Zika virus (ZIKV) is a re-emerging mosquito-borne flavivirus that affects humans and can cause severe neurological complications, including Guillain-Barré syndrome and microcephaly. Since 2007 there ...have been three large outbreaks; the last and larger spread in the Americas in 2015. Actually, ZIKV is circulating in the Americas, Southeast Asia, and the Pacific Islands, and represents a potential pandemic threat. Given the rapid ZIKV dissemination and the severe neurological and teratogenic sequelae associated with ZIKV infection, the development of a safe and efficacious vaccine is critical. In this study, we have developed and characterized the immunogenicity and efficacy of a novel ZIKV vaccine based on the highly attenuated poxvirus vector modified vaccinia virus Ankara (MVA) expressing the ZIKV prM and E structural genes (termed MVA-ZIKV). MVA-ZIKV expressed efficiently the ZIKV structural proteins, assembled in virus-like particles (VLPs) and was genetically stable upon nine passages in cell culture. Immunization of mice with MVA-ZIKV elicited antibodies that were able to neutralize ZIKV and induced potent and polyfunctional ZIKV-specific CD8
T cell responses that were mainly of an effector memory phenotype. Moreover, a single dose of MVA-ZIKV reduced significantly the viremia in susceptible immunocompromised mice challenged with live ZIKV. These findings support the use of MVA-ZIKV as a potential vaccine against ZIKV.
Induction of the endogenous innate immune system by interferon (IFN) triggers the expression of many proteins that serve like alarm bells in the body, activating an immune response. After a viral ...infection, one of the genes activated by IFN induction is the IFN-stimulated gene 15 (
), which encodes a ubiquitin-like protein that undergoes a reversible posttranslational modification (ISGylation). ISG15 protein can also act unconjugated, intracellularly and secreted, acting as a cytokine. Although ISG15 has an essential role in host defense responses to microbial infection, its role as an immunomodulator in the vaccine field remains to be defined. In this investigation, we showed that ISG15 exerts an immunomodulatory role in human immunodeficiency virus (HIV) vaccines. In mice, after priming with a DNA-ISG15 vector mixed with a DNA expressing HIV-1 gp120 (DNA-gp120), followed by a booster with a modified vaccinia virus Ankara (MVA) vector expressing HIV-1 antigens, both wild-type ISG15-conjugated (ISG15-wt) and mutant unconjugated (ISG15-mut) proteins act as immune adjuvants by increasing the magnitude and quality of HIV-1-specific CD8 T cells, with ISG15-wt providing better immunostimulatory activity than ISG15-mut. The HIV-1 Env-specific CD8 T cell responses showed a predominant T effector memory (TEM) phenotype in all groups. Moreover, the amount of DNA-gp120 used to immunize mice could be reduced 5-fold after mixing with DNA-ISG15 without affecting the potency and the quality of the HIV-1 Env-specific immune responses. Our study clearly highlights the potential use of the IFN-induced ISG15 protein as immune adjuvant to enhance immune responses to HIV antigens, suggesting that this molecule might be exploitable for prophylactic and therapeutic vaccine approaches against pathogens.
Our study described the potential role of ISG15 as an immunomodulatory molecule in the optimization of HIV/AIDS vaccine candidates. Using a DNA prime-MVA boost immunization protocol, our results indicated an increase in the potency and the quality of the HIV-1 Env-specific CD8 T cell response. These results highlight the adjuvant potency of ISG15 to elicit improved viral antigen presentation to the immune system, resulting in an enhanced HIV-1 vaccine immune response. The DNA-ISG15 vector could find applicability in the vaccine field in combination with other nucleic acid-based vector vaccines.
Hepatitis C virus (HCV) represents a major global health problem for which a vaccine is not available. Modified vaccinia virus Ankara (MVA)-HCV is a unique HCV vaccine candidate based in the modified ...vaccinia virus Ankara (MVA) vector expressing the nearly full-length genome of HCV genotype 1a that elicits CD8⁺ T-cell responses in mice. With the aim to improve the immune response of MVA-HCV and because of the importance of interferon (IFN) in HCV infection, we deleted in MVA-HCV the vaccinia virus (VACV)
gene, encoding an inhibitor of IFN-β that prevents activation of the interferon regulatory factors 3 and 7 (IRF3 and IRF7). The resulting vaccine candidate (MVA-HCV ΔC6L) expresses all HCV antigens and deletion of
had no effect on viral growth in permissive chicken cells. In human monocyte-derived dendritic cells, infection with MVA-HCV ΔC6L triggered severe down-regulation of IFN-β, IFN-β-induced genes, and cytokines in a manner similar to MVA-HCV, as defined by real-time polymerase chain reaction (PCR) and microarray analysis. In infected mice, both vectors had a similar profile of recruited immune cells and induced comparable levels of adaptive and memory HCV-specific CD8⁺ T-cells, mainly against p7 + NS2 and NS3 HCV proteins, with a T cell effector memory (TEM) phenotype. Furthermore, antibodies against E2 were also induced. Overall, our findings showed that while these vectors had a profound inhibitory effect on gene expression of the host, they strongly elicited CD8⁺ T cell and humoral responses against HCV antigens and to the virus vector. These observations add support to the consideration of these vectors as potential vaccine candidates against HCV.
