RESUMEN

Passive immunization is an effective option for treatment against hand, foot and mouth disease caused by EV71, especially with cross-neutralizing IgG monoclonal antibodies. In this study, an EV71-specific IgG2a antibody designated 5H7 was identified and characterized. 5H7 efficiently neutralizes the major EV71 genogroups (A, B4, C2, C4). The conformational epitope of 5H7 was mapped to the highly conserved amino acid position 74 on VP3 capsid protein using escape mutants. Neutralization with 5H7 is mediated by the inhibition of viral attachment, as revealed by virus-binding and post-attachment assays. In a competitive pull-down assay with SCARB2, 5H7 blocks the receptor-binding site on EV71 for virus neutralization. Passive immunization of chimeric 5H7 protected 100% of two-week-old AG129 mice from lethal challenge with an EV71 B4 strain for both prophylactic and therapeutic treatments. In contrast, 10D3, a previously reported neutralizing antibody that takes effect after virus attachment, could only confer prophylactic protection. These results indicate that efficient interruption of viral attachment is critical for effective therapeutic activity with 5H7. This report documents a novel universal neutralizing IgG antibody for EV71 therapeutics and reveals the underlying mechanism.

Asunto(s)

Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales/uso terapéutico , Enterovirus Humano A/inmunología , Infecciones por Enterovirus/inmunología , Infecciones por Enterovirus/terapia , Animales , Antígenos Virales/química , Antígenos Virales/genética , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Modelos Animales de Enfermedad , Enterovirus Humano A/genética , Enterovirus Humano A/fisiología , Infecciones por Enterovirus/prevención & control , Epítopos/química , Epítopos/genética , Epítopos/inmunología , Humanos , Inmunización Pasiva , Inmunoglobulina G/uso terapéutico , Ratones , Modelos Moleculares , Pruebas de Neutralización , Receptores Virales/inmunología , Acoplamiento Viral

RESUMEN

Enterovirus 71 (EV71) is a causative agent of hand, foot and mouth disease (HFMD). EV71 causes fever, rash, diarrhoea and, in some cases, acute encephalopathy/encephalitis, which can be fatal. No specific treatment is currently available for EV71 infection. Here, we conducted a cDNA library screen and identified acid ß-glucosidase 1 (GBA1; also known as ß-glucocerebrosidase) as an EV71 resistance factor. The anti-EV71 function of GBA1 was verified by gene transduction and knockdown experiments. Cerezyme, a molecular drug used to treat Gaucher's disease and having recombinant human GBA1 as the active ingredient, protected against EV71 infection. The anti-EV71 activity of GBA1 was bimodal: endogenous GBA1 restricted cell surface expression levels of scavenger receptor class B, member 2 (SCARB2), also known as lysosomal integral membrane protein 2 (LIMP-2), and exogenous recombinant GBA1 interfered with EV71 to interact with SCARB2 outside the cell. Thus, our findings suggest that GBA1 may represent a novel molecular target for the treatment of EV71 infection.

Asunto(s)

Antivirales/farmacología , Enterovirus Humano A/inmunología , Enterovirus Humano A/fisiología , Glucosilceramidasa/farmacología , Acoplamiento Viral , Línea Celular , Enfermedad de Boca, Mano y Pie/virología , Humanos , Proteínas de Membrana de los Lisosomas/metabolismo , Receptores Depuradores/metabolismo , Receptores Virales/metabolismo

RESUMEN

Asian seabass is an important food fish in Southeast Asia. Viral nervous necrosis (VNN) disease, triggered by nervous necrosis virus (NNV) infection, has caused mass mortality of Asian seabass larvae, resulting in enormous economic losses in the Asian seabass industry. In order to better understand the complex molecular interaction between Asian seabass and NNV, we investigated the transcriptome profiles of Asian seabass epithelial cells, which play an essential role in immune regulation, after NNV infection. Using the next generation sequencing (NGS) technology, we sequenced mRNA from eight samples (6, 12, 24, 48 h post-inoculation) of mock and NNV-infected Asian seabass epithelial cell line, respectively. Clean reads were de novo assembled into a transcriptome consisting of 89026 transcripts with a N50 of 2617 bp. Furthermore, 251 differentially expressed genes (DEGs) in response to NNV infection were identified. Top DEGs include protein asteroid homolog 1-like (ASTE1), receptor-transporting protein 3 (RTP3), heat shock proteins 30 (HSP30) and 70 (HSP70), Viperin, interferon regulatory factor 3 (IRF3) and other genes related to innate immunity. Our data suggest that abundant and diverse genes corresponding to NNV infection. The results of this study could also offer vital information not only for identification of novel genes involved in Asian seabass-NNV interaction, but also for our understanding of the molecular mechanism of Asian seabass' response to viral infection. In addition, 24807 simple sequence repeats (SSRs) were detected in the assembled transcriptome, providing valuable resources for studying genetic variations and accelerating quantitative trait loci (QTL) mapping for disease resistance in Asian seabass in the future.

