RESUMEN
Vaccines are a powerful medical intervention for preventing epidemic diseases. Efficient inactivated or protein vaccines typically rely on an effective adjuvant to elicit an immune response and boost vaccine activity. In this study, we investigated the adjuvant activities of combinations of Toll-like receptor 9 (TLR9) and stimulator of interferon genes (STING) agonists in a SARS-CoV-2 receptor binding domain protein vaccine. Adjuvants formulated with a TLR9 agonist, CpG-2722, with various cyclic dinucleotides (CDNs) that are STING agonists increased germinal center B cell response and elicited humoral immune responses in immunized mice. An adjuvant containing CpG-2722 and 2'3'-c-di-AM(PS)2 effectively boosted the immune response to both intramuscularly and intranasally administrated vaccines. Vaccines adjuvanted with CpG-2722 or 2'3'-c-di-AM(PS)2 alone were capable of inducing an immune response, but a cooperative adjuvant effect was observed when both were combined. CpG-2722 induced antigen-dependent T helper (Th)1 and Th17 responses, while 2'3'-c-di-AM(PS)2 induced a Th2 response. The combination of CpG-2722 and 2'3'-c-di-AM(PS)2 generated a distinct antigen-dependent Th response profile characterized by higher Th1 and Th17, but lower Th2 responses. In dendritic cells, CpG-2722 and 2'3'-c-di-AM(PS)2 showed a cooperative effect on inducing expression of molecules critical for T cell activation. CpG-2722 and 2'3'-c-di-AM(PS)2 have distinct cytokine inducing profiles in different cell populations. The combination of these two agonists enhanced the expression of cytokines for Th1 and Th17 responses and suppressed the expression of cytokines for Th2 response in these cells. Thus, the antigen-dependent Th responses observed in the animals immunized with different vaccines were shaped by the antigen-independent cytokine-inducing profiles of their adjuvant. The expanded targeting cell populations, the increased germinal center B cell response, and reshaped T helper responses are the molecular bases for the cooperative adjuvant effect of the combination of TLR9 and STING agonists.
Asunto(s)
COVID-19 , Vacunas , Animales , Ratones , Vacunas contra la COVID-19 , Receptor Toll-Like 9/agonistas , SARS-CoV-2 , Oligodesoxirribonucleótidos/farmacología , Adyuvantes Inmunológicos/farmacología , Adyuvantes Inmunológicos/química , Citocinas , Inmunidad , Centro GerminalRESUMEN
Post-translational modifications (PTMs), such as glycosylation and palmitoylation, are critical to protein folding, stability, intracellular trafficking, and function. Understanding regulation of PTMs of SARS-CoV-2 spike (S) protein could help the therapeutic drug design. Herein, the VSV vector was used to produce SARS-CoV-2 S pseudoviruses to examine the roles of the 611LYQD614 and cysteine-rich motifs in S protein maturation and virus infectivity. Our results show that 611LY612 mutation alters S protein intracellular trafficking and reduces cell surface expression level. It also changes S protein glycosylation pattern and decreases pseudovirus infectivity. The S protein contains four cysteine-rich clusters with clusters I and II as the main palmitoylation sites. Mutations of clusters I and II disrupt S protein trafficking from ER-to-Golgi, suppress pseudovirus production, and reduce spike-mediated membrane fusion activity. Taken together, glycosylation and palmitoylation orchestrate the S protein maturation processing and are critical for S protein-mediated membrane fusion and infection.
RESUMEN
An effective COVID-19 vaccine against broad SARS-CoV-2 variants is still an unmet need. In the study, the vesicular stomatitis virus (VSV)-based vector was used to express the SARS-CoV-2 Spike protein to identify better vaccine designs. The replication-competent of the recombinant VSV-spike virus with C-terminal 19 amino acid truncation (SΔ19 Rep) was generated. A single dose of SΔ19 Rep intranasal vaccination is sufficient to induce protective immunity against SARS-CoV-2 infection in hamsters. All the clones isolated from the SΔ19 Rep virus contained R682G mutation located at the Furin cleavage site. An additional S813Y mutation close to the TMPRSS2 cleavage site was identified in some clones. The enzymatic processing of S protein was blocked by these mutations. The vaccination of the R682G-S813Y virus produced a high antibody response against S protein and a robust S protein-specific CD8+ T cell response. The vaccinated animals were protected from the lethal SARS-CoV-2 (delta variant) challenge. The S antigen with resistance to enzymatic processes by Furin and TMPRSS2 will provide better immunogenicity for vaccine design.
