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Respiratory syncytial virus (RSV) remains the primary cause of lower respiratory tract infections, particularly in infants and the elderly. In this study, we employed reverse genetics to generate a chimeric influenza virus expressing neuraminidase-3F protein conjugate with three repeats of the RSV F protein protective epitope inserted into the NA gene of A/California/7/2009 ca (CA/AA ca), resulting in rFlu/RSV/NA-3F (hereafter, rFRN3). The expression of NA-3F protein was confirmed by Western blotting. The morphology and temperature-sensitive phenotype of rFRN3 were similar to CA/AA ca. Its immunogenicity and protective efficiency were evaluated in BALB/c mice and cotton rats. Intranasal administration of rFRN3 elicited robust humoral, cellular, and to some extent, mucosal immune responses. Compared to controls, rFRN3 protected animals from RSV infection, attenuated lung injury, and reduced viral titers in the nose and lungs post-RSV challenge. These results demonstrate that rFRN3 can trigger RSV-specific immune responses and thus exhibits potent protective efficacy. The "dual vaccine" approach of a cold-adapted influenza vector RSV vaccine will improve the prophylaxis of influenza and RSV infection. rFRN3 thus warrants further clinical investigations as a candidate RSV vaccine.
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Anticuerpos Antivirales , Vectores Genéticos , Ratones Endogámicos BALB C , Infecciones por Virus Sincitial Respiratorio , Vacunas contra Virus Sincitial Respiratorio , Sigmodontinae , Animales , Infecciones por Virus Sincitial Respiratorio/prevención & control , Infecciones por Virus Sincitial Respiratorio/inmunología , Vacunas contra Virus Sincitial Respiratorio/inmunología , Vacunas contra Virus Sincitial Respiratorio/administración & dosificación , Vacunas contra Virus Sincitial Respiratorio/genética , Femenino , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Ratones , Vectores Genéticos/genética , Vectores Genéticos/inmunología , Pulmón/virología , Pulmón/inmunología , Pulmón/patología , Administración Intranasal , Frío , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Modelos Animales de Enfermedad , Proteínas Virales de Fusión/inmunología , Proteínas Virales de Fusión/genética , Inmunidad Mucosa , Eficacia de las Vacunas , Carga ViralRESUMEN
Respiratory syncytial virus is the major cause of respiratory viral infections, particularly in infants, immunocompromised populations, and the elderly (over 65 years old), the prevention of RSV infection has become a priority. In this study, we generated a chimeric influenza virus, termed LAIV/RSV/HA-3F, using reverse genetics technology which contained three repeats of the RSV fusion protein neutralizing epitope site II to the N terminal in the background of the hemagglutinin (HA) gene of cold adapted influenza vaccine A/California/7/2009 ca. LAIV/RSV/HA-3F exhibited cold-adapted (ca) and attenuated (att) phenotype. BALB/c mice immunized intranasally with LAIV/RSV/HA-3F showed robust immunogenicity, inducing viral-specific antibody responses against both influenza and RSV, eliciting RSV-specific humoral, cellular and mucosal immune responses. LAIV/RSV/HA-3F also conferred protection as indicated by reduced viral titers and improved lung histopathological alterations against live RSV virus challenge. Mechanismly, single-cell RNA sequencing (scRNA-seq) and single-cell T cell antigen receptor (TCR) sequencing were employed to characterize the immune responses triggered by chimeric RSV vaccine, displaying that LAIV/RSV/HA-3F provided protection mainly via interferon-γ (IFN-γ). Moreover, we found that LAIV/RSV/HA-3F significantly inhibited viral replication in the challenged lung and protected against subsequent RSV challenge in cotton rats without causing lung disease. Taken together, our findings demonstrated that LAIV/RSV/HA-3F has potential as a promising bivalent vaccine with dual purpose candidate for the prevention of influenza and RSV, and preclinical and clinical studies warrant further investigations.