Asunto(s)

Lubina , Células Epiteliales/virología , Enfermedades de los Peces/genética , Enfermedades de los Peces/inmunología , Nodaviridae/fisiología , Infecciones por Virus ARN/veterinaria , Transcriptoma , Animales , Línea Celular , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Perfilación de la Expresión Génica/veterinaria , Regulación de la Expresión Génica , Repeticiones de Microsatélite/genética , Infecciones por Virus ARN/genética , Infecciones por Virus ARN/inmunología , ARN Mensajero/genética , ARN Mensajero/metabolismo

RESUMEN

Streptococcus pneumoniae produces pneumolysin toxin as a key virulence factor against host cells. Pneumolysin is a cholesterol-dependent cytolysin (CDC) toxin that forms lytic pores in host membranes and mediates pneumococcal disease pathogenesis by modulating inflammatory responses. Here, we show that pneumolysin, which is released during bacterial lysis, induces DNA double strand breaks (DSBs), as indicated by ataxia telangiectasia mutated (ATM)-mediated H2AX phosphorylation (γH2AX). Pneumolysin-induced γH2AX foci recruit mediator of DNA damage checkpoint 1 (MDC1) and p53 binding protein 1 (53BP1), to sites of DSBs. Importantly, results show that toxin-induced DNA damage precedes cell cycle arrest and causes apoptosis when DNA-dependent protein kinase (DNA-PK)-mediated non-homologous end joining is inhibited. Further, we observe that cells that were undergoing DNA replication harbored DSBs in greater frequency during pneumolysin treatment. This observation raises the possibility that DSBs might be arising as a result of replication fork breakdown. Additionally, neutralizing the oligomerization domain of pneumolysin with monoclonal antibody suppresses DNA damage and also cell cycle arrest, indicating that pneumolysin oligomerization is important for causing DNA damage. Taken together, this study reveals a previously unidentified ability of pneumolysin to induce cytotoxicity via DNA damage, with implications in the pathophysiology of S. pneumoniae infection.

Asunto(s)

Puntos de Control del Ciclo Celular/efectos de los fármacos , Roturas del ADN de Doble Cadena/efectos de los fármacos , Daño del ADN , Estreptolisinas/farmacología , Células A549 , Proteínas Adaptadoras Transductoras de Señales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/farmacología , Western Blotting , Proteínas de Ciclo Celular , Proteína Quinasa Activada por ADN/metabolismo , Histonas/metabolismo , Humanos , Microscopía Confocal , Proteínas Nucleares/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Recombinantes/farmacología , Streptococcus pneumoniae/metabolismo , Estreptolisinas/genética , Transactivadores/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo

RESUMEN

Vaccination is considered to be the most effective and economical strategy against pandemic influenza. Vaccine development for multiple highly pathogenic avian influenza viruses, for example, H5N1, is hindered by antigenic drift, especially in the hemagglutinin (HA) sequence, as well as the antigenic shift. Growing efforts have been made to generate universal pandemic influenza vaccines. As mainly shown in animal trials, cross-clade and heterosubtypic protection by these universal vaccines are generally elicited by either a broad antigen-specific antibody response or influenza-specific CD4+ and CD8+ T-cell responses. Strain selection, HA engineering and broad neutralizing antigen determination are major strategies to achieve universal and specific antibody response, while studies on other factors including vectors, adjuvants and administration routes aim for enhanced T-cell responses against diverse influenza subtypes. Prospectively, cost-effective universal vaccines developed based on these combined technologies are promising solutions for broad protection against influenza.

Asunto(s)

Protección Cruzada , Descubrimiento de Drogas/métodos , Vacunas contra la Influenza/inmunología , Vacunas contra la Influenza/aislamiento & purificación , Gripe Humana/prevención & control , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Modelos Animales de Enfermedad , Humanos , Linfocitos T/inmunología

RESUMEN

Enterovirus 71 (EV71) is a group of viruses that belongs to the Picornaviridae family, which also includes viruses such as polioviruses. EV71, together with coxsackieviruses, is widely known for its association with Hand Foot Mouth Disease (HFMD), which generally affects children age five and below. Besides HFMD, EV71 can also trigger more severe and life-threatening neurological conditions such as encephalitis. Considering the lack of a vaccine and antiviral drug against EV71, together with the increasing spread of these viruses, the development of such drugs and vaccines becomes the top priority in protecting our younger generations. This article, hence, reviews some of the recent progress in the formulations of anti-therapeutics and vaccine generation for EV71, covering (i) inactivated vaccines; (ii) baculovirus-expressed vaccines against EV71; (iii) human intravenous immunoglobulin (IVIg) treatment; and (iv) the use of monoclonal antibody therapy as a prevention and treatment for EV71 infections.

