RESUMO
Dengue is the most common vector-borne viral disease, causing nearly 400 million infections yearly. Currently there are no approved therapies. Antibody epitopes that elicit weak humoral responses may not be accessible by conventional B cell panning methods. To demonstrate an alternative strategy to generating a therapeutic antibody, we employed a non-immunodominant, but functionally relevant, epitope in domain III of the E protein, and engineered by structure-guided methods an antibody directed to it. The resulting antibody, Ab513, exhibits high-affinity binding to, and broadly neutralizes, multiple genotypes within all four serotypes. To assess therapeutic relevance of Ab513, activity against important human clinical features of dengue was investigated. Ab513 mitigates thrombocytopenia in a humanized mouse model, resolves vascular leakage, reduces viremia to nearly undetectable levels, and protects mice in a maternal transfer model of lethal antibody-mediated enhancement. The results demonstrate that Ab513 may reduce the public health burden from dengue.
Assuntos
Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/química , Vírus da Dengue/fisiologia , Dengue/terapia , Epitopos Imunodominantes/química , Sequência de Aminoácidos , Animais , Dengue/imunologia , Dengue/virologia , Vírus da Dengue/imunologia , Modelos Animais de Doenças , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Fagocitose , Engenharia de Proteínas , Receptores Fc/imunologia , Alinhamento de SequênciaRESUMO
BACKGROUND: Insufficient vaccine doses and the lack of therapeutic agents for yellow fever put global health at risk, should this virus emerge from sub-Saharan Africa and South America. METHODS: In phase 1a of this clinical trial, we assessed the safety, side-effect profile, and pharmacokinetics of TY014, a fully human IgG1 anti-yellow fever virus monoclonal antibody. In a double-blind, phase 1b clinical trial, we assessed the efficacy of TY014, as compared with placebo, in abrogating viremia related to the administration of live yellow fever vaccine (YF17D-204; Stamaril). The primary safety outcomes were adverse events reported 1 hour after the infusion and throughout the trial. The primary efficacy outcome was the dose of TY014 at which 100% of the participants tested negative for viremia within 48 hours after infusion. RESULTS: A total of 27 healthy participants were enrolled in phase 1a, and 10 participants in phase 1b. During phase 1a, TY014 dose escalation to a maximum of 20 mg per kilogram of body weight occurred in 22 participants. During phases 1a and 1b, adverse events within 1 hour after infusion occurred in 1 of 27 participants who received TY014 and in none of the 10 participants who received placebo. At least one adverse event occurred during the trial in 22 participants who received TY014 and in 8 who received placebo. The mean half-life of TY014 was approximately 12.8 days. At 48 hours after the infusion, none of the 5 participants who received the starting dose of TY014 of 2 mg per kilogram had detectable YF17D-204 viremia; these participants remained aviremic throughout the trial. Viremia was observed at 48 hours after the infusion in 2 of 5 participants who received placebo and at 72 hours in 2 more placebo recipients. Symptoms associated with yellow fever vaccine were less frequent in the TY014 group than in the placebo group. CONCLUSIONS: This phase 1 trial of TY014 did not identify worrisome safety signals and suggested potential clinical benefit, which requires further assessment in a phase 2 trial. (Funded by Tysana; ClinicalTrials.gov number, NCT03776786.).