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Anticuerpos Antivirales , Epítopos , Vacunas contra la Influenza , Ratones Endogámicos BALB C , Infecciones por Virus Sincitial Respiratorio , Sigmodontinae , Proteínas Virales de Fusión , Animales , Infecciones por Virus Sincitial Respiratorio/prevención & control , Infecciones por Virus Sincitial Respiratorio/inmunología , Vacunas contra la Influenza/inmunología , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/genética , Ratones , Proteínas Virales de Fusión/inmunología , Proteínas Virales de Fusión/genética , Epítopos/inmunología , Epítopos/genética , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre , Femenino , Virus Sincitiales Respiratorios/inmunología , Virus Sincitiales Respiratorios/genética , Vacunas contra Virus Sincitial Respiratorio/inmunología , Vacunas contra Virus Sincitial Respiratorio/administración & dosificación , Pulmón/virología , Pulmón/inmunología , Pulmón/patología , Humanos , Frío , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/sangre , Virus Sincitial Respiratorio Humano/inmunología , Virus Sincitial Respiratorio Humano/genética , Infecciones por Orthomyxoviridae/prevención & control , Infecciones por Orthomyxoviridae/inmunologíaRESUMEN
Human metapneumovirus (HMPV) is a leading cause of acute respiratory tract infections in infants and children. Currently, no approved HMPV vaccine is available. We developed a novel recombinant influenza virus, which carried partial HMPV F protein (HMPV-F) epitopes, utilizing reverse genetics. The novel single-stranded RNA virus, termed rFLU-HMPV/F-NA, was synthesized in the neuraminidase (NA) fragment of influenza virus A/PuertoRico/8/34 (PR8). The morphological characteristics of rFLU-HMPV/F-NA were consistent with the wild-type flu virus. The virus could passage in specific pathogen-free (SPF) chicken embryos for at least five consecutive generations with haemagglutinin (HA) titres of 28-9 or 8-9LogTCID50/mL. BALB/c mice were intranasally immunized at 21-day intervals with 104 TCID50 (low-dose group) or 106 TCID50 (high-dose group) rFLU-HMPV/F-NA, and PBS or PR8 vaccine was used for the control group. rFLU-HMPV/F-NA induced robust humoral, mucosal, and cellular immune responses in vivo in a dose-dependent manner. More importantly, wt clinical HMPV isolate challenge studies showed that rFLU-HMPV/F-NA provided significant immune protection against HMPV infection compared to the PBS or PR8 vaccine control group, as shown by improved histopathological changes and reduced viral titres in the lungs of immunized mice post-challenge. These findings demonstrate that rFLU-HMPV/F-NA has potential as a promising HMPV candidate vaccine and warrants further investigation into its control of HMPV infection.
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Human inactivated rabies virus (RABV) vaccines have been widely used worldwide over 30 years. The mechanisms of humoral immunity elicited by previously reported rabies candidate vaccines have been fully investigated, but little is known about the cellular immunity profiles. Herein, the recombinant RABV rLBNSE-IL-33 overexpressing the mouse interleukin-33 (IL-33) proliferated well in Neuro-2a cells and had no effects with the parent virus on growth kinetic in vitro and viral pathogenicity in mice. The rLBNSE-IL-33 experienced more antigen presentations by MHC-II on DCs and activated more CD4+ T cells which helped recruit more CD19+CD40+ B cells in blood and promote rapid and robust IgG1 antibodies responses at initial infection stage compared with the parent rLBNSE strain. Simultaneously, the rLBNSE-IL-33 were also presented by MHC-I to CD8+ T cells which contributed to produce high levels of IgG2a. The rLBNSE-IL-33 elicited significantly high levels of RABV-specific IFN-γ secreting memory CD4+ T cells, more RABV-specific IL-4 and IFN-γ secreting memory CD8+ T cells in spleens at early infection stage in mice. Altogether, overexpression of IL-33 in rLBNSE-IL-33 enhanced early antigen presentation, markedly promote CD4+, memory CD4+ and CD8+ T cells-mediated responses and provided a 100 % protection from lethal RABV challenge in mice. These findings provided an alternative novel therapy and vaccine strategy in future.
