RESUMEN
We present a live-attenuated RNA hybrid vaccine technology that uses an RNA vaccine delivery vehicle to deliver in vitro-transcribed, full-length, live-attenuated viral genomes to the site of vaccination. This technology allows ready manufacturing in a cell-free environment, regardless of viral attenuation level, and it promises to avoid many safety and manufacturing challenges of traditional live-attenuated vaccines. We demonstrate this technology through development and testing of a live-attenuated RNA hybrid vaccine against Chikungunya virus (CHIKV), comprised of an in vitro-transcribed, highly attenuated CHIKV genome delivered by a highly stable nanostructured lipid carrier (NLC) formulation as an intramuscular injection. We demonstrate that single-dose immunization of immunocompetent C57BL/6 mice results in induction of high CHIKV-neutralizing antibody titers and protection against mortality and footpad swelling after lethal CHIKV challenge.
Asunto(s)
Anticuerpos Neutralizantes/sangre , Fiebre Chikungunya/prevención & control , Virus Chikungunya/genética , Lípidos/química , Vacunas de ARNm/administración & dosificación , Animales , Anticuerpos Antivirales/sangre , Fiebre Chikungunya/inmunología , Virus Chikungunya/inmunología , Chlorocebus aethiops , Modelos Animales de Enfermedad , Composición de Medicamentos , Femenino , Genoma Viral , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Nanoestructuras , Células Vero , Vacunas Virales/administración & dosificación , Vacunas Virales/química , Vacunas Virales/inmunología , Vacunas de ARNm/química , Vacunas de ARNm/inmunologíaRESUMEN
Rubella vaccination induces widely variable immune responses in vaccine recipients. While rubella vaccination is effective at inducing immunity to rubella infection in most subjects, up to 5% of individuals do not achieve or maintain long-term protective immunity. To expand upon our previous work identifying genetic polymorphisms that are associated with these interindividual differences in humoral immunity to rubella virus, we performed a genome-wide association study in a large cohort of 1843 subjects to discover single-nucleotide polymorphisms (SNPs) associated with rubella virus-specific cellular immune responses. We identified SNPs in the Wilms tumor protein gene (WT1) that were significantly associated (P < 5 × 10-8) with interindividual variations in rubella-specific interleukin 6 secretion from subjects' peripheral blood mononuclear cells postvaccination. No SNPs were found to be significantly associated with variations in rubella-specific interferon-γ secretion. Our findings demonstrate that genetic polymorphisms in the WT1 gene in subjects of European ancestry are associated with interindividual differences in rubella virus-specific cellular immunity after measles-mumps-rubella II vaccination.
Asunto(s)
Variación Biológica Poblacional , Inmunidad Celular , Individualidad , Vacuna contra el Sarampión-Parotiditis-Rubéola/inmunología , Polimorfismo de Nucleótido Simple , Virus de la Rubéola/inmunología , Proteínas WT1/genética , Adolescente , Adulto , Niño , Estudios de Cohortes , Femenino , Estudio de Asociación del Genoma Completo , Humanos , Masculino , Vacuna contra el Sarampión-Parotiditis-Rubéola/administración & dosificación , Población Blanca , Adulto JovenRESUMEN
We describe a new method to accomplish multiplexed, absolute protein quantification in a targeted fashion. The approach draws upon the recently developed neutron encoding (NeuCode) metabolic labeling strategy and parallel reaction monitoring (PRM). Since PRM scanning relies upon high-resolution tandem mass spectra for targeted protein quantification, incorporation of multiple NeuCode labeled peptides permits high levels of multiplexing that can be accessed from high-resolution tandem mass spectra. Here we demonstrate this approach in cultured cells by monitoring a viral infection and the corresponding viral protein production over many infection time points in a single experiment. In this context the NeuCode PRM combination affords up to 30 channels of quantitative information in a single MS experiment.
Asunto(s)
Proteínas/análisis , Línea Celular Tumoral , Humanos , Masculino , Espectrometría de Masas en TándemRESUMEN
Type I interferons (IFN-I) are critical mediators of innate control of viral infections but also drive the recruitment of inflammatory cells to sites of infection, a key feature of severe coronavirus disease 2019. Here, IFN-I signaling was modulated in rhesus macaques (RMs) before and during acute SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection using a mutated IFN-α2 (IFN-modulator; IFNmod), which has previously been shown to reduce the binding and signaling of endogenous IFN-I. IFNmod treatment in uninfected RMs was observed to induce a modest up-regulation of only antiviral IFN-stimulated genes (ISGs); however, in SARS-CoV-2-infected RMs, IFNmod reduced both antiviral and inflammatory ISGs. IFNmod treatment resulted in a potent reduction in SARS-CoV-2 viral loads both in vitro in Calu-3 cells and in vivo in bronchoalveolar lavage (BAL), upper airways, lung, and hilar lymph nodes of RMs. Furthermore, in SARS-CoV-2-infected RMs, IFNmod treatment potently reduced inflammatory cytokines, chemokines, and CD163+ MRC1- inflammatory macrophages in BAL and expression of Siglec-1 on circulating monocytes. In the lung, IFNmod also reduced pathogenesis and attenuated pathways of inflammasome activation and stress response during acute SARS-CoV-2 infection. Using an intervention targeting both IFN-α and IFN-ß pathways, this study shows that, whereas early IFN-I restrains SARS-CoV-2 replication, uncontrolled IFN-I signaling critically contributes to SARS-CoV-2 inflammation and pathogenesis in the moderate disease model of RMs.