Assuntos
Anticorpos Monoclonais Humanizados/administração & dosagem , Vacina contra Febre Amarela , Febre Amarela/tratamento farmacológico , Vírus da Febre Amarela/imunologia , Adulto , Anticorpos Monoclonais Humanizados/efeitos adversos , Anticorpos Monoclonais Humanizados/farmacocinética , Relação Dose-Resposta a Droga , Método Duplo-Cego , Meia-Vida , Humanos , Estimativa de Kaplan-Meier , Viremia/tratamento farmacológico , Febre Amarela/virologia , Vírus da Febre Amarela/efeitos dos fármacosRESUMO
The expansion of the geographical footprint of dengue viruses (DENVs) and their mosquito vectors have affected more than half of the global population, including older adults who appear to show elevated risk of severe dengue. Despite this epidemiological trend, how aging contributes to increased dengue pathogenesis is poorly understood. A limitation has been the lack of useful in vitro experimental approaches; cell lines commonly used for infection studies are immortal and hence do not age. Cell strains such as WI-38 and MRC-5 with diploid genomes do age with in vitro passaging, but these cell strains were isolated decades ago and are now mostly highly passaged. Here, we show that reprogramming of cell strains with finite life span into induced pluripotent stem cells (iPSCs), followed by conversion back into terminally differentiated cells, can be an approach to derive genetically identical cells at different stages of aging. The iPSC-derived differentiated cells were susceptible to wild-type DENV infection and produced greater levels of type I interferon expression with increased passaging, despite similar levels of infection. In contrast, infection with the attenuated DENV-2 PDK53 and YF17D-204 strains showed reduced and increased levels of infection with increasing passages, respectively; the latter could be clinically pertinent, as YF17D-204 vaccination in older adults is associated with increased risk of severe adverse outcome. The differences in infection susceptibility and host response collectively suggest the potential of iPSC-derived cell strains as a genetically controlled approach to understanding how aging impacts viral pathogenesis. IMPORTANCE Aging has been a risk factor for poor clinical outcome in several infectious diseases, including dengue. However, age-dependent responses to dengue and other flaviviral infection or vaccination have remained incompletely understood due partly to lack of suitable laboratory tools. We thus developed an in vitro approach to examine age-related changes in host response to flaviviral infection. Notably, this approach uses cell strains with diploid rather than aneuploidic genomes, which are unstable. Conversion of these cells into iPSCs ensures sustainability of this resource, and reprogramming back into terminally differentiated cells would, even with a limited number of passages, produce cells at different stages of aging for infection studies. Our findings suggest that this in vitro system has the potential to serve as a genetically controlled approach to define the age-related response to flavivirus infection.
Assuntos
Infecções por Flavivirus/metabolismo , Infecções por Flavivirus/virologia , Flavivirus/fisiologia , Interações Hospedeiro-Patógeno , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/virologia , Fatores Etários , Diferenciação Celular , Células Cultivadas , Senescência Celular/genética , Senescência Celular/imunologia , Dengue/virologia , Vírus da Dengue , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , MasculinoRESUMO
Dengue virus (DENV) is a global health threat, causing repeated epidemics throughout the tropical world. While low herd immunity levels to any one of the four antigenic types of DENV predispose populations to outbreaks, viral genetic determinants that confer greater fitness for epidemic spread is an important but poorly understood contributor of dengue outbreaks. Here we report that positive epistasis between the coding and noncoding regions of the viral genome combined to elicit an epidemiologic fitness phenotype associated with the 1994 DENV2 outbreak in Puerto Rico. We found that five amino acid substitutions in the NS5 protein reduced viral genomic RNA (gRNA) replication rate to achieve a more favorable and relatively more abundant subgenomic flavivirus RNA (sfRNA), a byproduct of host 5'-3' exoribonuclease activity. The resulting increase in sfRNA relative to gRNA levels not only inhibited type I interferon (IFN) expression in infected cells through a previously described mechanism, but also enabled sfRNA to compete with gRNA for packaging into infectious particles. We suggest that delivery of sfRNA to new susceptible cells to inhibit type I IFN induction before gRNA replication and without the need for further de novo sfRNA synthesis could form a "preemptive strike" strategy against DENV.