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Vacunas Antirrábicas , Virus de la Rabia , Rabia , Humanos , Animales , Ratones , Rabia/prevención & control , Interleucina-33 , Presentación de Antígeno , Linfocitos T CD8-positivos , Anticuerpos Antivirales , Antígenos Virales , Inmunidad CelularRESUMEN
Electrochemical Immunosensing (EI) combines electrochemical analysis and immunology principles and is characterized by its simplicity, rapid detection, high sensitivity, and specificity. EI has become an important approach in various fields, such as clinical diagnosis, disease prevention and treatment, environmental monitoring, and food safety. However, EI multi-component detection still faces two major bottlenecks: first, the lack of cost-effective and portable detection platforms; second, the difficulty in eliminating batch differences and accurately decoupling signals from multiple analytes. With the gradual maturation of biochip technology, high-throughput analysis and portable detection utilizing the advantages of miniaturized chips, high sensitivity, and low cost have become possible. Meanwhile, Artificial Intelligence (AI) enables accurate decoupling of signals and enhances the sensitivity and specificity of multi-component detection. We believe that by evaluating and analyzing the characteristics, benefits, and linkages of EI, biochip, and AI technologies, we may considerably accelerate the development of EI multi-component detection. Therefore, we propose three specific prospects: first, AI can enhance and optimize the performance of the EI biochips, addressing the issue of multi-component detection for portable platforms. Second, the AI-enhanced EI biochips can be widely applied in home care, medical healthcare, and other areas. Third, the cross-fusion and innovation of EI, biochip, and AI technologies will effectively solve key bottlenecks in biochip detection, promoting interdisciplinary development. However, challenges may arise from AI algorithms that are difficult to explain and limited data access. Nevertheless, we believe that with technological advances and further research, there will be more methods and technologies to overcome these challenges.
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Objective: Influenza B virus (IBV) is highly contagious, spreads rapidly, and causes seasonal epidemic respiratory disease in the human population, especially in immunocompromised people and young children. Clinical manifestations in this high-risk population are often more severe than in immunocompetent hosts and sometimes atypical. Therefore, rapid, and accurate detection of IBV is important. Methods: An amplified luminescent proximity homogeneous assay linked immunosorbent assay (AlphaLISA) was developed for detection of IBV by optimizing the ratio of IBV antibody-labeled receptor beads, streptavidin-conjugated donor beads and biotinylated IBV antibody, as well as the optimal temperature and time conditions for incubation. Assay sensitivity, specificity and reproducibility were evaluated. A total of 228 throat swab samples and inactivated influenza B virus were tested by AlphaLISA and lateral flow colloidal gold-based immunoassay (LFIA). Results: AlphaLISA produced the best results for detection of inactivated influenza B virus when IBV antibody-labeled acceptor beads were 50 µg/ mL, streptavidin-conjugated donor beads were 40 µg/mL, and biotinylated IBV antibody was 0.5 µg/mL at 37°C for 15-10 min. Under these conditions, AlphaLISA had a limit of detection of 0.24 ng/mL for the detection of influenza B nucleoprotein, did not cross react with other common respiratory viruses, and showed good reproducibility with inter-assay coefficient of variation (CV) and intra-assay CV < 5%. The results of 228 clinical throat swab samples showed good agreement between AlphaLISA and LFIA (Kappa = 0.982), and AlphaLISA showed better sensitivity than LFIA for detecting inactivated influenza B virus. Conclusion: AlphaLISA showed higher sensitivity and throughput in the detection of IBV and can be used for IBV diagnosis and epidemic control.
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Vaccination is a cost-effective medical intervention. Inactivated whole virusor large protein fragments-based severe acute respiratory syndrome coronavirus (SARS-CoV-2) vaccines have high unnecessary antigenic load to induce allergenicity and/orreactogenicity, which can be avoided by peptide vaccines of short peptide fragments that may induce highly targeted immune response. However, epitope identification and peptide delivery remain the major obstacles in developing peptide vaccines. Here, a multi-source data integrated linear B-cell epitope screening strategy is presented and a linear B-cell epitope enriched hotspot region is identified in Spike protein, from which a monomeric peptide vaccine (Epitope25) is developed and applied to subcutaneously immunize wildtype BALB/c mice. Indirect ELISA assay reveals specific and dose-dependent binding between Epitope25 and serum IgG antibodies from immunized mice. The neutralizing activity of sera from vaccinated mice is validated by pseudo and live SARS-CoV-2 wild-type strain neutralization assays. Then a dissolvable microneedle array (DMNA) is developed to pain-freely deliver Epitope25. Compared with intramuscular injection, DMNA and subcutaneous injection elicit neutralizing activities against SARS-CoV-2 wild-type strain as demonstrated by live SARS-CoV-2 virus neutralization assay. No obvious damages are found in major organs of immunized mice. This study may lay the foundation for developing linear B-cell epitope-based vaccines against SARS-CoV-2.
