H7N9 del Virus de la Influenza A , Vacunas contra la Influenza/efectos adversos , Masculino , Persona de Mediana Edad , ARN Viral/administración & dosificación , Adulto JovenRESUMEN
The HCV cell culture system, consisting of the JFH-1 strain and HuH-7 cells, has been broadly used to assess the complete HCV life cycle in cultured cells. However, being able to use multiple HCV strains in such a system is vital for future studies of this virus. We recently established a novel HCV cell culture system using another HCV genotype 2a strain, J6CF, which replicates in chimpanzees but not in cultured cells. We identified effective cell culture-adaptive mutations and established a replication-competent J6CF strain with minimum modifications in cultured cells. The strategy of how we established the replication-competent HCV strain and how we identified the effective cell culture-adaptive mutations is described here and could prove useful for establishing other replication-competent HCV strains.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Hepacivirus/genética , Hepatitis C/virología , Mutación , Transfección/métodos , Cultivo de Virus/métodos , Línea Celular , Genotipo , Hepacivirus/fisiología , Hepatitis C/patología , Humanos , ARN Viral/administración & dosificación , ARN Viral/genética , Replicación ViralRESUMEN
Adjuvants potentiate and direct the type of immunity elicited during vaccination. However, there is a shortage of adjuvants that elicit robust type-1 immunity required for the control of intracellular pathogens, including viruses. RNA derived from Sendai virus defective viral genomes (DVGs) stimulates RIG-I-like receptor signaling leading to type-1 immunity during infection. Here, we investigated whether a 268nt DVG-derived oligonucleotide (DDO) functions as a strong type-1 immunity-inducing adjuvant during vaccination against influenza virus. We show that DDO induces robust IgG2c antibody production when used in an inactivated influenza A virus (IAV) vaccine. Additionally, DDO induces Th1 and CD8+ T-cell responses able to protect against heterosubtypic IAV challenge. Interestingly, DDO synergized with AddaVax and skewed the immune response towards type-1 immunity. The adjuvancy of DDO alone and in synergy with AddaVax was heavily dependent on type I interferon signaling. Our data support a critical role for type I interferon in the induction of type-1 immune responses during vaccination and demonstrate that DDO is a type-1 immunity orienting vaccine adjuvant that can be used alone or in synergy with other adjuvants.
Asunto(s)
Anticuerpos Antivirales/sangre , Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Interferón Tipo I/metabolismo , ARN Viral/administración & dosificación , Virus Sendai/química , Linfocitos T/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/aislamiento & purificación , Animales , Modelos Animales de Enfermedad , Femenino , Vacunas contra la Influenza/administración & dosificación , Masculino , Ratones Endogámicos C57BL , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/prevención & control , ARN Viral/aislamiento & purificación , Vacunas de Productos Inactivados/administración & dosificación , Vacunas de Productos Inactivados/inmunologíaRESUMEN
Most current Ebola virus (EBOV) vaccine candidates are based on viral vectors, some of which cause side effects or require complex manufacturing. Modified mRNA vaccines are easily produced, safe, and are highly immunogenic. We developed 2 mRNA vaccines based on the EBOV envelope glycoprotein, which differed by the nature of signal peptide for improved glycoprotein post-translational translocation. The mRNAs were formulated with lipid nanoparticles to facilitate delivery. Vaccination of guinea pigs induced EBOV-specific IgG and neutralizing antibody responses and 100% survival after EBOV infection. The efficacy of our mRNA vaccine combined with preclinical safety data supports testing in clinical studies.
Asunto(s)
Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Vacunas contra el Virus del Ébola/administración & dosificación , Vacunas contra el Virus del Ébola/inmunología , Fiebre Hemorrágica Ebola/prevención & control , ARN Mensajero/administración & dosificación , ARN Viral/administración & dosificación , Adyuvantes Inmunológicos/administración & dosificación , Animales , Modelos Animales de Enfermedad , Portadores de Fármacos/administración & dosificación , Femenino , Cobayas , Inmunoglobulina G/sangre , Liposomas/administración & dosificación , Análisis de SupervivenciaRESUMEN
The Zika virus (ZIKV) outbreak in the Americas and South Pacific poses a significant burden on human health because of ZIKV's neurotropic effects in the course of fetal development. Vaccine candidates against ZIKV are coming online, but immunological tools to study anti-ZIKV responses in preclinical models, particularly T cell responses, remain sparse. We deployed RNA nanoparticle technology to create a vaccine candidate that elicited ZIKV E protein-specific IgG responses in C57BL/6 mice as assayed by ELISA. Using this tool, we identified a unique H-2Db-restricted epitope to which there was a CD8+ T cell response in mice immunized with our modified dendrimer-based RNA nanoparticle vaccine. These results demonstrate that this approach can be used to evaluate new candidate antigens and identify immune correlates without the use of live virus.