Assuntos
Regiões 3' não Traduzidas/genética , Vírus da Dengue/genética , Dengue/virologia , Proteínas não Estruturais Virais/genética , Células A549 , Dengue/epidemiologia , Epistasia Genética , Exorribonucleases , Técnicas de Inativação de Genes , Genoma Viral , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Interferon Tipo I/metabolismo , Proteínas Associadas aos Microtúbulos , Mutação , Porto Rico/epidemiologia , RNA Guia de Cinetoplastídeos/metabolismo , Replicação ViralRESUMO
A self-transcribing and replicating RNA (STARR)-based vaccine (LUNAR-COV19) has been developed to prevent SARS-CoV-2 infection. The vaccine encodes an alphavirus-based replicon and the SARS-CoV-2 full-length spike glycoprotein. Translation of the replicon produces a replicase complex that amplifies and prolongs SARS-CoV-2 spike glycoprotein expression. A single prime vaccination in mice led to robust antibody responses, with neutralizing antibody titers increasing up to day 60. Activation of cell-mediated immunity produced a strong viral antigen-specific CD8+ T lymphocyte response. Assaying for intracellular cytokine staining for interferon (IFN)γ and interleukin-4 (IL-4)-positive CD4+ T helper (Th) lymphocytes as well as anti-spike glycoprotein immunoglobulin G (IgG)2a/IgG1 ratios supported a strong Th1-dominant immune response. Finally, single LUNAR-COV19 vaccination at both 2 µg and 10 µg doses completely protected human ACE2 transgenic mice from both mortality and even measurable infection following wild-type SARS-CoV-2 challenge. Our findings collectively suggest the potential of LUNAR-COV19 as a single-dose vaccine.
Assuntos
Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , Vacinas contra COVID-19/administração & dosagem , COVID-19/prevenção & controle , SARS-CoV-2/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Sintéticas/administração & dosagem , Alphavirus/genética , Alphavirus/imunologia , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/imunologia , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , COVID-19/imunologia , COVID-19/patologia , COVID-19/virologia , Vacinas contra COVID-19/biossíntese , Vacinas contra COVID-19/genética , Vacinas contra COVID-19/imunologia , Feminino , Expressão Gênica , Humanos , Imunidade Celular/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Interferon gama/genética , Interferon gama/imunologia , Interleucina-4/genética , Interleucina-4/imunologia , Camundongos , Camundongos Transgênicos , Replicon/imunologia , SARS-CoV-2/imunologia , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th1/virologia , Transgenes , Resultado do Tratamento , Vacinação/métodos , Vacinas Sintéticas/biossíntese , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas de mRNARESUMO
Dengue virus (DENV) has been found to replicate in lymphoid organs such as the lymph nodes, spleen, and liver in post-mortem analysis. These organs are known to have low oxygen levels (~0.5-4.5% O2) due to the vascular anatomy. However, how physiologically low levels of oxygen affect DENV infection via hypoxia-induced changes in the immune response remains unknown. Here, we show that monocytes adapted to 3% O2 show greater susceptibility to antibody-dependent enhancement of DENV infection. Low oxygen level induces HIF1α-dependent upregulation of fragment crystallizable gamma receptor IIA (FcγRIIA) as well as HIF1α-independent alterations in membrane ether lipid concentrations. The increased FcγRIIA expression operates synergistically with altered membrane composition, possibly through increase membrane fluidity, to increase uptake of DENV immune complexes for enhanced infection. Our findings thus indicate that the increased viral burden associated with secondary DENV infection is antibody-dependent but hypoxia-induced and suggest a role for targeting hypoxia-induced factors for anti-dengue therapy.
Assuntos
Anticorpos Antivirais/metabolismo , Anticorpos Facilitadores , Vírus da Dengue/crescimento & desenvolvimento , Dengue/patologia , Hipóxia , Monócitos/virologia , Membrana Celular/metabolismo , Células Cultivadas , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Metabolismo dos Lipídeos , Receptores de IgG/metabolismoRESUMO
Viruses must evade the host innate defenses for replication and dengue is no exception. During secondary infection with a heterologous dengue virus (DENV) serotype, DENV is opsonized with sub- or nonneutralizing antibodies that enhance infection of monocytes, macrophages, and dendritic cells via the Fc-gamma receptor (FcγR), a process termed antibody-dependent enhancement of DENV infection. However, this enhancement of DENV infection is curious as cross-linking of activating FcγRs signals an early antiviral response by inducing the type-I IFN-stimulated genes (ISGs). Entry through activating FcγR would thus place DENV in an intracellular environment unfavorable for enhanced replication. Here we demonstrate that, to escape this antiviral response, antibody-opsonized DENV coligates leukocyte Ig-like receptor-B1 (LILRB1) to inhibit FcγR signaling for ISG expression. This immunoreceptor tyrosine-based inhibition motif-bearing receptor recruits Src homology phosphatase-1 to dephosphorylate spleen tyrosine kinase (Syk). As Syk is a key intermediate of FcγR signaling, LILRB1 coligation resulted in reduced ISG expression for enhanced DENV replication. Our findings suggest a unique mechanism for DENV to evade an early antiviral response for enhanced infection.