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COVID-19 , Vacunas Virales , Humanos , Animales , Ratones , SARS-CoV-2 , Glicoproteínas de Membrana , Proteínas del Envoltorio Viral , Epítopos de Linfocito B , Vacunas contra la COVID-19 , Glicoproteína de la Espiga del Coronavirus , Anticuerpos Antivirales , Pruebas de Neutralización , COVID-19/prevención & control , Vacunas de SubunidadRESUMEN
Huoxiang Zhengqi Oral Liquid (HZOL) is a classic Chinese patent medicine used in China for more than 1,000 years in treating gastrointestinal and respiratory diseases. Clinically applied HZOL in early respiratory disease stages can reduce the proportion of lung infection patients that progress to severe acute lung injury (ALI). However, few pharmacological studies evaluated its level of protection against ALI. We explored mechanisms of HZOL against ALI by employing network pharmacology, molecular docking, and rat experiments. Firstly, network pharmacology prediction and published biological evaluation of active ingredients of HZOL suggested that HZOL exerted the protective effect in treating ALI mainly in the areas of regulation of cell adhesion, immune response, and inflammatory response and closely related to the NF-κB pathway. Secondly, molecular docking results demonstrated that imperatorin and isoimperatorin combined well with targets in the NF-κB pathway. Finally, ALI rats induced by lipopolysaccharides (LPS) were used to validate prediction after pretreatment with HZOL for 2 weeks. Results confirmed that lung and colon injury occurred in ALI rats. Furthermore, HZOL exerts anti-inflammatory effects on LPS-induced ALI and gut injury by repairing lung and colon pathology, reducing and alleviating pulmonary edema, inhibiting abnormal enhancement of thymus and spleen index, modulating hematologic indices, and increasing levels of total short-chain fatty acids (SCFAs) in the cecum. Additionally, abnormal accumulation of inflammatory cytokines IL-6, IL-1ß, TNF-α, and IFN-γ in serum and bronchoalveolar lavage fluid was significantly reduced after pretreating with HZOL. Furthermore, HZOL downregulated the expression of TLR4, CD14, and MyD88 and phosphorylation of NF-κB p65 in lung tissue. Altogether, HZOL was found to exert an anti-inflammatory effect regulation by increasing levels of SCFAs, inhibiting the accumulation of inflammatory cytokines, and attenuating the activation of the TLR4/NF-κB p65 pathway. Our study provided experimental evidences for the application of HZOL in preventing and treating ALI.
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Lesión Pulmonar Aguda , FN-kappa B , Animales , Ratas , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/tratamiento farmacológico , Lesión Pulmonar Aguda/metabolismo , Antiinflamatorios/farmacología , Citocinas/metabolismo , Lipopolisacáridos/farmacología , Pulmón/patología , Simulación del Acoplamiento Molecular , FN-kappa B/metabolismo , Transducción de Señal , Receptor Toll-Like 4/metabolismo , Ácidos Grasos Volátiles/metabolismoRESUMEN
Respiratory viral infections, such as coronavirus disease of 2019 (COVID-19) and influenza, cause significant morbidity and mortality and have become a worldwide public health concern with tremendous economic and societal burdens. Vaccination is a major strategy for preventing infections. However, some new vaccines have an unmet need for impairing responses in certain individuals, especially COVID-19 vaccines, despite ongoing vaccine and adjuvant research. Here, we evaluated the effectiveness of Astragalus polysaccharide (APS), a bioactive polysaccharide extracted from the traditional Chinese herb Astragalus membranaceus as an immune adjuvant to regulate the efficacy of influenza split vaccine (ISV) and recombinant severe acute respiratory syndrome (SARS)-Cov-2 vaccine in mice. Our data indicated that APS as an adjuvant can facilitate the induction of high levels of hemagglutination inhibition (HAI) titer and specific antibody immunoglobulin G (IgG) and confer protection against the lethal challenge of influenza A viruses, including increased survival and amelioration of weight loss in mice immunized with the ISV. RNA sequencing (RNA-seq) analysis revealed that the NF-κB and Fc gamma R-mediated phagocytosis signaling pathways are essential for the immune response of mice immunized with the recombinant SARS-Cov-2 vaccine (RSV). Another important finding was that bidirectional immunomodulation of APS on cellular and humoral immunity was observed, and APS-adjuvant-induced antibodies persisted at a high level for at least 20 weeks. These findings suggest that APS is a potent adjuvant for influenza and COVID-19 vaccines, and has the advantages of bidirectional immunoregulation and persistent immunity.