Asunto(s)
COVID-19 , Interferón Tipo I , Animales , Interferón Tipo I/farmacología , SARS-CoV-2 , Macaca mulatta , Replicación Viral , Antivirales/farmacología , Antivirales/uso terapéutico , Inflamación/tratamiento farmacológicoRESUMEN
mRNA vaccines were the first to be authorized for use against SARS-CoV-2 and have since demonstrated high efficacy against serious illness and death. However, limitations in these vaccines have been recognized due to their requirement for cold storage, short durability of protection, and lack of access in low-resource regions. We have developed an easily-manufactured, potent self-amplifying RNA (saRNA) vaccine against SARS-CoV-2 that is stable at room temperature. This saRNA vaccine is formulated with a nanostructured lipid carrier (NLC), providing stability, ease of manufacturing, and protection against degradation. In preclinical studies, this saRNA/NLC vaccine induced strong humoral immunity, as demonstrated by high pseudovirus neutralization titers to the Alpha, Beta, and Delta variants of concern and induction of bone marrow-resident antibody-secreting cells. Robust Th1-biased T-cell responses were also observed after prime or homologous prime-boost in mice. Notably, the saRNA/NLC platform demonstrated thermostability when stored lyophilized at room temperature for at least 6 months and at refrigerated temperatures for at least 10 months. Taken together, this saRNA delivered by NLC represents a potential improvement in RNA technology that could allow wider access to RNA vaccines for the current COVID-19 and future pandemics.
RESUMEN
We conducted a large genome-wide association study (GWAS) of the immune responses to primary smallpox vaccination in a combined cohort of 1,653 subjects. We did not observe any polymorphisms associated with standard vaccine response outcomes (e.g., neutralizing antibody, T cell ELISPOT response, or T cell cytokine production); however, we did identify a cluster of SNPs on chromosome 5 (5q31.2) that were significantly associated (p-value: 1.3 x 10-12 - 1.5x10-36) with IFNα response to in vitro poxvirus stimulation. Examination of these SNPs led to the functional testing of rs1131769, a non-synonymous SNP in TMEM173 causing an Arg-to-His change at position 232 in the STING protein-a major regulator of innate immune responses to viral infections. Our findings demonstrate differences in the ability of the two STING variants to phosphorylate the downstream intermediates TBK1 and IRF3 in response to multiple STING ligands. Further downstream in the STING pathway, we observed significantly reduced expression of type I IFNs (including IFNα) and IFN-response genes in cells carrying the H232 variant. Subsequent molecular modeling of both alleles predicted altered ligand binding characteristics between the two variants, providing a potential mechanism underlying differences in inter-individual responses to poxvirus infection. Our data indicate that possession of the H232 variant may impair STING-mediated innate immunity to poxviruses. These results clarify prior studies evaluating functional effects of genetic variants in TMEM173 and provide novel data regarding genetic control of poxvirus immunity.
Asunto(s)
Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Inmunidad Innata , Proteínas de la Membrana/genética , Polimorfismo de Nucleótido Simple , Infecciones por Poxviridae/genética , Infecciones por Poxviridae/inmunología , Poxviridae/inmunología , Alelos , Susceptibilidad a Enfermedades , Efecto Fundador , Expresión Génica , Estudio de Asociación del Genoma Completo , Genotipo , Humanos , Inmunidad Innata/genética , Fenómenos Inmunogenéticos , Ligandos , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Fosforilación , Infecciones por Poxviridae/virología , Regiones Promotoras Genéticas , Unión Proteica , Conformación Proteica , Relación Estructura-ActividadRESUMEN
Background: Sex differences in immune responses to influenza vaccine may impact efficacy across populations. Methods: In a cohort of 138 older adults (50-74 years old), we measured influenza A/H1N1 antibody titers, B-cell ELISPOT response, PBMC transcriptomics, and PBMC cell compositions at 0, 3, and 28 days post-immunization with the 2010/11 seasonal inactivated influenza vaccine. Results: We identified higher B-cell ELISPOT responses in females than males. Potential mechanisms for sex effects were identified in four gene clusters related to T, NK, and B cells. Mediation analysis indicated that sex-dependent expression in T and NK cell genes can be partially attributed to higher CD4+ T cell and lower NK cell fractions in females. We identified strong sex effects in 135 B cell genes whose expression correlates with ELISPOT measures, and found that cell subset differences did not explain the effect of sex on these genes' expression. Post-vaccination expression of these genes, however, mediated 41% of the sex effect on ELISPOT responses. Conclusions: These results improve our understanding of sexual dimorphism in immunity and influenza vaccine response.