Assuntos
Anticorpos Facilitadores/fisiologia , Antígenos CD/metabolismo , Vírus da Dengue/metabolismo , Dengue/fisiopatologia , Receptores Imunológicos/metabolismo , Anticorpos Facilitadores/imunologia , Western Blotting , Linhagem Celular , Dengue/imunologia , Vírus da Dengue/fisiologia , Humanos , Receptor B1 de Leucócitos Semelhante a Imunoglobulina , Análise em Microsséries , RNA Interferente Pequeno/genética , Receptores de IgG/metabolismoRESUMO
Dengue (DEN) represents the most serious arthropod-borne viral disease. DEN clinical manifestations range from mild febrile illness to life-threatening hemorrhage and vascular leakage. Early epidemiological observations reported that infants born to DEN-immune mothers were at greater risk to develop the severe forms of the disease upon infection with any serotype of dengue virus (DENV). From these observations emerged the hypothesis of antibody-dependent enhancement (ADE) of disease severity, whereby maternally acquired anti-DENV antibodies cross-react but fail to neutralize DENV particles, resulting in higher viremia that correlates with increased disease severity. Although in vitro and in vivo experimental set ups have indirectly supported the ADE hypothesis, direct experimental evidence has been missing. Furthermore, a recent epidemiological study has challenged the influence of maternal antibodies in disease outcome. Here we have developed a mouse model of ADE where DENV2 infection of young mice born to DENV1-immune mothers led to earlier death which correlated with higher viremia and increased vascular leakage compared to DENV2-infected mice born to dengue naïve mothers. In this ADE model we demonstrated the role of TNF-α in DEN-induced vascular leakage. Furthermore, upon infection with an attenuated DENV2 mutant strain, mice born to DENV1-immune mothers developed lethal disease accompanied by vascular leakage whereas infected mice born to dengue naïve mothers did no display any clinical manifestation. In vitro ELISA and ADE assays confirmed the cross-reactive and enhancing properties towards DENV2 of the serum from mice born to DENV1-immune mothers. Lastly, age-dependent susceptibility to disease enhancement was observed in mice born to DENV1-immune mothers, thus reproducing epidemiological observations. Overall, this work provides direct in vivo demonstration of the role of maternally acquired heterotypic dengue antibodies in the enhancement of dengue disease severity and offers a unique opportunity to further decipher the mechanisms involved.
Assuntos
Anticorpos Antivirais/imunologia , Dengue/imunologia , Imunidade Materno-Adquirida , Animais , Cricetinae , Reações Cruzadas/imunologia , Dengue/patologia , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Mutantes , Gravidez , Índice de Gravidade de Doença , Fator de Necrose Tumoral alfa/imunologiaRESUMO
The interaction of antibodies, dengue virus (DENV), and monocytes can result in either immunity or enhanced virus infection. These opposing outcomes of dengue antibodies have hampered dengue vaccine development. Recent studies have shown that antibodies neutralize DENV by either preventing virus attachment to cellular receptors or inhibiting viral fusion intracellularly. However, whether the antibody blocks attachment or fusion, the resulting immune complexes are expected to be phagocytosed by Fc gamma receptor (FcγR)-bearing cells and cleared from circulation. This suggests that only antibodies that are able to block fusion intracellularly would be able to neutralize DENV upon FcγR-mediated uptake by monocytes whereas other antibodies would have resulted in enhancement of DENV replication. Using convalescent sera from dengue patients, we observed that neutralization of the homologous serotypes occurred despite FcγR-mediated uptake. However, FcγR-mediated uptake appeared to be inhibited when neutralized heterologous DENV serotypes were used instead. We demonstrate that this inhibition occurred through the formation of viral aggregates by antibodies in a concentration-dependent manner. Aggregation of viruses enabled antibodies to cross-link the inhibitory FcγRIIB, which is expressed at low levels but which inhibits FcγR-mediated phagocytosis and hence prevents antibody-dependent enhancement of DENV infection in monocytes.