Asunto(s)

Antivirales/aislamiento & purificación , Descubrimiento de Drogas/métodos , Enterovirus Humano A/efectos de los fármacos , Enterovirus Humano A/inmunología , Enfermedad de Boca, Mano y Pie/tratamiento farmacológico , Enfermedad de Boca, Mano y Pie/prevención & control , Vacunas Virales/aislamiento & purificación , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Antivirales/uso terapéutico , Antivirales/farmacología , Descubrimiento de Drogas/tendencias , Humanos , Inmunoglobulinas Intravenosas/uso terapéutico , Vacunas de Productos Inactivados/inmunología , Vacunas de Productos Inactivados/aislamiento & purificación , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/aislamiento & purificación , Vacunas Virales/inmunología

RESUMEN

Influenza outbreaks by highly pathogenic avian influenza viruses, such as H5N1, have caused significant morbidity and mortality in poultry and mammals. Though the risk of community-level spread of highly pathogenic avian influenza (HPAI) remains to be low since the transmission of influenza virus from animals to humans is not common, HPAI may acquire changes in receptor-binding specificity that lead to human to human transmission. Cases of sporadic infection in humans have been increasing every year and the continuous spread has posed a threat to animal and human health. Vaccination remains to be the most effective strategy to prevent and control the spread of influenza viruses. Other than conventional inactivated whole virus vaccine, several vaccine strategies have been adopted in order to universally defend against H5N1s infection, including subunit vaccine, viral vector-based vaccine, chimeric epitope vaccine strains and HA stalk-based vaccine.

Asunto(s)

Subtipo H5N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Gripe Humana/inmunología , Gripe Humana/prevención & control , Contraindicaciones , Humanos , Subtipo H5N1 del Virus de la Influenza A/genética , Gripe Humana/transmisión

RESUMEN

Avian influenza A H7N7/NL/219/03 virus creates a serious pandemic threat to human health because it can transmit directly from domestic poultry to humans and from human to human. Our previous vaccine study reported that mice when immunized intranasally (i.n) with live Bac-HA were protected from lethal H7N7/NL/219/03 challenge, whereas incomplete protection was obtained when administered subcutaneously (s.c) due to the fact that H7N7 is a poor inducer of neutralizing antibodies. Interestingly, our recent vaccine studies reported that mice when vaccinated subcutaneously with Bac-HA (H7N9) was protected against both H7N9 (A/Sh2/2013) and H7N7 virus challenge. HA1 region of both H7N7 and H7N9 viruses are differ at 15 amino acid positions. Among those, we selected three amino acid positions (T143, T198 and I211) in HA1 region of H7N7. These amino acids are located within or near the receptor binding site. Following the selection, we substituted the amino acid at these three positions with amino acids found on H7N9HA wild-type. In this study, we evaluate the impact of amino acid substitutions in the H7N7 HA-protein on the immunogenicity. We generated six mutant constructs from wild-type influenza H7N7HA cDNA by site directed mutagenesis, and individually expressed mutant HA protein on the surface of baculovirus (Bac-HAm) and compared their protective efficacy of the vaccines with Bac-H7N7HA wild-type (Bac-HA) by lethal H7N7 viral challenge in a mouse model. We found that mice immunized subcutaneously with Bac-HAm constructs T143A or T198A-I211V or I211V-T143A serum showed significantly higher hemagglutination inhibition and neutralization titer against H7N7 and H7N9 viruses when compared to Bac-HA vaccinated mice groups. We also observed low level of lung viral titer, negligible weight loss and complete protection against lethal H7N7 viral challenge. Our results indicated that amino acid substitution at position 143 or 211 improve immunogenicity of H7N7HA vaccine against H7N7/NL/219/03 virus.

Asunto(s)

Sustitución de Aminoácidos/inmunología , Formación de Anticuerpos/inmunología , Subtipo H7N7 del Virus de la Influenza A/inmunología , Infecciones por Orthomyxoviridae/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Línea Celular , Femenino , Inmunización/métodos , Subtipo H7N9 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Ratones , Ratones Endogámicos BALB C , Células Sf9 , Spodoptera , Vacunación/métodos