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COVID-19 , Vacunas contra la Influenza , Gripe Humana , Animales , Ratones , Humanos , Vacunas contra la COVID-19 , Anticuerpos Antivirales , COVID-19/prevención & control , SARS-CoV-2 , Adyuvantes Inmunológicos/farmacología , Adyuvantes Farmacéuticos , Inmunidad Humoral , Polisacáridos/farmacologíaRESUMEN
(1) Background: With the resurgence of brucellosis epidemics in China in recent years, the chances of a brucella coinfection with other common respiratory pathogens, such as the influenza virus, have increased dramatically. However, little is known about the pathogenicity or the mechanisms of brucella and influenza coinfections. (2) Methods: To clarify the interventions in the early stages of lung damage due to brucella and influenza coinfections, we evaluated the effect of the coinfection on disease progression and mortality using a coinfection model in WT mice and NLRP6-/- mice, and we verified the function of NLRP6 in infection and proinflammation. (3) Results: The coinfection induced significant respiratory symptoms, weight loss, and a high mortality rate in WT mice. Influenza in the coinfection group significantly increased brucella proliferation in a synergistic manner. Meanwhile, a histological examination showed severe lung tissue destruction and excessive inflammatory responses in coinfected WT animals, and the expression of NLRP6 and IL-18 was dramatically increased in the lung tissues. Furthermore, NLRP6 deletion attenuated lung injuries and inflammation, a reduced bacterial load, and decreased IL-18 protein expression. (4) Conclusions: Our findings indicated that NLRP6 plays a critical role and might be a promising potential therapeutic target for brucella-influenza coinfections.
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REV-ERBα is a member of the nuclear receptor superfamily of transcription factors that aids in the regulation of many diseases. However, the prospect of using REV-ERBα for anti-influenza virus treatment remains poorly described, and there is an urgent need to develop effective anti-influenza agents due to the emergence of drug-resistant influenza viruses. In this study, eight SR9009 analogues were designed, synthesized, and evaluated for their biological activities against multiple influenza virus strains (H1N1, H3N2, adamantane- and oseltamivir-resistant H1N1 and influenza B virus), using ribavirin as the positive control. SR9009 and its analogues showed low micromolar or submicromolar EC50 values and exhibited modestly improved antiviral potency compared to that of ribavirin. In particular, compound 5a possessed the most potent inhibitory activity (EC50 = 0.471, 0.644, 1.644, 0.712 and 0.661 µM for A/PR/8/34, A/WSN/33, A/Wisconsin/67/2005, B/Yamagata/16/88 and Hebei/SWL1/2006, respectively). Cotransfection assays showed that all synthesized derivatives efficaciously suppressed transcription driven by the Bmal1 promoter. Mechanistic study results indicated that 5a efficiently inhibited IAV replication and interfered with the ealry stage of influenza virus life cycle. In addition, we found that 5a upregulated the key antiviral interferon-stimulated genes MxA, OAS2 and CH25H. Further in-depth transcriptome analysis revealed a series of upregulated genes that may contribute to the antiviral activities of 5a. These findings may provide an important direction for the development of new host-targeted broad-spectrum antiviral agents.
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Adamantano , Subtipo H1N1 del Virus de la Influenza A , Factores de Transcripción ARNTL/farmacología , Adamantano/farmacología , Antivirales/farmacología , Subtipo H3N2 del Virus de la Influenza A , Interferones/farmacología , Oseltamivir/farmacología , Pirrolidinas , Ribavirina/farmacología , TiofenosRESUMEN
Although tremendous effort has been exerted to elucidate the pathogenesis of severe COVID-19 cases, the detailed mechanism of moderate cases, which accounts for 90% of all patients, remains unclear yet, partly limited by lacking the biopsy tissues. Here, we established the COVID-19 infection model in cynomolgus macaques (CMs), monitored the clinical and pathological features, and analyzed underlying pathogenic mechanisms at early infection stage by performing proteomic and metabolomic profiling of lung tissues and sera samples from COVID-19 CMs models. Our data demonstrated that innate immune response, neutrophile and platelet activation were mainly dysregulated in COVID-19 CMs. The symptom of neutrophilia, lymphopenia and massive "cytokines storm", main features of severe COVID-19 patients, were greatly weakened in most of the challenged CMs, which are more semblable as moderate patients. Thus, COVID-19 model in CMs is rational to understand the pathogenesis of moderate COVID-19 and may be a candidate model to assess the safety and efficacy of therapeutics and vaccines against SARS-CoV-2 infection.