Development of a safe and efficacious vaccine against the HIV/AIDS pandemic remains a major scientific goal. We previously described an HIV/AIDS vaccine based on the modified vaccinia virus Ankara ...(MVA) expressing HIV-1 gp120 and Gag-Pol-Nef (GPN) of clade B (termed MVA-B), which showed moderate immunogenicity in phase I prophylactic and therapeutic clinical trials. Here, to improve the immunogenicity of MVA-B, we generated a novel recombinant virus, MVA-B ΔA40R, by deleting in the MVA-B genome the vaccinia virus (VACV)
gene, which encodes a protein with unknown immune function. The innate immune responses triggered by MVA-B ΔA40R in infected human macrophages, in comparison to parental MVA-B, revealed an increase in the mRNA expression levels of interferon (IFN)-β, IFN-induced genes, and chemokines. Compared to priming with DNA-B (a mixture of DNA-gp120 plus DNA-GPN) and boosting with MVA-B, mice immunized with a DNA-B/MVA-B ΔA40R regimen induced higher magnitude of adaptive and memory HIV-1-specific CD4+ and CD8+ T-cell immune responses that were highly polyfunctional, mainly directed against Env. and of an effector memory phenotype, together with enhanced levels of antibodies against HIV-1 gp120. Reintroduction of the A40R gene into the MVA-B ΔA40R genome (virus termed MVA-B ΔA40R-rev) promoted in infected cells high mRNA and protein A40 levels, with A40 protein localized in the cell membrane. MVA-B ΔA40R-rev significantly reduced mRNA levels of IFN-β and of several other innate immune-related genes in infected human macrophages. In immunized mice, MVA-B ΔA40R-rev reduced the magnitude of the HIV-1-specific CD4+ and CD8+ T cell responses compared to MVA-B ΔA40R. These results revealed an immunosuppressive role of the A40 protein, findings relevant for the optimization of poxvirus vectors as vaccines.
Hepatitis C is a liver disease caused by the hepatitis C virus (HCV) affecting 71 million people worldwide with no licensed vaccines that prevent infection. Here, we have generated four novel ...alphavirus-based DNA-launched self-amplifying RNA replicon (DREP) vaccines expressing either structural core-E1-E2 or nonstructural p7-NS2-NS3 HCV proteins of genotype 1a placed under the control of an alphavirus promoter, with or without an alphaviral translational enhancer (grouped as DREP-HCV or DREP-e-HCV, respectively). DREP vectors are known to induce cross-priming and further stimulation of immune responses through apoptosis, and here we demonstrate that they efficiently trigger apoptosis-related proteins in transfected cells. Immunization of mice with the DREP vaccines as the priming immunization followed by a heterologous boost with a recombinant modified vaccinia virus Ankara (MVA) vector expressing the nearly full-length genome of HCV (MVA-HCV) induced potent and long-lasting HCV-specific CD4
and CD8
T cell immune responses that were significantly stronger than those of a homologous MVA-HCV prime/boost immunization, with the DREP-e-HCV/MVA-HCV combination the most immunogenic regimen. HCV-specific CD4
and CD8
T cell responses were highly polyfunctional, had an effector memory phenotype, and were mainly directed against E1-E2 and NS2-NS3, respectively. Additionally, DREP/MVA-HCV immunization regimens induced higher antibody levels against HCV E2 protein than homologous MVA-HCV immunization. Collectively, these results provided an immunization protocol against HCV by inducing high levels of HCV-specific T cell responses as well as humoral responses. These findings reinforce the combined use of DREP-based vectors and MVA-HCV as promising prophylactic and therapeutic vaccines against HCV.