RESUMEN

Streptococcus pneumoniae is a leading cause of pneumonia and one of the most common causes of death globally. The impact of S. pneumoniae on host molecular processes that lead to detrimental pulmonary consequences is not fully understood. Here, we show that S. pneumoniae induces toxic DNA double-strand breaks (DSBs) in human alveolar epithelial cells, as indicated by ataxia telangiectasia mutated kinase (ATM)-dependent phosphorylation of histone H2AX and colocalization with p53-binding protein (53BP1). Furthermore, results show that DNA damage occurs in a bacterial contact-independent fashion and that Streptococcus pyruvate oxidase (SpxB), which enables synthesis of H2O2, plays a critical role in inducing DSBs. The extent of DNA damage correlates with the extent of apoptosis, and DNA damage precedes apoptosis, which is consistent with the time required for execution of apoptosis. Furthermore, addition of catalase, which neutralizes H2O2, greatly suppresses S. pneumoniae-induced DNA damage and apoptosis. Importantly, S. pneumoniae induces DSBs in the lungs of animals with acute pneumonia, and H2O2 production by S. pneumoniae in vivo contributes to its genotoxicity and virulence. One of the major DSBs repair pathways is nonhomologous end joining for which Ku70/80 is essential for repair. We find that deficiency of Ku80 causes an increase in the levels of DSBs and apoptosis, underscoring the importance of DNA repair in preventing S. pneumoniae-induced genotoxicity. Taken together, this study shows that S. pneumoniae-induced damage to the host cell genome exacerbates its toxicity and pathogenesis, making DNA repair a potentially important susceptibility factor in people who suffer from pneumonia.

Asunto(s)

Apoptosis , Daño del ADN , Peróxido de Hidrógeno/metabolismo , Alveolos Pulmonares/metabolismo , Streptococcus pneumoniae/metabolismo , Animales , Reparación del ADN , Células Epiteliales/patología , Femenino , Ratones , Ratones Endogámicos BALB C , Alveolos Pulmonares/citología , Streptococcus pneumoniae/patogenicidad , Virulencia

RESUMEN

UNLABELLED: Highly pathogenic avian influenza virus subtype H5N1 continues to be a severe threat to public health, as well as the poultry industry, because of its high lethality and antigenic drift rate. Neutralizing monoclonal antibodies (MAbs) can serve as a useful tool for preventing, treating, and detecting H5N1. In the present study, humanized H5 antibody 8A8 was developed from a murine H5 MAb. Both the humanized and mouse MAbs presented positive activity in hemagglutination inhibition (HI), virus neutralization, and immunofluorescence assays against a wide range of H5N1 strains. Interestingly, both human and murine 8A8 antibodies were able to detect H5 in Western blot assays under reducing conditions. Further, by sequencing of escape mutants, the conformational epitope of 8A8 was found to be located within the receptor binding domain (RBD) of H5. The linear epitope of 8A8 was identified by Western blotting of overlapping fragments and substitution mutant forms of HA1. Reverse genetic H5N1 strains with individual mutations in either the conformational or the linear epitope were generated and characterized in a series of assays, including HI, postattachment, and cell-cell fusion inhibition assays. The results indicate that for 8A8, virus neutralization mediated by RBD blocking relies on the conformational epitope while binding to the linear epitope contributes to the neutralization by inhibiting membrane fusion. Taken together, the results of this study show that a novel humanized H5 MAb binds to two types of epitopes on HA, leading to virus neutralization via two mechanisms. IMPORTANCE: Recurrence of the highly pathogenic avian influenza virus subtype H5N1 in humans and poultry continues to be a serious public health concern. Preventive and therapeutic measures against influenza A viruses have received much interest in the context of global efforts to combat the current and future pandemics. Passive immune therapy is considered to be the most effective and economically prudent preventive strategy against influenza virus besides vaccination. It is important to develop a humanized neutralizing monoclonal antibody (MAb) against all of the clades of H5N1. For the first time, we report in this study that a novel humanized H5 MAb binds to two types of epitopes on HA, leading to virus neutralization via two mechanisms. These findings further deepen our understanding of influenza virus neutralization.

Asunto(s)

Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Subtipo H5N1 del Virus de la Influenza A/inmunología , Animales , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales Humanizados/metabolismo , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Antivirales/metabolismo , Western Blotting , Mapeo Epitopo , Técnica del Anticuerpo Fluorescente , Pruebas de Inhibición de Hemaglutinación , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Humanos , Subtipo H5N1 del Virus de la Influenza A/genética , Subtipo H5N1 del Virus de la Influenza A/fisiología , Ratones Endogámicos BALB C , Proteínas Mutantes/genética , Pruebas de Neutralización , Unión Proteica , Análisis de Secuencia de ADN , Acoplamiento Viral/efectos de los fármacos , Internalización del Virus/efectos de los fármacos