Asunto(s)
COVID-19 , Furina , SARS-CoV-2 , Serina Endopeptidasas , Animales , COVID-19/inmunología , COVID-19/prevención & control , COVID-19/virología , Vacunas contra la COVID-19 , Furina/genética , Furina/metabolismo , Humanos , Inmunidad Celular , SARS-CoV-2/inmunología , Serina Endopeptidasas/genética , Serina Endopeptidasas/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
Increasing evidence suggests that elderly people with dementia are vulnerable to the development of severe coronavirus disease 2019 (COVID-19). In Alzheimer's disease (AD), the major form of dementia, ß-amyloid (Aß) levels in the blood are increased; however, the impact of elevated Aß levels on the progression of COVID-19 remains largely unknown. Here, our findings demonstrate that Aß1-42, but not Aß1-40, bound to various viral proteins with a preferentially high affinity for the spike protein S1 subunit (S1) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the viral receptor, angiotensin-converting enzyme 2 (ACE2). These bindings were mainly through the C-terminal residues of Aß1-42. Furthermore, Aß1-42 strengthened the binding of the S1 of SARS-CoV-2 to ACE2 and increased the viral entry and production of IL-6 in a SARS-CoV-2 pseudovirus infection model. Intriguingly, data from a surrogate mouse model with intravenous inoculation of Aß1-42 show that the clearance of Aß1-42 in the blood was dampened in the presence of the extracellular domain of the spike protein trimers of SARS-CoV-2, whose effects can be prevented by a novel anti-Aß antibody. In conclusion, these findings suggest that the binding of Aß1-42 to the S1 of SARS-CoV-2 and ACE2 may have a negative impact on the course and severity of SARS-CoV-2 infection. Further investigations are warranted to elucidate the underlying mechanisms and examine whether reducing the level of Aß1-42 in the blood is beneficial to the fight against COVID-19 and AD.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Enzima Convertidora de Angiotensina 2/metabolismo , Fragmentos de Péptidos/metabolismo , SARS-CoV-2/enzimología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Células A549 , Enfermedad de Alzheimer/complicaciones , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/química , Animales , COVID-19/complicaciones , COVID-19/metabolismo , Chlorocebus aethiops , Humanos , Interleucina-6/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Fragmentos de Péptidos/química , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Células Vero , Internalización del VirusRESUMEN
HIV-1 protease autoprocessing liberates the free mature protease from its Gag-Pol polyprotein precursor through a series of highly regulated autoproteolysis reactions. Herein, we report the development and validation (Z' ≥ 0.50) of a cell-based functional assay for high-throughput screening (HTS) of autoprocessing inhibitors using fusion precursors in combination with AlphaLISA (amplified luminescent proximity homogeneous assay ELISA). Through pilot screening of a collection of 130 known protease inhibitors, the AlphaLISA assay confirmed all 11 HIV protease inhibitors in the library capable of suppressing precursor autoprocessing at low micromolar concentrations. Meanwhile, other protease inhibitors had no impact on precursor autoprocessing. We next conducted HTS of ~23,000 compounds but found no positive hits. Such high selectivity is advantageous for large-scale HTS campaigns and as anticipated based on assay design because a positive hit needs simultaneously to be nontoxic, cell permeable, and inhibiting precursor autoprocessing. Furthermore, AlphaLISA quantification of fusion precursors carrying mutations known to cause resistance to HIV protease inhibitors faithfully recapitulated the reported resistance, suggesting that precursor autoprocessing is a critical step contributing to drug resistance. Taken together, this reported AlphaLISA platform will provide a useful tool for drug discovery targeting HIV-1 protease autoprocessing and for quantification of PI resistance.
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Descubrimiento de Drogas/métodos , Resistencia a Medicamentos/efectos de los fármacos , Inhibidores de la Proteasa del VIH/análisis , Proteasa del VIH/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Proteasa del VIH/efectos de los fármacos , Humanos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , ProteolisisRESUMEN
Flaviviruses accumulate abundant subgenomic RNA (sfRNA) in infected cells. It has been reported that sfRNA results from stalling of host 5'-to-3' exoribonuclease XRN1 at the highly structured RNA of the 3' untranslated region (UTR). Although XRN1 digestion of a 3'-terminal 800-nt RNA could stall at a position to generate the sfRNA in vitro, we found that knocking out XRN1 had no effect on the accumulation of sfRNA in Japanese encephalitis virus (JEV) infected cells. Mutagenesis studies revealed that the stemloop II (SLII) at the 3' UTR is required for the accumulation of sfRNA. According to the results of an in vitro RNA-dependent RNA polymerase (RdRp) assay, the (-)10431-10566 RNA fragment, containing the putative promoter on the antigenome for the sfRNA transcription, binds to RdRp protein and exhibits a strong promoter activity. Taken together, our results indicate that the JEV sfRNA could be transcribed initially and then be trimmed by XRN1 or other unidentified exoribonucleases.