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COVID-19 , SARS-CoV-2 , Animales , Vacunas contra la COVID-19 , Humanos , Macaca fascicularis , ProteómicaAsunto(s)
Vacunas contra la COVID-19/inmunología , COVID-19/prevención & control , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Enzima Convertidora de Angiotensina 2/química , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Células CHO , COVID-19/inmunología , Vacunas contra la COVID-19/genética , Cricetulus , Fragmentos Fc de Inmunoglobulinas/genética , Macaca fascicularis , Ratones , Ratones Endogámicos BALB C , Simulación del Acoplamiento Molecular , Dominios Proteicos/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
In face of the everlasting battle toward COVID-19 and the rapid evolution of SARS-CoV-2, no specific and effective drugs for treating this disease have been reported until today. Angiotensin-converting enzyme 2 (ACE2), a receptor of SARS-CoV-2, mediates the virus infection by binding to spike protein. Although ACE2 is expressed in the lung, kidney, and intestine, its expressing levels are rather low, especially in the lung. Considering the great infectivity of COVID-19, we speculate that SARS-CoV-2 may depend on other routes to facilitate its infection. Here, we first discover an interaction between host cell receptor CD147 and SARS-CoV-2 spike protein. The loss of CD147 or blocking CD147 in Vero E6 and BEAS-2B cell lines by anti-CD147 antibody, Meplazumab, inhibits SARS-CoV-2 amplification. Expression of human CD147 allows virus entry into non-susceptible BHK-21 cells, which can be neutralized by CD147 extracellular fragment. Viral loads are detectable in the lungs of human CD147 (hCD147) mice infected with SARS-CoV-2, but not in those of virus-infected wild type mice. Interestingly, virions are observed in lymphocytes of lung tissue from a COVID-19 patient. Human T cells with a property of ACE2 natural deficiency can be infected with SARS-CoV-2 pseudovirus in a dose-dependent manner, which is specifically inhibited by Meplazumab. Furthermore, CD147 mediates virus entering host cells by endocytosis. Together, our study reveals a novel virus entry route, CD147-spike protein, which provides an important target for developing specific and effective drug against COVID-19.
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Basigina/genética , COVID-19/genética , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Animales , Basigina/inmunología , COVID-19/inmunología , COVID-19/patología , COVID-19/virología , Interacciones Huésped-Patógeno/inmunología , Humanos , Pulmón/inmunología , Pulmón/patología , Pulmón/virología , Ratones , Pandemias , Unión Proteica/inmunología , Dominios Proteicos/genética , Dominios Proteicos/inmunología , SARS-CoV-2/patogenicidad , Glicoproteína de la Espiga del Coronavirus/genética , Internalización del VirusRESUMEN
The ongoing coronavirus disease 2019 (COVID-19) pandemic has prioritized the development of small-animal models for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We adapted a clinical isolate of SARS-CoV-2 by serial passaging in the respiratory tract of aged BALB/c mice. The resulting mouse-adapted strain at passage 6 (called MASCp6) showed increased infectivity in mouse lung and led to interstitial pneumonia and inflammatory responses in both young and aged mice after intranasal inoculation. Deep sequencing revealed a panel of adaptive mutations potentially associated with the increased virulence. In particular, the N501Y mutation is located at the receptor binding domain (RBD) of the spike protein. The protective efficacy of a recombinant RBD vaccine candidate was validated by using this model. Thus, this mouse-adapted strain and associated challenge model should be of value in evaluating vaccines and antivirals against SARS-CoV-2.
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Betacoronavirus/inmunología , Infecciones por Coronavirus/prevención & control , Modelos Animales de Enfermedad , Ratones , Pandemias/prevención & control , Neumonía Viral/prevención & control , Vacunas Virales/inmunología , Administración Intranasal , Enzima Convertidora de Angiotensina 2 , Animales , Betacoronavirus/genética , Betacoronavirus/patogenicidad , COVID-19 , Vacunas contra la COVID-19 , Infecciones por Coronavirus/inmunología , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inmunogenicidad Vacunal , Pulmón/virología , Enfermedades Pulmonares Intersticiales/virología , Ratones Endogámicos BALB C , Ratones Transgénicos , Mutación , Peptidil-Dipeptidasa A/genética , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/genética , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunología , Vacunas Virales/administración & dosificación , Virulencia/genéticaRESUMEN
Each influenza pandemic was caused at least partly by avian- and/or swine-origin influenza A viruses (IAVs). The timing of and the potential IAVs involved in the next pandemic are currently unpredictable. We aim to build machine learning (ML) models to predict human-adaptive IAV nucleotide composition. A total of 217,549 IAV full-length coding sequences of the PB2 (polymerase basic protein-2), PB1, PA (polymerase acidic protein), HA (hemagglutinin), NP (nucleoprotein), and NA (neuraminidase) segments were decomposed for their codon position-based mononucleotides (12 nts) and dinucleotides (48 dnts). A total of 68,742 human sequences and 68,739 avian sequences (1:1) were resampled to characterize the human adaptation-associated (d)nts with principal component analysis (PCA) and other ML models. Then, the human adaptation of IAV sequences was predicted based on the characterized (d)nts. Respectively, 9, 12, 11, 13, 10 and 9 human-adaptive (d)nts were optimized for the six segments. PCA and hierarchical clustering analysis revealed the linear separability of the optimized (d)nts between the human-adaptive and avian-adaptive sets. The results of the confusion matrix and the area under the receiver operating characteristic curve indicated a high performance of the ML models to predict human adaptation of IAVs. Our model performed well in predicting the human adaptation of the swine/avian IAVs before and after the 2009 H1N1 pandemic. In conclusion, we identified the human adaptation-associated genomic composition of IAV segments. ML models for IAV human adaptation prediction using large IAV genomic data sets can facilitate the identification of key viral factors that affect virus transmission/pathogenicity. Most importantly, it allows the prediction of pandemic influenza.