RESUMEN

Invertebrates lack true adaptive immunity and it solely depends on the primitive immunity called innate immunity. However, various innate immune molecules and mechanisms are identified in shrimp that plays potential role against invading bacterial, fungal and viral pathogens. Perceiving the shrimp innate immune mechanisms will contribute in developing effective vaccine strategies against major shrimp pathogens. Hence this review intends to explore the innate immune molecules of shrimp with suitable experimental evidences together with the evolution of "specific immune priming" of invertebrates. In addition, we have emphasized on the development of an effective vaccine strategy against major shrimp pathogen, white spot syndrome virus (WSSV). The baculovirus displayed rVP28 (Bac-VP28), a major envelope protein of WSSV was utilized to study its vaccine efficacy by oral route. A significant advantage of this baculovirus expression cassette is the use of WSSV-immediate early 1 (ie1) promoter that derived the abundant expression of rVP28 protein at the early stage of the infection in insect cell. The orally vaccinated shrimp with Bac-VP28 transduced successfully in the shrimp cells as well as provided highest survival rate. In support to our vaccine efficacy we analysed Pattern Recognition Proteins (PRPs) ß-1,3 glucan lipopolysaccharides (LGBP) and STAT gene profiles in the experimental shrimp. Indeed, the vaccination of shrimp with Bac-VP28 demonstrated some degree of specificity with enhanced survival rate when compared to control vaccination with Bac-wt. Hence it is presumed that the concept of "specific immune priming" in relevant to shrimp immunity is possible but may not be common to all shrimp pathogens.

RESUMEN

BACKGROUND: The rapid evolution of new sublineages of H5N1 influenza poses the greatest challenge in control of H5N1 infection by currently existing vaccines. To overcome this, an MVAtor vector expressing three H5HA antigens A/Vietnam/1203/04, A/Indonesia/669/06 and A/Anhui/01/05 (MVAtor-tri-HA vector) was developed to elicit broad cross-protection against diverse clades by covering amino acid variations in the major neutralizing epitopes of HA among H5N1 subtypes. METHODS: BALB/c mice and guinea pigs were immunized i.m. with 8×107 TCID50/animal of MVAtor-tri-HA vector. The immunogenicity and cross-protective immunity of the MVAtor-tri-HA vector was evaluated against diverse clades of H5N1 strains. RESULTS: The results showed that mice immunized with MVAtor-tri-HA vector induced robust cross-neutralizing immunity to diverse H5N1 clades. In addition, the MVAtor-tri-HA vector completely protected against 10 MLD50 of a divergent clade of H5N1 infection (clade 7). Importantly, the serological surveillance of post-vaccinated guinea pig sera demonstrated that MVAtor-tri-HA vector was able to elicit strong cross-clade neutralizing immunity against twenty different H5N1 strains from six clades that emerged between 1997 and 2012. CONCLUSIONS: The present findings revealed that incorporation of carefully selected HA genes from divergent H5N1 strains within a single vector could be an effective approach in developing a vaccine with broad coverage to prevent infection during a pandemic situation.

Asunto(s)

Subtipo H5N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Línea Celular , Protección Cruzada , Reacciones Cruzadas/inmunología , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Orden Génico , Vectores Genéticos/genética , Cobayas , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Humanos , Inmunización , Subtipo H5N1 del Virus de la Influenza A/genética , Ratones , Infecciones por Orthomyxoviridae/prevención & control , Infecciones por Orthomyxoviridae/virología , Virus Vaccinia/genética

RESUMEN

The outbreak of human infections with avian-origin H7N9 influenza has raised global concerns about a potential human pandemic. Therefore, the generation of simple and reliable newer vaccines is high priority for pandemic preparedness. In this study, we aimed to develop a recombinant vaccine by expressing HA of H7N9 (A/Shanghai/2/2013) on the surface of baculovirus (BacHA). Further, live or inactive form of BacHA (H7N9) vaccine was immunized twice either intranasally or subcutaneously into mice. The immunogenicity and cross-protective efficacy of the BacHA (H7N9) vaccine was assessed against H7N9 or H7N7 subtype challenge. The results showed that mice immunized subcutaneously with adjuvanted inactive BacHA (H7N9) induced robust cross-neutralizing antibody responses against H7 subtypes (H7N9, H7N7 and H7N3) compared to subcutaneous or intranasal immunization of live BacHA. In contrast, mice immunized intranasally with live BacHA stimulated higher HA-specific mucosal IgA levels in the upper airways, the port of virus entry. Also, intranasal immunization of BacHA of either H7N9 or H7N7 completely protected against 5 MLD50 of both H7N9 and H7N7 infections. An overall study revealed that intranasal administration of HA expressed on the baculovirus envelope is alternative way to prime the immune system against influenza infection during a pandemic situation.