HCV represents a global health problem as more than 71 million people are chronically infected worldwide. Direct-acting antiviral agents can cure HCV infection in most patients, but due to the high cost of these agents and the emergence of resistant mutants, they do not represent a feasible and affordable strategy to eradicate the virus. Therefore, a vaccine is an urgent goal that requires efforts to understand the correlates of protection for HCV clearance. Here, we describe for the first time the generation of novel vaccines against HCV based on alphavirus DNA replicons expressing HCV antigens. We demonstrate that potent T cell immune responses, as well as humoral immune responses, against HCV can be achieved in mice by using a combined heterologous prime/boost immunization protocol consisting of the administration of alphavirus replicon DNA vectors as the priming immunization followed by a boost with a recombinant modified vaccinia virus Ankara vector expressing HCV antigens.
Hepatitis C virus (HCV) represents a major global health challenge and an efficient vaccine is urgently needed. Many HCV vaccination strategies employ recombinant versions of the viral E2 ...glycoprotein. However, recombinant E2 readily forms disulfide-bonded aggregates that might not be optimally suited for vaccines. Therefore, we have designed an E2 protein in which we strategically changed eight cysteines to alanines (E2.C8A). E2.C8A formed predominantly monomers and virtually no aggregates. Furthermore, E2.C8A also interacted more efficiently with broadly neutralizing antibodies than conventional E2. We used mice to evaluate different prime/boost immunization strategies involving a modified vaccinia virus Ankara (MVA) expressing the nearly full-length genome of HCV (MVA-HCV) in combination with either the E2 aggregates or the E2.C8A monomers. The combined MVA-HCV/E2 aggregates prime/boost strategy markedly enhanced HCV-specific effector memory CD4+ T cell responses and antibody levels compared to MVA-HCV/MVA-HCV. Moreover, the aggregated form of E2 induced higher levels of anti-E2 antibodies in vaccinated mice than E2.C8A monomers. These antibodies were cross-reactive and mainly of the IgG1 isotype. Our findings revealed how two E2 viral proteins that differ in their capacity to form aggregates are able to enhance to different extent the HCV-specific cellular and humoral immune responses, either alone or in combination with MVA-HCV. These combined protocols of MVA-HCV/E2 could serve as a basis for the development of a more effective HCV vaccine.
Highly attenuated poxviral vectors, such as modified vaccinia virus ankara (MVA), are promising vaccine candidates against several infectious diseases. One of the approaches developed to enhance the ...immunogenicity of poxvirus vectors is increasing the promoter strength and accelerating during infection production levels of heterologous antigens. Here, we have generated and characterized the biology and immunogenicity of an optimized MVA-based vaccine candidate against HIV/AIDS expressing HIV-1 clade B gp120 protein under the control of a novel synthetic late/early optimized (LEO) promoter (LEO160 promoter; with a spacer length of 160 nucleotides), termed MVA-LEO160-gp120. In infected cells, MVA-LEO160-gp120 significantly increased the expression levels of HIV-1 gp120 mRNA and protein, compared to the clinical vaccine MVA-B vector expressing HIV-1 gp120 under the control of the commonly used synthetic early/late promoter. When mice were immunized with a heterologous DNA-prime/MVA-boost protocol, the immunization group DNA-gp120/MVA-LEO160-gp120 induced an enhancement in the magnitude of gp120-specific CD4
and CD8
T-cell responses, compared to DNA-gp120/MVA-B; with most of the responses being mediated by the CD8
T-cell compartment, with a T effector memory phenotype. DNA-gp120/MVA-LEO160-gp120 also elicited a trend to a higher magnitude of gp120-specific CD4
T follicular helper cells, and modest enhanced levels of antibodies against HIV-1 gp120. These findings revealed that this new optimized vaccinia virus promoter could be considered a promising strategy in HIV/AIDS vaccine design, confirming the importance of early expression of heterologous antigen and its impact on the antigen-specific immunogenicity elicited by poxvirus-based vectors.
