RESUMEN
Enterovirus 71 (EV71) and coxsackieviruses are the major causative agents of hand, foot, and mouth disease (HFMD) outbreaks worldwide and have a significant socioeconomic impact, particularly in Asia. Formalin-inactivated (FI) EV71 vaccines evaluated in human clinical trials in China, Taiwan, and Singapore were found to be safe and to elicit strong neutralizing antibody responses against EV71 currently circulating in Asia. The results from 3 different phase 3 clinical trials performed in young children (6-60 months) indicate that the efficacy of FI-EV71 vaccines is >90% against EV71-related HFMDs and >80% against EV71-associated serious diseases, but the vaccines did not protect against coxsackievirus A16 infections. Here we discuss the critical factors affecting EV71 vaccine product registration, including clinical epidemiology, antigenic shift issues in cross-protection and vaccine strain selection, standardized animal models for potency testing, and cost-effective manufacturing processes for potential incorporation of FI-EV71 vaccine into Expanded Programme on Immunization vaccines.
Asunto(s)
Enterovirus Humano A/inmunología , Enfermedad de Boca, Mano y Pie/prevención & control , Enfermedad de Boca, Mano y Pie/virología , Vacunas Virales/administración & dosificación , Vacunas Virales/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Variación Antigénica , Ensayos Clínicos Fase III como Asunto , Protección Cruzada , Modelos Animales de Enfermedad , Enfermedad de Boca, Mano y Pie/epidemiología , Humanos , Resultado del Tratamiento , Vacunas Virales/aislamiento & purificaciónRESUMEN
Enterovirus71 (EV71) is now recognized as an emerging neurotropic virus in Asia and one major causative agent of hand-foot-mouth diseases (HFMD). However potential animal models for vaccine development are limited to young mice. In this study, we used an adeno-associated virus (AAV) vector to introduce the human EV71 receptors P-selectin glycoprotein ligand-1 (hPSGL1) or a scavenger receptor class-B member-2 (hSCARB2) into adult ICR mice to change their susceptibility to EV71 infection. Mice were administered AAV-hSCARB2 or AAV-hPSGL1 through intravenous and oral routes. After three weeks, expression of human SCARB2 and PSGL1 was detected in various organs. After infection with EV71, we found that the EV71 viral load in AAV-hSCARB2- or AAV-hPSGL1-transduced mice was higher than that of the control mice in both the brain and intestines. The presence of EV71 viral particles in tissues was confirmed using immunohistochemistry analysis. Moreover, inflammatory cytokines were induced in the brain and intestines of AAV-hSCARB2- or AAV-hPSGL1-transduced mice after EV71 infection but not in wild-type mice. However, neurological disease was not observed in these animals. Taken together, we successfully infected adult mice with live EV71 and induced local inflammation using an AAV delivery system.
Asunto(s)
Dependovirus/genética , Enterovirus/genética , Inflamación/genética , Transfección/métodos , Animales , Encéfalo/metabolismo , Química Encefálica , Línea Celular , Citocinas/análisis , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Intestinos/química , Proteínas de Membrana de los Lisosomas/genética , Proteínas de Membrana de los Lisosomas/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos ICR , Receptores Depuradores/genética , Receptores Depuradores/metabolismoRESUMEN
Enterovirus 71 (EV71) has caused epidemics of hand, foot and mouth diseases in Asia during the past decades and no vaccine is available. A formalin-inactivated EV71 candidate vaccine (EV71vac) based on B4 subgenotype has previously been developed and found to elicit strong neutralizing antibody responses in mice and humans. In this study, we evaluated the long-term immunogenicity and safety of this EV71vac in a non-human primate model. Juvenile macaques were immunized at 0, 3 and 6 weeks either with 10 or 5 µg doses of EV71vac formulated with AlPO4 adjuvant, or PBS as control. During the 56 weeks of studies, no fever nor local redness and swelling at sites of injections was observed in the immunized macaques. After single immunization, 100% seroconversion based on 4-fold increased in neutralization titer (Nt) was detected in EV71vac immunized monkeys but not PBS controls. A dose-dependent IgG antibody response was observed in monkeys receiving EV71vac immunization. The Nt of EV71vac immunized macaques had reached the peak after 3 vaccinations, then decreased gradually; however, the GMT of neutralizing antibody in the EV71vac immunized macaques were still above 100 at the end of the study. Correspondingly, both dose- and time-dependent interferon-γ and CD4+ T cell responses were detected in monkeys receiving EV71vac. Interestingly, similar to human responses, the dominant T cell epitopes of macaques were identified mainly in VP2 and VP3 regions. In addition, strong cross-neutralizing antibodies against most EV71 subgenotypes except some C2 and C4b strains, and Coxsackievirus A16 were observed. In summary, our results indicate that EV71vac elicits dose-dependent T-cell and antibody responses in macaques that could be a good animal model for evaluating the long-term immune responses elicited by EV71 vaccines.