Assuntos
Dengue/imunologia , Receptores de IgG/sangue , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Complexo Antígeno-Anticorpo/sangue , Linhagem Celular , Dengue/virologia , Vírus da Dengue/classificação , Vírus da Dengue/imunologia , Humanos , Técnicas In Vitro , Monócitos/imunologia , Monócitos/virologia , Fagocitose , RNA Interferente Pequeno/genética , Receptores de IgG/antagonistas & inibidores , Receptores de IgG/genética , Sorotipagem , TransfecçãoRESUMO
Dengue viruses (DENVs), like all viruses, evolve to perpetuate transmission of their species in their hosts. However, how DENV genetics influences dengue disease outbreaks remains poorly understood. Here, we examined isolates of the South Pacific dengue virus type 2 (DENV-2) that emerged in the 1970s and caused major dengue outbreaks in islands in this region until it reached Tonga, where only a few mild cases were reported. Phylogenetically, the DENV-2 strain isolated in Tonga segregated into a clade different from those clades infecting populations in other South Pacific islands. We found that this epidemiological observation could be explained by a single histidine-to-arginine substitution in position 86 of the premembrane (prM) protein of the Tonga DENV-2 strain. This mutation attenuated viral protein translation in mammalian cells but not in midgut cells of the mosquito vector Aedes aegypti. In mammalian cells, the prM mutation resulted in reduced translation of the viral genome and subsequent reduced virus replication. In contrast, in mosquito midgut cells, the prM mutation conferred a selective infection advantage, possibly because of the positively charged arginine residue introduced by the mutation. These findings provide molecular insights into the year-long silent transmission of attenuated DENV-2 in Tonga during the 1970s dengue outbreak in the South Pacific.
Assuntos
Aedes , Vírus da Dengue , Mutação , Replicação Viral , Vírus da Dengue/genética , Vírus da Dengue/fisiologia , Replicação Viral/genética , Animais , Humanos , Mutação/genética , Aedes/virologia , Dengue/virologia , Dengue/transmissão , Filogenia , Linhagem Celular , Biossíntese de ProteínasRESUMO
Vaccination has successfully controlled several infectious diseases although better vaccines remain desirable. Host response to vaccination studies have identified correlates of vaccine immunogenicity that could be useful to guide development and selection of future vaccines. However, it remains unclear whether these findings represent mere statistical correlations or reflect functional associations with vaccine immunogenicity. Functional associations, rather than statistical correlates, would offer mechanistic insights into vaccine-induced adaptive immunity. Through a human experimental study to test the immunomodulatory properties of metformin, an anti-diabetic drug, we chanced upon a functional determinant of neutralizing antibodies. Although vaccine viremia is a known correlate of antibody response, we found that in healthy volunteers with no detectable or low yellow fever 17D viremia, metformin-treated volunteers elicited higher neutralizing antibody titers than placebo-treated volunteers. Transcriptional and metabolomic analyses collectively showed that a brief course of metformin, started 3 days prior to YF17D vaccination and stopped at 3 days after vaccination, expanded oxidative phosphorylation and protein translation capacities. These increased capacities directly correlated with YF17D neutralizing antibody titers, with reduced reactive oxygen species response compared to placebo-treated volunteers. Our findings thus demonstrate a functional association between cellular respiration and vaccine-induced humoral immunity and suggest potential approaches to enhancing vaccine immunogenicity.
Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , Metformina , Vacina contra Febre Amarela , Humanos , Vacina contra Febre Amarela/imunologia , Vacina contra Febre Amarela/administração & dosagem , Metformina/farmacologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Imunogenicidade da Vacina , Febre Amarela/prevenção & controle , Febre Amarela/imunologia , Adulto , Masculino , FemininoRESUMO
BACKGROUND: In 2001 and 2002, fatal myocarditis resulted in the sudden deaths of four, two adult and two juvenile, orang utans out of a cohort of 26 in the Singapore Zoological Gardens. METHODS: Of the four orang utans that underwent post-mortem examination, virus isolation was performed from the tissue homogenates of the heart and lung obtained from the two juvenile orang utans in Vero cell cultures. The tissue culture fluid was examined using electron microscopy. Reverse transcription and polymerase chain reaction with Encephalomyocarditis virus (EMCV)-specific primers targeting the gene regions of VP3/VP1 and 3D polymerase (3Dpol) confirmed the virus genus and species. The two EMCV isolates were sequenced and phylogenetic analyses of the virus genes performed. Serological testing on other animal species in the Singapore Zoological Gardens was also conducted. RESULTS: Electron microscopy of the two EMCV isolates, designated Sing-M100-02 and Sing-M105-02, revealed spherical viral particles of about 20 to 30 nm, consistent with the size and morphology of members belonging to the family Picornaviridae. In addition, infected-Vero cells showed positive immunoflorescence staining with antiserum to EMCV. Sequencing of the viral genome showed that the two EMCV isolates were 99.9% identical at the nucleotide level, indicating a similar source of origin. When compared with existing EMCV sequences in the VP1 and 3Dpol gene regions, the nucleotide divergence were at a maximum of 38.8% and 23.6% respectively, while the amino acid divergence were at a maximum of 33.9% and 11.3% respectively. Phylogenetic analyses of VP1 and 3Dpol genes further grouped the Sing-M100-02 and Sing-M105-02 isolates to themselves, away from existing EMCV lineages. This strongly suggested that Sing-M100-02 and Sing-M105-02 isolates are highly divergent variants of EMCV. Apart from the two deceased orang utans, a serological survey conducted among other zoo animals showed that a number of other animal species had neutralizing antibodies to Sing-M105-02 isolate, indicating that the EMCV variant has a relatively wide host range. CONCLUSIONS: The etiological agent responsible for the fatal myocarditis cases among two of the four orang utans in the Singapore Zoological Gardens was a highly divergent variant of EMCV. This is the first report of an EMCV infection in Singapore and South East Asia.
Assuntos
Vírus da Encefalomiocardite/classificação , Vírus da Encefalomiocardite/isolamento & purificação , Pongo/virologia , Animais , Animais de Zoológico , Chlorocebus aethiops , Análise por Conglomerados , Vírus da Encefalomiocardite/genética , Genoma Viral , Coração/virologia , Pulmão/virologia , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Filogenia , RNA Viral/genética , Análise de Sequência de DNA , Homologia de Sequência , Singapura , Células Vero , Proteínas Virais/genética , Vírion/ultraestrutura , Cultura de VírusRESUMO
Aberrant cellular bioenergetics has detrimental consequences in host cells. For instance, pathogenic Zika virus strains can suppress mitochondria respiration and glycolytic functions, disrupting cellular bioenergetics that leads to apoptosis. Herein, we describe methods for flavivirus propagation, titering and infection, cell preparation, and procedures for mitochondrial and glycolytic stress tests. The protocol enables assessment of cellular respiration and glycolytic flux in flavivirus-infected cells. For complete details on the use and execution of this protocol, please refer to Yau et al. (2021).