Asunto(s)

Protección Cruzada , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Subtipo H7N9 del Virus de la Influenza A/inmunología , Gripe Humana/inmunología , Animales , Anticuerpos Antivirales/inmunología , Baculoviridae/genética , Baculoviridae/metabolismo , Femenino , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Glicoproteínas Hemaglutininas del Virus de la Influenza/administración & dosificación , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Humanos , Subtipo H7N3 del Virus de la Influenza A/inmunología , Subtipo H7N3 del Virus de la Influenza A/fisiología , Subtipo H7N7 del Virus de la Influenza A/inmunología , Subtipo H7N7 del Virus de la Influenza A/fisiología , Subtipo H7N9 del Virus de la Influenza A/genética , Subtipo H7N9 del Virus de la Influenza A/fisiología , Gripe Humana/prevención & control , Gripe Humana/virología , Ratones , Ratones Endogámicos BALB C

RESUMEN

BACKGROUND: Enterovirus 71 (EV-71) is a neurotropic virus causing Hand, Foot and Mouth Disease (HFMD) in infants and children under the age of five. It is a major concern for public health issues across Asia-Pacific region. The most effective way to control the disease caused by EV-71 is by vaccination thus a novel vaccine is urgently needed. Inactivated EV-71 induces a strong, virus-neutralizing antibody response in animal models, protecting them against a lethal EV-71 challenge and it has been shown to elicit cross-neutralizing antibodies in human trials. Hence, the large-scale production of purified EV-71 is required for vaccine development, diagnosis and clinical trials. METHODS: CIM® Monolith columns are single-piece columns made up of poly(glycidyl methacrylate co-ethylene dimethacrylate) as support matrix. They are designed as porous channels rather than beads with different chemistries for different requirements. As monolithic columns have a high binding capacity, flow rate and resolution, a CIM® DEAE-8f tube monolithic column was selected for purification in this study. The EV-71 infected Rhabdomyosarcoma (RD) cell supernatant was concentrated using 8% PEG 8000 in the presence of 400 mM sodium chloride. The concentrated virus was purified by weak anion exchange column using 50 mM HEPES + 1 M sodium chloride as elution buffer. RESULTS: Highly pure viral particles were obtained at a concentration of 350 mM sodium chloride as confirmed by SDS-PAGE and electron microscopy. Presence of viral proteins VP1, VP2 and VP3 was validated by western blotting. The overall process achieved a recovery of 55%. CONCLUSIONS: EV-71 viral particles of up to 95% purity can be recovered by a single step ion-exchange chromatography using CIM-DEAE monolithic columns and 1 M sodium chloride as elution buffer. Moreover, this method is scalable to purify several litres of virus-containing supernatant, using industrial monolithic columns with a capacity of up to 8 L such as CIM® cGMP tube monolithic columns.

Asunto(s)

Cromatografía por Intercambio Iónico/métodos , Enterovirus Humano A/aislamiento & purificación , Virología/métodos , Línea Celular Tumoral , Humanos

RESUMEN

Low pathogenic influenza viruses of H6 hemagglutinin (HA) subtype have a high prevalence among aquatic and domestic birds and have caused outbreaks in poultry worldwide. The first human infection with wild avian influenza H6N1 virus was reported in Taiwan and these subtype viruses may continue to evolve and accumulate changes which increasing the potential risk of human-to-human transmission. To develop a vaccine against influenza viruses of the H6 subtype, we displayed the HA gene on the baculovirus surface (Bac-HA), and studied its vaccine efficacy against a lethal challenge with mouse-adapted RG-H6(Shorebird) virus carrying the H6 HA gene from A/shorebird/DE/12/2004 (H6N8) virus and 7 genes from A/Puerto Rico/8/1934 (H1N1) virus. Immunization with 256 HA units of Bac-HA via the intranasal route triggered HA-specific serum and mucosal antibodies in mice besides increased HA inhibition titers compared to mice immunized subcutaneously. Moreover, we observed an increase in cellular immune response (IL-4) and improved in vitro neutralization activity in the mice immunized intranasally with live Bac-HA compared to mice immunized with inactivated influenza virus (IV). Interestingly, Bac-HA intranasal immunized mice showed one fold higher neutralization titer against heterologous H6 influenza virus compared to inactivated IV immunized mice. In addition, the live Bac-HA, administered through either immunization route, as well as the adjuvanted inactivated Bac-HA, administered subcutaneously, conferred 100% protection to mice challenged with homologous mouse-adapted RG-H6(Shorebird) virus. The reduction in viral titers and extend of histopathological changes of Bac-HA immunized mice lungs further demonstrated the protective efficacy of Bac-HA. Hence, the recombinant baculovirus subunit vaccine is an alternative candidate against H6 subtypes that could be propagated and administered with minimal biosafety concerns.