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive memory loss and cognitive decline that has been associated with an accumulation in the brain of intracellular ...neurofibrillary tangles (NFTs) formed by hyperphosphorylated tau protein, and extracellular senile plaques formed by β-amyloid peptides. Currently, there is no cure for AD and after the failure of anti β-amyloid therapies, active and passive tau immunotherapeutic approaches have been developed in order to prevent, reduce or ideally reverse the disease. Vaccination is one of the most effective approaches to prevent diseases and poxviruses, particularly modified vaccinia virus Ankara (MVA), are one of the most promising viral vectors used as vaccines against several human diseases. Thus, we present here the generation and characterization of the first MVA vectors expressing human tau genes; the full-length 4R2N tau protein or a 3RC tau fragment containing 3 tubulin-binding motifs and the C-terminal region (termed MVA-Tau4R2N and MVA-Tau3RC, respectively). Both MVA-Tau recombinant viruses efficiently expressed the human tau 4R2N or 3RC proteins in cultured cells, being detected in the cytoplasm of infected cells and co-localized with tubulin. These MVA-Tau vaccines impacted the innate immune responses with a differential recruitment of innate immune cells to the peritoneal cavity of infected mice. However, no tau-specific T cell or humoral immune responses were detected in vaccinated mice. Immunization of transgenic P301S mice, a mouse model for tauopathies, with a DNA-Tau prime/MVA-Tau boost approach showed no significant differences in the hyperphosphorylation of tau, motor capacity and survival rate, when compared to non-vaccinated mice. These findings showed that a well-established and potent protocol of T and B cell activation based on DNA/MVA prime/boost regimens using DNA and MVA vectors expressing tau full-length 4R2N or 3RC proteins is not sufficient to trigger tau-specific T and B cell immune responses and to induce a protective effect against tauopathy in this P301S murine model. In the pursuit of AD vaccines, our results highlight the need for novel optimized tau immunogens and additional modes of presentation of tau protein to the immune system.
Los sistemas de uso del suelo con leñosas perennes, como cacaotales, mitigan el cambio climático, al capturar CO2 atmosférico. El objetivo del estudio fue estimar la fijación de carbono en biomasa ...total en los sistemas de producción de cacao, dominantes del Tolima, Colombia. El estudio, se realizó en Rovira y Falan, empleando un diseño experimental, completamente al azar, con seis sistemas de producción de cacao: monocultivo, sistema agroforestal (SAF) con maderables, SAF con aguacate, SAF con cítricos, SAF con frutales, y SAF con maderables y frutales y tres repeticiones. Se establecieron dos parcelas de muestreo rectangulares, de 1.000m² por unidad de muestreo, donde se identificaron y midieron los árboles con dap ≥ 10cm (diámetro del tronco a la altura del pecho -dap- y altura total -ht). Se establecieron dos sub-parcelas de 256m², por parcela principal, para medir arbustos de cacao: ht y diámetro del tronco, a 30 cm de altura (D30). Se emplearon modelos alométricos, para estimar la biomasa. El SAF con maderables y frutales y SAF con frutales presentaron la mayor biomasa total (122,0 y 72,5t/ ha). En el SAF con maderables y frutales, se evidenció el mayor almacenamiento de carbono, con 61,0t C/ha, mientras que la mayor tasa de fijación de carbono fue de 17,7t/ha/ año, para el SAF con cítricos. Los resultados sugieren que la producción de cacao, que incluyen otras especies vegetales, diversifica la producción e incrementa la seguridad alimentaria y la generación de servicios ambientales, tal como la captura de carbono atmosférico.
Abstract
Background
Response to targeted therapy varies between patients for largely unknown reasons. Here, we developed and applied an integrative platform using mass spectrometry imaging (MSI), ...phosphoproteomics, and multiplexed tissue imaging for mapping drug distribution, target engagement, and adaptive response to gain insights into heterogeneous response to therapy.
Methods
Patient-derived xenograft (PDX) lines of glioblastoma were treated with adavosertib, a Wee1 inhibitor, and tissue drug distribution was measured with MALDI-MSI. Phosphoproteomics was measured in the same tumors to identify biomarkers of drug target engagement and cellular adaptive response. Multiplexed tissue imaging was performed on sister sections to evaluate spatial co-localization of drug and cellular response. The integrated platform was then applied on clinical specimens from glioblastoma patients enrolled in the phase 1 clinical trial.
Results
PDX tumors exposed to different doses of adavosertib revealed intra- and inter-tumoral heterogeneity of drug distribution and integration of the heterogeneous drug distribution with phosphoproteomics and multiplexed tissue imaging revealed new markers of molecular response to adavosertib. Analysis of paired clinical specimens from patients enrolled in the phase 1 clinical trial informed the translational potential of the identified biomarkers in studying patient’s response to adavosertib.
Conclusions
The multimodal platform identified a signature of drug efficacy and patient-specific adaptive responses applicable to preclinical and clinical drug development. The information generated by the approach may inform mechanisms of success and failure in future early phase clinical trials, providing information for optimizing clinical trial design and guiding future application into clinical practice.
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