Assuntos
Flavivirus , Infecção por Zika virus , Zika virus , Metabolismo Energético , Glicólise , Humanos , Mitocôndrias/metabolismo , Infecção por Zika virus/metabolismoRESUMO
The four dengue viruses (DENVs) have evolved multiple mechanisms to ensure its survival. Among these mechanisms is the ability to regulate its replication rate, which may contribute to avoiding premature immune activation that limit infection dissemination: DENVs associated with dengue epidemics have shown slower replication rate than pre-epidemic strains. Correspondingly, wild-type DENVs replicate more slowly than their clinically attenuated derivatives. To understand how DENVs 'make haste slowly', we generated and screened for DENV2 mutants with accelerated replication that also induced high type-I interferon (IFN) expression in infected cells. We chanced upon a single NS2B-I114T amino acid substitution, in an otherwise highly conserved amino acid residue. Accelerated DENV2 replication damaged host DNA as mutant infection was dependent on host DNA damage repair factors, namely RAD21, EID3 and NEK5. DNA damage induced cGAS/STING signalling and activated early type-I IFN response that inhibited infection dissemination. Unexpectedly, STING activation also supported mutant DENV replication in infected cells through STING-induced autophagy. Our findings thus show that DENV NS2B has multi-faceted role in controlling DENV replication rate and immune evasion and suggest that the dual role of STING in supporting virus replication within infected cells but inhibiting infection dissemination could be particularly advantageous for live attenuated vaccine development.
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Vírus da Dengue , Interferon Tipo I , Evasão da Resposta Imune , Replicação Viral , Interferon Tipo I/genética , Transdução de SinaisRESUMO
Coronavirus disease-19 (Covid-19) pandemic have demonstrated the importantance of vaccines in disease prevention. Self-amplifying mRNA vaccines could be another option for disease prevention if demonstrated to be safe and immunogenic. Phase 1 of this randomized, double-blinded, placebo-controlled trial (N = 42) assessed the safety, tolerability, and immunogenicity in healthy young and older adults of ascending levels of one-dose ARCT-021, a self-amplifying mRNA vaccine against Covid-19. Phase 2 (N = 64) tested two-doses of ARCT-021 given 28 days apart. During phase 1, ARCT-021 was well tolerated up to one 7.5 µg dose and two 5.0 µg doses. Local solicited AEs, namely injection-site pain and tenderness were more common in ARCT-021vaccinated, while systemic solicited AEs, mainly fatigue, headache and myalgia were reported in 62.8% and 46.4% of ARCT-021 and placebo recipients, respectively. Seroconversion rate for anti-S IgG was 100% in all cohorts, except for the 1 µg one-dose in younger adults and the 7.5 µg one-dose in older adults. Anti-S IgG and neutralizing antibody titers showed a general increase with increasing dose, and overlapped with titers in Covid-19 convalescent patients. T-cell responses were also observed in response to stimulation with S-protein peptides. Taken collectively, ARCT-021 is immunogenic and has favorable safety profile for further development.
RESUMO
Dengue poses a significant burden of individual health, health systems and the economy in dengue endemic regions. As such, dengue vaccine development has been an active area of research. Previous studies selected attenuated vaccine candidates based on plaque size. However, these candidates led to mixed safety outcome in clinical trials, suggesting it is insufficiently informative as an indicator of dengue virus (DENV) attenuation. In this study, we examined the genome diversity of wild-type DENVs and their attenuated derivatives developed by Mahidol University and tested in phase 1 clinical trials. We found that the attenuated DENVs, in particular the strain under clinical development by Takeda Vaccines, DENV2 PDK53, showed significantly higher genome diversity than its wild-type parent, DENV2 16681. The determinant of genomic diversity was intrinsic to the PDK53 genome as infectious clone of PDK53 showed greater genomic diversity after a single in vitro passage compared to 16681 infectious clone. Similar trends were observed with attenuated DENV1 and DENV4, both of which were shown to be attenuated clinically, but not DENV3 that was not adequately attenuated clinically. Taken together, evidence presented here suggests that genome diversity could be developed into a marker of DENV attenuation.
RESUMO
The global spread of SARS-CoV-2 has made millions ill with COVID-19 and even more from the economic fallout of this pandemic. Our quest to test new therapeutics and vaccines require small animal models that replicate disease phenotypes seen in COVID-19 cases. Rodent models of SARS-CoV-2 infection thus far have shown mild to moderate pulmonary disease; mortality, if any, has been associated with prominent signs of central nervous system (CNS) infection and dysfunction. Here we describe the isolation of SARS-CoV-2 variants with propensity for either pulmonary or CNS infection. Using a wild-type SARS-CoV-2 isolated from a COVID-19 patient, we first found that infection was lethal in transgenic mice expressing the human angiotensin I-converting enzyme 2 (hACE2). Fortuitously, full genome sequencing of SARS-CoV-2 from the brain and lung of these animals showed genetic differences. Likewise, SARS-CoV-2 isolates from brains and lungs of these also showed differences in plaque morphology. Inoculation of these brain and lung SARS-CoV-2 isolates into new batch of hACE2 mice intra-nasally resulted in lethal CNS and pulmonary infection, respectively. Collectively, our study suggests that genetic variants of SARS-CoV-2 could be used to replicate specific features of COVID-19 for the testing of potential vaccines or therapeutics.