Asunto(s)

Hemaglutininas/inmunología , Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Gripe Humana/prevención & control , Proteínas Virales/inmunología , Animales , Anticuerpos Antivirales/inmunología , Baculoviridae/genética , Baculoviridae/metabolismo , Femenino , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Hemaglutininas/administración & dosificación , Hemaglutininas/genética , Humanos , Inmunización , Virus de la Influenza A/clasificación , Virus de la Influenza A/genética , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/genética , Gripe Humana/inmunología , Gripe Humana/virología , Interleucina-4/inmunología , Ratones , Ratones Endogámicos BALB C , Proteínas Virales/administración & dosificación , Proteínas Virales/genética

RESUMEN

Invertebrates lack true adaptive immunity and it solely depends on the primitive immunity called innate immunity. However, various innate immune molecules and mechanisms are identified in shrimp that plays potential role against invading bacterial, fungal and viral pathogens. Perceiving the shrimp innate immune mechanisms will contribute in developing effective vaccine strategies against major shrimp pathogens. Hence this review intends to explore the innate immune molecules of shrimp with suitable experimental evidences together with the evolution of "specific immune priming" of invertebrates. In addition, we have emphasized on the development of an effective vaccine strategy against major shrimp pathogen, white spot syndrome virus (WSSV). The baculovirus displayed rVP28 (Bac-VP28), a major envelope protein of WSSV was utilized to study its vaccine efficacy by oral route. A significant advantage of this baculovirus expression cassette is the use of WSSV-immediate early 1 (ie1) promoter that derived the abundant expression of rVP28 protein at the early stage of the infection in insect cell. The orally vaccinated shrimp with Bac-VP28 transduced successfully in the shrimp cells as well as provided highest survival rate. In support to our vaccine efficacy we analysed Pattern Recognition Proteins (PRPs) ß-1,3 glucan lipopolysaccharides (LGBP) and STAT gene profiles in the experimental shrimp. Indeed, the vaccination of shrimp with Bac-VP28 demonstrated some degree of specificity with enhanced survival rate when compared to control vaccination with Bac-wt. Hence it is presumed that the concept of "specific immune priming" in relevant to shrimp immunity is possible but may not be common to all shrimp pathogens.

Asunto(s)

Penaeidae/inmunología , Virus del Síndrome de la Mancha Blanca 1/inmunología , Inmunidad Adaptativa , Animales , Acuicultura , Baculoviridae/genética , Evolución Biológica , Humanos , Inmunidad Innata , Penaeidae/virología , Vacunación

RESUMEN

Hand, foot and mouth disease caused by enterovirus 71(EV71) leads to the majority of neurological complications and death in young children. While putative inactivated vaccines are only now undergoing clinical trials, no specific treatment options exist yet. Ideally, EV71 specific intravenous immunoglobulins could be developed for targeted treatment of severe cases. To date, only a single universally neutralizing monoclonal antibody against a conserved linear epitope of VP1 has been identified. Other enteroviruses have been shown to possess major conformational neutralizing epitopes on both the VP2 and VP3 capsid proteins. Hence, we attempted to isolate such neutralizing antibodies against conformational epitopes for their potential in the treatment of infection as well as differential diagnosis and vaccine optimization. Here we describe a universal neutralizing monoclonal antibody that recognizes a conserved conformational epitope of EV71 which was mapped using escape mutants. Eight escape mutants from different subgenogroups (A, B2, B4, C2, C4) were rescued; they harbored three essential mutations either at amino acid positions 59, 62 or 67 of the VP3 protein which are all situated in the "knob" region. The escape mutant phenotype could be mimicked by incorporating these mutations into reverse genetically engineered viruses showing that P59L, A62D, A62P and E67D abolish both monoclonal antibody binding and neutralization activity. This is the first conformational neutralization epitope mapped on VP3 for EV71.

Asunto(s)

Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Proteínas de la Cápside/química , Proteínas de la Cápside/inmunología , Enterovirus Humano A/inmunología , Infecciones por Enterovirus/inmunología , Secuencia de Aminoácidos , Animales , Encéfalo/patología , Encéfalo/virología , Proteínas de la Cápside/genética , Chlorocebus aethiops , Epítopos/química , Epítopos/genética , Epítopos/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Alineación de Secuencia , Células Vero