Asunto(s)
Regiones no Traducidas 3' , Virus de la Encefalitis Japonesa (Especie)/genética , Encefalitis Japonesa/virología , Genoma Viral , ARN Viral/genética , Línea Celular , Virus de la Encefalitis Japonesa (Especie)/fisiología , Encefalitis Japonesa/genética , Encefalitis Japonesa/metabolismo , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Flavivirus/genética , Flavivirus/metabolismo , Regulación Viral de la Expresión Génica , Técnicas de Inactivación de Genes , Interacciones Huésped-Patógeno , Humanos , ARN Viral/metabolismo , Replicación ViralRESUMEN
HIV-1 protease autoprocessing is responsible for liberation of free mature protease (PR) from the Gag-Pol polyprotein precursor. A cell-based model system was previously developed to examine the autoprocessing mechanism of fusion precursors carrying the p6*-PR miniprecursor sandwiched between various proteins or epitopes. We here report that precursor autoprocessing is context-dependent as its activity and outcomes can be modulated by sequences upstream of p6*-PR. This was exemplified by the 26aa maltose binding protein (MBP) signal peptide (SigP) when placed at the N-terminus of a fusion precursor. The mature PRs released from SigP-carrying precursors are resistant to self-degradation whereas those released from SigP-lacking fusion precursors are prone to self-degradation. A H69D mutation in PR abolished autoprocessing of SigP-containing fusion precursors whereas it only partially suppressed autoprocessing of fusion precursors lacking SigP. An autoprocessing deficient GFP fusion precursor with SigP exhibited a subcellular distribution pattern distinct from the one without it in transfected HeLa cells. Furthermore, a SigP fusion precursor carrying a substitution at the P1 position released the mature PR and PR-containing fragments that were different from those released from the precursor carrying the same mutation but lacking SigP. We also examined autoprocessing outcomes in viral particles produced by a NL4-3 derived proviral construct and demonstrated the existence of several PR-containing fragments along with the mature PR. Some of these resembled the SigP precursor autoprocessing outcomes. This finding of context-dependent modulation reveals the complexity of precursor autoprocessing regulation that most likely accompanies sequence variation imposed by the evolution of the upstream Gag moiety.
Asunto(s)
Precursores Enzimáticos/metabolismo , Proteasa del VIH/metabolismo , VIH-1/enzimología , Secuencia de Aminoácidos , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Células HEK293 , Proteasa del VIH/química , Proteasa del VIH/genética , Humanos , MutaciónRESUMEN
Viral replication depends on host proteins to supply energy and replication accessories for the sufficient production of viral progeny. In this study, we identified fructose-bisphosphate aldolase A as a binding partner of Japanese encephalitis virus (JEV) untranslated regions (UTRs) on the antigenome via RNA affinity capture and mass spectrometry. Direct interaction of aldolase A with JEV RNAs was confirmed by gel mobility shift assay and colocalization with active replication of double-stranded RNA in JEV-infected cells. Infection of JEV caused an increase in aldolase A expression of up to 33%. Knocking down aldolase A reduced viral translation, genome replication, and viral production significantly. Furthermore, JEV infection consumed 50% of cellular ATP, and the ATP level decreased by 70% in the aldolase A-knockdown cells. Overexpression of aldolase A in aldolase A-knockdown cells increased ATP levels significantly. Taken together, these results indicate that JEV replication requires aldolase A and consumes ATP. This is the first report of direct involvement of a host metabolic enzyme, aldolase A protein, in JEV replication.
Asunto(s)
Adenosina Trifosfato/biosíntesis , Virus de la Encefalitis Japonesa (Especie)/fisiología , Fructosa-Bifosfato Aldolasa/antagonistas & inhibidores , Fructosa-Bifosfato Aldolasa/metabolismo , Células HEK293 , Humanos , Replicación ViralRESUMEN
Replication of the Japanese encephalitis virus (JEV) genome depends on host factors for successfully completing their life cycles; to do this, host factors have been recruited and/or relocated to the site of viral replication. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a cellular metabolic protein, was found to colocalize with viral RNA-dependent RNA polymerase (NS5) in JEV-infected cells. Subcellular fractionation further indicated that GAPDH remained relatively constant in the cytosol, while increasing at 12 to 24 hours postinfection (hpi) and decreasing at 36 hpi in the nuclear fraction of infected cells. In contrast, the redistribution patterns of GAPDH were not observed in the uninfected cells. Co-immunoprecipitation of GAPDH and JEV NS5 protein revealed no direct protein-protein interaction; instead, GAPDH binds to the 3' termini of plus- and minus-strand RNAs of JEV by electrophoretic mobility shift assays. Accordingly, GAPDH binds to the minus strand more efficiently than to the plus strand of JEV RNAs. This study highlights the findings that infection of JEV changes subcellular localization of GAPDH suggesting that this metabolic enzyme may play a role in JEV replication.