Asunto(s)
Inmunidad , Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Gripe Humana/inmunología , Gripe Humana/prevención & control , Factores de Edad , Anciano , Anticuerpos Antivirales/inmunología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Biomarcadores , Ensayo de Immunospot Ligado a Enzimas , Femenino , Evaluación Geriátrica , Humanos , Inmunidad Celular , Inmunidad Humoral , Gripe Humana/genética , Leucocitos Mononucleares/inmunología , Masculino , Persona de Mediana Edad , Factores Sexuales , Linfocitos T/inmunología , Linfocitos T/metabolismo , VacunaciónRESUMEN
PBMC transcriptomes after influenza vaccination contain valuable information about factors affecting vaccine responses. However, distilling meaningful knowledge out of these complex datasets is often difficult and requires advanced data mining algorithms. We investigated the use of the data-driven Weighted Gene Correlation Network Analysis (WGCNA) gene clustering method to identify vaccine response-related genes in PBMC transcriptomic datasets collected from 138 healthy older adults (ages 50-74) before and after 2010-2011 seasonal trivalent influenza vaccination. WGCNA separated the 14,197 gene dataset into 15 gene clusters based on observed gene expression patterns across subjects. Eight clusters were strongly enriched for genes involved in specific immune cell types and processes, including B cells, T cells, monocytes, platelets, NK cells, cytotoxic T cells, and antiviral signaling. Examination of gene cluster membership identified signatures of cellular and humoral responses to seasonal influenza vaccination, as well as pre-existing cellular immunity. The results of this study illustrate the utility of this publically available analysis methodology and highlight genes previously associated with influenza vaccine responses (e.g., CAMK4, CD19), genes with functions not previously identified in vaccine responses (e.g., SPON2, MATK, CST7), and previously uncharacterized genes (e.g. CORO1C, C8orf83) likely related to influenza vaccine-induced immunity due to their expression patterns.
Asunto(s)
Perfilación de la Expresión Génica , Inmunidad Celular , Inmunidad Humoral , Factores Inmunológicos/biosíntesis , Vacunas contra la Influenza/inmunología , Anciano , Biología Computacional , Femenino , Voluntarios Sanos , Humanos , Factores Inmunológicos/genética , Vacunas contra la Influenza/administración & dosificación , Masculino , Persona de Mediana EdadRESUMEN
Smallpox has shaped human history, from the earliest human civilizations well into the 20th century. With high mortality rates, rapid transmission, and serious long-term effects on survivors, smallpox was a much-feared disease. The eradication of smallpox represents an unprecedented medical victory for the lasting benefit of human health and prosperity. Concerns remain, however, about the development and use of the smallpox virus as a biological weapon, which necessitates the need for continued vaccine development. Smallpox vaccine development is thus a much-reviewed topic of high interest. This review focuses on the current state of smallpox vaccines and their context in biodefense efforts.
Asunto(s)
Bioterrorismo , Erradicación de la Enfermedad , Vacuna contra Viruela/inmunología , Vacuna contra Viruela/aislamiento & purificación , Viruela/prevención & control , Animales , Modelos Animales de Enfermedad , HumanosRESUMEN
The virus/host interaction is a complex interplay between pro- and anti-viral factors that ultimately determines the spread or halt of virus infections in tissues. This interplay develops over multiple rounds of infection. The purpose of this study was to determine how cellular-level processes combine to impact the spatial spread of infection. We measured the kinetics of virus replication (VSV), antiviral paracrine signal upregulation and secretion, spatial spread of virus and paracrine antiviral signaling, and inhibition of virus production in antiviral-exposed A549 human lung epithelial cells. We found that initially infected cells released antiviral signals 4-to-7h following production of virus. However, the subsequent rapid dissemination of signal and fast induction of a robust and persistent antiviral state ultimately led to a suppression of infection spread. This work shows how cellular responses to infection and activation of antiviral responses can integrate to ultimately control infection spread across host cell populations.