RESUMEN

UNLABELLED: In a screen for ribavirin resistance, a novel high-fidelity variant of human enterovirus 71 (EV71) with the single amino acid change L123F in its RNA-dependent RNA polymerase (RdRp or 3D) was identified. Based on the crystal structure of EV71 RdRp, L123 locates at the entrance of the RNA template binding channel, which might form a fidelity checkpoint. EV71 RdRp-L123F variants generated less progeny in a guanidine resistance assay and virus populations with lower mutation frequencies in cell culture passage due to their higher replication fidelity. However, compared with wild-type viruses, they did not show growth defects. In vivo infections further revealed that high-fidelity mutations L123F and G64R (previously reported) negatively impacted EV71 fitness and greatly reduced viral pathogenicity alone or together in AG129 mice. Interestingly, a variant with double mutations, RG/B4-G64R/L123F (where RG/B4 is an EV71 genotype B4 virus constructed by reverse genetics [RG])showed higher fidelity in vitro and less virulence in vivo than any one of the above two single mutants. The 50% lethal dose (LD50) of the double mutant increased more than 500 times compared with the LD50 of wild-type RG/B4 in mice. The results indicated that these high-fidelity variants exhibited an attenuated pathogenic phenotype in vivo and offer promise as a live attenuated EV71 vaccine. IMPORTANCE: The error-prone nature of the RNA-dependent RNA polymerase (RdRp) of RNA viruses during replication results in quasispecies and aids survival of virus populations under a wide range of selective pressures. Virus variants with higher replication fidelity exhibit lower genetic diversity and attenuated pathogenicity in vivo. Here, we identified a novel high-fidelity mutation L123F in the RdRp of human enterovirus 71 (EV71). We further elucidated that EV71 variants with the RdRp-L123F mutation and/or the previously identified high-fidelity mutation RdRp-G64R were attenuated in an AG129 mouse model. As EV71 has emerged as a serious worldwide health threat, especially in developing countries in the Asia-Pacific region, we urgently need EV71 vaccines. Learning from the poliovirus vaccination, we prefer live attenuated EV71 vaccines to inactivated EV71 vaccines in order to effectively control EV71 outbreaks at low cost. Our results imply a new means of attenuating EV71 and reducing its mutation rate at the same time.

Asunto(s)

Enterovirus Humano A/enzimología , Enterovirus Humano A/patogenicidad , Infecciones por Enterovirus/patología , Infecciones por Enterovirus/virología , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo , Animales , Antivirales/farmacología , Línea Celular , Modelos Animales de Enfermedad , Farmacorresistencia Viral , Enterovirus Humano A/efectos de los fármacos , Enterovirus Humano A/genética , Humanos , Dosificación Letal Mediana , Ratones , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación Missense , Conformación Proteica , ARN Polimerasa Dependiente del ARN/química , Ribavirina/farmacología , Pase Seriado , Análisis de Supervivencia , Virulencia

RESUMEN

H5N1 HPAI virus continues to be a severe threat for public health, as well as for the poultry industry, due to its high mortality and antigenic drift rate. There is no monovalent vaccine available which provides broad protection against those major circulating strains. In the present study, a monovalent H5 vaccine strain was developed with antigenic sequence analysis and epitope mutations. H5 from Indonesia strain (A/Indonesia/CDC669/2006) was used as backbone sequence. Three amino acids were mutated to express immunogenic epitopes from other circulating H5N1s in the backbone. RG influenza virus expressing the epitope-chimeric H5 can react in HI with multiple H5 monoclonal antibodies which fail to neutralize wild type CDC669. High titers in HI and virus neutralization against different clades H5N1s (clade 1, 2, 4 and 7) were detected using sera from mice immunized with the epitope-chimeric H5N1. The monovalent vaccine with RG-epitope-chimeric H5N1 protected mice from lethal challenge with H5N1s of different clades, including clade 1.0, 2.1, 2.2 and 2.3. This study indicates that the broad immune response elicited by this single H5N1 virus allows it to be a promising candidate for a monovalent H5 universal vaccine.

Asunto(s)

Protección Cruzada , Epítopos/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Subtipo H5N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Modelos Animales de Enfermedad , Epítopos/genética , Femenino , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Subtipo H5N1 del Virus de la Influenza A/genética , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/genética , Ratones Endogámicos BALB C , Infecciones por Orthomyxoviridae/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Análisis de Supervivencia , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología

RESUMEN

The rapid evolution of new sublineages of H5N1 influenza in Asia poses the greatest challenge in vaccine development for pre-pandemic preparedness. To overcome the antigenic diversity of H5N1 strains, multiple vaccine strains can be designed based on the distribution of neutralizing epitopes in the globular head of H5 hemagglutinin (HA). Recently, we selected two different HAs of H5N1 strains based on the neutralizing epitopes and reactivity with different neutralizing antibodies. The HAs of selected vaccine strains were individually expressed on the baculovirus envelope (bivalent-BacHA) with its native antigenic configuration. Further, oral delivery of live bivalent-BacHA elicited broadly reactive humoral, mucosal and cell-mediated immune responses and showed complete protection against antigenically distinct H5N1 strains in mice. The strategy for the vaccine strain selection, vaccine design and route of administration will provide an idea for development of a widely protective vaccine against highly pathogenic H5N1 for pre-pandemic preparedness.

Asunto(s)

Protección Cruzada , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Subtipo H5N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Animales , Femenino