Assuntos
COVID-19/patologia , Modelos Animais de Doenças , Pulmão/patologia , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Animais , Encéfalo/patologia , Encéfalo/virologia , COVID-19/metabolismo , COVID-19/mortalidade , COVID-19/virologia , Feminino , Humanos , Pulmão/virologia , Camundongos , Camundongos Transgênicos , Peptidil Dipeptidase A/metabolismoRESUMO
BACKGROUND: The coronavirus disease-19 (COVID-19) pandemic has cost lives and economic hardships globally. Various studies have found a number of different factors, such as hyperinflammation and exhausted/suppressed T cell responses to the etiological SARS coronavirus-2 (SARS-CoV-2), being associated with severe COVID-19. However, sieving the causative from associative factors of respiratory dysfunction has remained rudimentary. METHODS: We postulated that the host responses causative of respiratory dysfunction would track most closely with disease progression and resolution and thus be differentiated from other factors that are statistically associated with but not causative of severe COVID-19. To track the temporal dynamics of the host responses involved, we examined the changes in gene expression in whole blood of 6 severe and 4 non-severe COVID-19 patients across 15 different timepoints spanning the nadir of respiratory function. FINDINGS: We found that neutrophil activation but not type I interferon signaling transcripts tracked most closely with disease progression and resolution. Moreover, transcripts encoding for protein phosphorylation, particularly the serine-threonine kinases, many of which have known T cell proliferation and activation functions, were increased after and may thus contribute to the upswing of respiratory function. Notably, these associative genes were targeted by dexamethasone, but not methylprednisolone, which is consistent with efficacy outcomes in clinical trials. INTERPRETATION: Our findings suggest neutrophil activation as a critical factor of respiratory dysfunction in COVID-19. Drugs that target this pathway could be potentially repurposed for the treatment of severe COVID-19. FUNDING: This study was sponsored in part by a generous gift from The Hour Glass. EEO and JGL are funded by the National Medical Research Council of Singapore, through the Clinician Scientist Awards awarded by the National Research Foundation of Singapore.
Assuntos
COVID-19/patologia , Ativação Linfocitária/imunologia , Ativação de Neutrófilo/imunologia , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Adulto , Idoso , Progressão da Doença , Reposicionamento de Medicamentos , Feminino , Expressão Gênica/genética , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Neutrófilos/imunologia , Estudos Prospectivos , Linfócitos T/imunologiaRESUMO
Zika virus (ZIKV) is an Aedes-mosquito-borne flavivirus that causes debilitating congenital and developmental disorders. Improved understanding of ZIKV pathogenesis could assist efforts to fill the therapeutic and vaccine gap. We use several ZIKV strains, including a pair differing by a single phenylalanine-to-leucine substitution (M-F37L) in the membrane (M) protein, coupled with unbiased genomics to demarcate the border between attenuated and pathogenic infection. We identify infection-induced metabolic dysregulation as a minimal set of host alterations that differentiates attenuated from pathogenic ZIKV strains. Glycolytic rewiring results in impaired oxidative phosphorylation and mitochondrial dysfunction that trigger inflammation and apoptosis in pathogenic but not attenuated ZIKV strains. Critically, pyruvate supplementation prevents cell death, in vitro, and rescues fetal development in ZIKV-infected dams. Our findings thus demonstrate dysregulated metabolism as an underpinning of ZIKV pathogenicity and raise the potential of pyruvate supplementation in expectant women as a prophylaxis against congenital Zika syndrome.