Asunto(s)
Puntos de Control del Ciclo Celular , Interacciones Huésped-Patógeno , Comunicación Paracrina , Fenómenos Fisiológicos de los Virus , Replicación Viral , Células A549 , Antivirales/metabolismo , Células Cultivadas , Citocinas/metabolismo , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunidad InnataRESUMEN
In addition to host genetic and environmental factors, variations in immune responses to vaccination are influenced by demographic variables, such as race and sex. The influence of genetic race and sex on measles vaccine responses is not well understood, yet important for the development of much-needed improved measles vaccines with lower failure rates. We assessed associations between genetically defined race and sex with measles humoral and cellular immunity after measles vaccination in three independent and geographically distinct cohorts totaling 2872 healthy racially diverse children, older adolescents, and young adults. We found no associations between biological sex and either humoral or cellular immunity to measles vaccine, and no correlation between humoral and cellular immunity in these study subjects. Genetically defined race was, however, significantly associated with both measles vaccine-induced humoral and cellular immune responses, with subjects genetically classified as having African-American ancestry demonstrating significantly higher antibody and cell-mediated immune responses relative to subjects of Caucasian ancestry. This information may be useful in designing novel measles vaccines that are optimally effective across human genetic backgrounds.
Asunto(s)
Inmunidad Celular/genética , Inmunidad Humoral/genética , Vacuna Antisarampión/uso terapéutico , Sarampión/prevención & control , Adolescente , Adulto , Negro o Afroamericano , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Niño , Femenino , Humanos , Masculino , Sarampión/etnología , Factores Sexuales , Población Blanca , Adulto JovenRESUMEN
The spread of acute respiratory viral infections is controlled by type I and III interferon (IFN) signaling. While the mechanisms of type I IFN signaling have been studied in detail, features that distinguish type III IFN signaling remain poorly understood. Type III IFNs play an essential role in limiting infections of intestinal and respiratory epithelial surfaces; however, type III IFNs have been shown to activate similar genes to type I IFNs, raising the question of how these IFNs differ and their signals interact. We measured the kinetics of type I and III IFN activation, functional stability, and downstream antiviral responses on A549 human lung epithelial cells. Similar kinetics were found for transcriptional upregulation and secretion of type I and III IFNs in response to infection by an RNA virus, peaking at 12 h postinfection, and both protein types had similar stabilities with functional half-lives extending beyond 2 days. Both IFNs activated potent cellular antiviral responses; however, responses to type III IFNs were delayed by 2-6 h relative to type I IFN responses. Combined treatments with type I and III IFNs produced enhanced antiviral effects, and quantitative analysis of these data with a Bliss interaction model provides evidence for independence of type I and III IFN downstream signaling pathways. This novel synergistic interaction has therapeutic implications for treatment of respiratory virus infections.
Asunto(s)
Antivirales/farmacología , Interferón Tipo I/farmacología , Virus ARN/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Virosis/tratamiento farmacológico , Línea Celular Tumoral , Células Epiteliales/efectos de los fármacos , Humanos , Cinética , Transcripción Genética/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Gene order is often highly conserved within taxonomic groups, such that organisms with rearranged genomes tend to be less fit than wild type gene orders, and suggesting natural selection favors genome architectures that maximize fitness. But it is unclear whether rearranged genomes hinder adaptability: capacity to evolutionarily improve in a new environment. Negative-sense non-segmented RNA viruses (order Mononegavirales) have specific genome architecture: 3' UTR - core protein genes - envelope protein genes - RNA-dependent RNA-polymerase gene - 5' UTR. To test how genome architecture affects RNA virus evolution, we examined vesicular stomatitis virus (VSV) variants with the nucleocapsid (N) gene moved sequentially downstream in the genome. Because RNA polymerase stuttering in VSV replication causes greater mRNA production in upstream genes, N gene translocation toward the 5' end leads to stepwise decreases in N transcription, viral replication and progeny production, and also impacts the activation of type 1 interferon mediated antiviral responses. We evolved VSV gene-order variants in two prostate cancer cell lines: LNCap cells deficient in innate immune response to viral infection, and PC-3 cells that mount an IFN stimulated anti-viral response to infection. We observed that gene order affects phenotypic adaptability (reproductive growth; viral suppression of immune function), especially on PC-3 cells that strongly select against virus infection. Overall, populations derived from the least-fit ancestor (most-altered N position architecture) adapted fastest, consistent with theory predicting populations with low initial fitness should improve faster in evolutionary time. Also, we observed correlated responses to selection, where viruses improved across both hosts, rather than suffer fitness trade-offs on unselected hosts. Whole genomics revealed multiple mutations in evolved variants, some of which were conserved across selective environments for a given gene order.