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Adaptación Biológica/genética , Virus de la Influenza A/genética , Aprendizaje Automático , Proteínas Virales/genética , Interacciones Huésped-Patógeno , HumanosRESUMEN
Staphylococcal enterotoxin B (SEB) produced by the Staphylococcus aureus bacteriumis most commonly associated with food poisoning and is known to also cause toxic shock syndrome. Currently, no approved vaccine or specific drug is available to treat SEB intoxication. In this study, we fabricated dissolving microneedles (MNs) loaded with recombinant SEB (rSEB) protein, and evaluated its characteristics, including dissolution profile, protein particle size, insertion depth, antigen retention time in vivo, and skin irritation. Our results showed that rSEB protein-loaded dissolving MNs made of chondroitin sulfate (2%) and trehalose (0.8%) could easily penetrate into the mouse skin within 5â¯min. The rSEB particle size was unchanged before and after MN fabrication. The skin penetration depth of the MNs was 260⯵m. Moreover, the MNs also significantly extended the antigen retention time in vivo. rSEB protein-loaded dissolving MNs also triggered slight erythema at the beginning of administration, but this erythema disappeared within a few hours. More importantly, we investigated the immunogenicity and protective efficacy of rSEB protein-loaded dissolving MNs. Challenge studies in mice revealed that mice in full-dose MN group had a high level of SEB specific antibody response thatprovided100% protection against a lethal SEB toxin challenge. However, there was only 60% protection observed in mice that were in the half-dose MN (dose sparing) group. We also determined the pathological alterations in the tissues of the immunized mice. Taken together, these dissolving MNs may present a promising transcutaneous immunization strategy for treating SEB intoxication.
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Enterotoxinas/administración & dosificación , Inmunización/métodos , Microinyecciones/instrumentación , Agujas , Infecciones Estafilocócicas/prevención & control , Administración Cutánea , Animales , Anticuerpos Antibacterianos/sangre , Enterotoxinas/genética , Enterotoxinas/inmunología , Femenino , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/inmunología , Piel/efectos de los fármacos , Piel/patología , Infecciones Estafilocócicas/inmunología , Staphylococcus aureus , PorcinosRESUMEN
BACKGROUND: Hepatitis B virus (HBV) is considered highly prevalent in West Africa. However, major gaps in surveillance exist in Sierra Leone. Although healthcare workers (HCWs) are at high risk for HBV infection, little is known about the prevalence and knowledge of hepatitis B among HCWs in Sierra Leone. METHODS: A cross-sectional study of all HCWs at the No. 34 Military Hospital located in Freetown, Sierra Leone, was conducted from March 20 to April 10, 2017. Whole blood was collected and screened for HBV markers using a one-step rapid immunochromatographic test with positive samples tested for HBV DNA. Additionally, questionnaires assessing self-reported knowledge of HBV infections were administered to all participants. Data were processed and analyzed using SPSS (version 17.0) software. RESULTS: A total of 211 HCWs were included in this study with a median age of 39.0 years (range: 18-59). Of the participating HCWs, 172 (81.5%) participants were susceptible (all markers negative), 21(10.0%) were current HBV (HBsAg positive) and nine (4.3%) were considered immune because of past infection (HBsAg negative and anti-HBc positive; anti-HBs positive). Additionally, nine (4.3%) participants displayed immunity to the virus as a result of prior hepatitis B vaccination (only anti-HBs positive). Of the 21 HCWs with positive HBsAg, 13 (61.9%) had detectable HBV DNA. There was a significantly lower risk for current HBV infection among HCWs older than 39 years (OR 0.337, p = 0.046). In addition, only 14 (6.6%), 73 (34.6%) and 82 (38.9%) participants in this survey had adequate knowledge about the clinical outcome, routes of transmission, and correct preventive measures of HBV infection, respectively. CONCLUSIONS: HCWs in Sierra Leone lacked adequate knowledge of the hepatitis B virus. Additionally, the low coverage rate of hepatitis B vaccination among HCWs fails to meet WHO recommendations, leaving many of the sampled HCWs susceptible to infection. This study reaffirms the need for more intensive training for HCWs in addition to strengthening vaccination programmes to protect HCWs against HBV in Sierra Leone.