Asunto(s)
Anticuerpos Antivirales/sangre , Linfocitos T CD8-positivos/inmunología , ARN Viral/inmunología , Vacunas Virales/inmunología , Infección por el Virus Zika/prevención & control , Virus Zika/inmunología , Animales , Dendrímeros/administración & dosificación , Dendrímeros/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática , Epítopos de Linfocito T/inmunología , Inmunoglobulina G/sangre , Ratones Endogámicos C57BL , Nanopartículas/administración & dosificación , ARN Viral/administración & dosificación , ARN Viral/aislamiento & purificación , Vacunas Virales/administración & dosificación , Vacunas Virales/aislamiento & purificaciónRESUMEN
Zika virus (ZIKV) has recently emerged as a pandemic associated with severe neuropathology in newborns and adults. There are no ZIKV-specific treatments or preventatives. Therefore, the development of a safe and effective vaccine is a high priority. Messenger RNA (mRNA) has emerged as a versatile and highly effective platform to deliver vaccine antigens and therapeutic proteins. Here we demonstrate that a single low-dose intradermal immunization with lipid-nanoparticle-encapsulated nucleoside-modified mRNA (mRNA-LNP) encoding the pre-membrane and envelope glycoproteins of a strain from the ZIKV outbreak in 2013 elicited potent and durable neutralizing antibody responses in mice and non-human primates. Immunization with 30 µg of nucleoside-modified ZIKV mRNA-LNP protected mice against ZIKV challenges at 2 weeks or 5 months after vaccination, and a single dose of 50 µg was sufficient to protect non-human primates against a challenge at 5 weeks after vaccination. These data demonstrate that nucleoside-modified mRNA-LNP elicits rapid and durable protective immunity and therefore represents a new and promising vaccine candidate for the global fight against ZIKV.
Asunto(s)
ARN Mensajero/administración & dosificación , ARN Mensajero/química , Vacunas Virales/inmunología , Infección por el Virus Zika/prevención & control , Virus Zika/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Antígenos Virales/genética , Antígenos Virales/inmunología , Femenino , Glicoproteínas/genética , Glicoproteínas/inmunología , Inyecciones Intradérmicas , Macaca mulatta/inmunología , Macaca mulatta/virología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Nanopartículas/administración & dosificación , Nanopartículas/química , Estabilidad del ARN , ARN Mensajero/genética , ARN Viral/administración & dosificación , ARN Viral/química , ARN Viral/genética , Factores de Tiempo , Vacunación , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Vacunas Virales/administración & dosificación , Virus Zika/química , Virus Zika/genética , Infección por el Virus Zika/inmunologíaRESUMEN
Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous substances into the brain. These exogenous substances may induce injurious impact or constitute therapeutic strategy. The most common delivery methods of exogenous substances into the brain are based on systemic deliveries, such as subcutaneous or intravenous routes. Although commonly used, these approaches have several limitations, including low delivery efficacy into the brain. In contrast, surgical methods that locally deliver substances into the CNS are more specific and prevent the uptake of the exogenous substances by other organs. Several surgical methods for CNS delivery are available; however, they tend to be very traumatic. Here, we describe a mouse infusion microsurgery technique, which effectively delivers substances into the brain via the internal carotid artery, with minimal trauma and no interference with normal CNS functionality.
Asunto(s)
Arteria Carótida Interna/cirugía , Preparaciones Farmacéuticas/administración & dosificación , Analgésicos/administración & dosificación , Animales , Antiinflamatorios/administración & dosificación , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Enfermedades del Sistema Nervioso Central/tratamiento farmacológico , Enfermedades del Sistema Nervioso Central/patología , Enfermedades del Sistema Nervioso Central/veterinaria , VIH/genética , Inyecciones Intravenosas , Ratones , Microscopía Fluorescente , Microcirugia , ARN Viral/administración & dosificaciónRESUMEN
Hepatitis E virus (HEV) is one of the prime causes of acute viral hepatitis, and chronic hepatitis E is increasingly recognized as an important problem in the transplant setting. Nevertheless, the fundamental understanding of the biology of HEV replication is limited and there are few therapeutic options. The development of such therapies is partially hindered by the lack of a robust and convenient animal model. We propose the infection of athymic nude rats with the rat HEV strain LA-B350 as such a model. A cDNA clone, pLA-B350, was constructed and the infectivity of its capped RNA transcripts was confirmed in vitro and in vivo Furthermore, a subgenomic replicon, pLA-B350/luc, was constructed and validated for in vitro antiviral studies. Interestingly, rat HEV proved to be less sensitive to the antiviral activity of α-interferon, ribavirin and mycophenolic acid than genotype 3 HEV (a strain that infects humans). As a proof-of-concept, part of the C-terminal polymerase sequence of pLA-B350/luc was swapped with its genotype 3 HEV counterpart: the resulting chimeric replicon replicated with comparable efficiency as the wild-type construct, confirming that LA-B350 strain is amenable to humanization (replacement of certain sequences or motifs by their counterparts from human HEV strains). Finally, ribavirin effectively inhibited LA-B350 replication in athymic nude rats, confirming the suitability of the rat model for antiviral studies.