Asunto(s)
Conocimientos, Actitudes y Práctica en Salud , Personal de Salud/estadística & datos numéricos , Hepatitis B/epidemiología , Adolescente , Adulto , Estudios Transversales , Femenino , Hepatitis B/prevención & control , Antígenos de Superficie de la Hepatitis B/sangre , Vacunas contra Hepatitis B , Virus de la Hepatitis B/patogenicidad , Humanos , Masculino , Persona de Mediana Edad , Prevalencia , Sierra Leona/epidemiología , Encuestas y Cuestionarios , Vacunación/estadística & datos numéricosRESUMEN
Human adenoviruses (HAdVs) are prevalent in pediatric and adult patients with severe acute respiratory disease (ARD). To date, there have been no widely used HAdV vaccines available. In this report, we developed a cold-adapted attenuated influenza virus, termed rg HAdV-Flu ca, carrying epitopes from HAdV hexon protein in the backbone of the ca influenza vaccine neuraminidase (NA) gene using reverse genetics. Rg HAdV-Flu ca virus exhibited a cold-adapted (ca) phenotype, and its morphological characteristics were observed using electron microscopy. Moreover, BALB/c mice were immunized intranasally (i.n.) with 105, 106 or 107 TCID50 rg HAdV-Flu ca. Results showed a specific, robust antibody response against influenza and HAdV in a dose-dependent manner. More importantly, potent humoral, mucosal and cellular immune responses protected against subsequent wild-type HAdV-3 or HAdV-7 challenges, as determined by a significant decrease in viral titers and a noticeable alleviation of histopathological alterations in the lung tissue of challenged mice. These findings demonstrate that rg HAdV-Flu ca warrants attention as a potential vaccine candidate against HAdV infection.
Asunto(s)
Adenovirus Humanos/inmunología , Vacunas contra la Influenza/inmunología , Orthomyxoviridae/inmunología , Infecciones por Adenoviridae/prevención & control , Adenovirus Humanos/genética , Administración Intranasal , Animales , Anticuerpos Antivirales/sangre , Epítopos/genética , Epítopos/inmunología , Inmunidad Celular , Inmunidad Mucosa , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/genética , Vacunas contra la Influenza/aislamiento & purificación , Ratones Endogámicos BALB C , Microscopía Electrónica de Transmisión , Orthomyxoviridae/genética , Infecciones por Orthomyxoviridae/prevención & control , Genética Inversa , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/aislamiento & purificación , Virión/ultraestructura , VirosisRESUMEN
Vaccination is the most effective method of preventing the spread of the influenza virus. However, the traditional intramuscular (IM) immunization causes fear, pain, and cross infection. In contrast, needle-free (NF) immunization is quick and easy for medical personnel and painless and safe for patients. In this study, we assessed the safety and protective efficacy of NF intradermal (ID) immunization with the influenza H7N9 split vaccine (Anhui H7N9/PR8). A preliminary safety evaluation showed that ID immunization with 15 µg of the H7N9 influenza vaccine was not toxic in rats. Moreover, the antigen was metabolized more rapidly after ID than after IM immunization, as determined by in vivo imaging, and ID immunization accelerated the generation of a specific immune response. Additionally, ID immunization with a 20% dose of the H7N9 split vaccine Anhui H7N9/PR8 offered complete protection against lethal challenge by the live H7N9 virus. Taken together, our findings suggest that NF ID immunization with the H7N9 influenza vaccine induces effective protection, has a good safety profile, requires little antigen, and elicits an immune response more rapidly than does IM immunization. This approach may be used to improve the control of influenza H7N9 outbreaks.