Asunto(s)
Enterovirus Humano A/inmunología , Macaca/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Formaldehído , Linfocitos T/inmunologíaRESUMEN
Enterovirus 71 (EV71), a positive-stranded RNA virus, is the major cause of hand, foot, and mouth disease (HFMD) with severe neurological symptoms. Antiviral type I interferon (alpha/beta interferon [IFN-α/ß]) responses initiated from innate receptor signaling are inhibited by EV71-encoded proteases. It is less well understood whether EV71-induced apoptosis provides a signal to activate type I interferon responses as a host defensive mechanism. In this report, we found that EV71 alone cannot activate Toll-like receptor 9 (TLR9) signaling, but supernatant from EV71-infected cells is capable of activating TLR9. We hypothesized that TLR9-activating signaling from plasmacytoid dendritic cells (pDCs) may contribute to host defense mechanisms. To test our hypothesis, Flt3 ligand-cultured DCs (Flt3L-DCs) from both wild-type (WT) and TLR9 knockout (TLR9KO) mice were infected with EV71. More viral particles were produced in TLR9KO mice than by WT mice. In contrast, alpha interferon (IFN-α), monocyte chemotactic protein 1 (MCP-1), tumor necrosis factor-alpha (TNF-α), IFN-γ, interleukin 6 (IL-6), and IL-10 levels were increased in Flt3L-DCs from WT mice infected with EV71 compared with TLR9KO mice. Seven-day-old TLR9KO mice infected with a non-mouse-adapted EV71 strain developed neurological lesion-related symptoms, including hind-limb paralysis, slowness, ataxia, and lethargy, but WT mice did not present with these symptoms. Lung, brain, small intestine, forelimb, and hind-limb tissues collected from TLR9KO mice exhibited significantly higher viral loads than equivalent tissues collected from WT mice. Histopathologic damage was observed in brain, small intestine, forelimb, and hind-limb tissues collected from TLR9KO mice infected with EV71. Our findings demonstrate that TLR9 is an important host defense molecule during EV71 infection. Importance: The host innate immune system is equipped with pattern recognition receptors (PRRs), which are useful for defending the host against invading pathogens. During enterovirus 71 (EV71) infection, the innate immune system is activated by pathogen-associated molecular patterns (PAMPs), which include viral RNA or DNA, and these PAMPs are recognized by PRRs. Toll-like receptor 3 (TLR3) and TLR7/8 recognize viral nucleic acids, and TLR9 senses unmethylated CpG DNA or pathogen-derived DNA. These PRRs stimulate the production of type I interferons (IFNs) to counteract viral infection, and they are the major source of antiviral alpha interferon (IFN-α) production in pDCs, which can produce 200- to 1,000-fold more IFN-α than any other immune cell type. In addition to PAMPs, danger-associated molecular patterns (DAMPs) are known to be potent activators of innate immune signaling, including TLR9. We found that EV71 induces cellular apoptosis, resulting in tissue damage; the endogenous DNA from dead cells may activate the innate immune system through TLR9. Therefore, our study provides new insights into EV71-induced apoptosis, which stimulates TLR9 in EV71-associated infections.
Asunto(s)
Enterovirus Humano A/aislamiento & purificación , Infecciones por Enterovirus/prevención & control , Receptor Toll-Like 9/fisiología , Animales , Secuencia de Bases , Células Cultivadas , Citocinas/metabolismo , Cartilla de ADN , Enterovirus Humano A/fisiología , Ensayo de Inmunoadsorción Enzimática , Interferón Tipo I/biosíntesis , Ratones , Ratones Noqueados , FN-kappa B/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor Toll-Like 9/genética , Replicación ViralRESUMEN
BACKGROUND: Enterovirus 71 (EV71) has caused several epidemics of hand, foot and mouth diseases (HFMD) in Asia. No effective EV71 vaccine is available. A randomized and open-label phase I clinical study registered with ClinicalTrials.gov #NCT01268787, aims to evaluate the safety, reactogenicity and immunogenicity of a formalin-inactivated EV71 vaccine candidate (EV71vac) at 5- and 10-µg doses. In this study we report the cross-neutralizing antibody responses from each volunteer against different subgenotypes of EV71 and CVA16. METHODS: Sixty eligible healthy adults were recruited and vaccinated. Blood samples were obtained on day 0, 21 and 42 and tested against B1, B4, B5, C2, C4A, C4B and CVA16 for cross-neutralizing antibody responses. RESULTS: The immunogenicity of both 5- and 10- µg doses were found to be very similar. Approximately 45% of the participants had <8 pre-vaccination neutralization titers (Nt) against the B4 vaccine strain. After the first EV71vac immunization, 95% of vaccinees have >4-fold increase in Nt, but there was no further increase in Nt after the second dose. EV71vac induced very strong cross-neutralizing antibody responses in >85% of volunteers without pre-existing Nt against subgenotype B1, B5 and C4A. EV71vac elicited weak cross-neutralizing antibody responses (â¼20% of participants) against a C4B and Coxsackie virus A16. Over 90% of vaccinated volunteers did not develop cross-neutralizing antibody responses (Nt<8) against a C2 strain. EV71vac can boost and significantly enhance the neutralizing antibody responses in volunteers who already had pre-vaccination antibodies against EV71 and/or CVA16. CONCLUSION: EV71vac is efficient in eliciting cross-neutralizing antibody responses against EV71 subgenotypes B1, B4, B5, and C4A, and provides the rationale for its evaluation in phase II clinical trials. TRIAL REGISTRATION: ClinicalTrials.gov NCT01268787.
Asunto(s)
Anticuerpos Neutralizantes/uso terapéutico , Enterovirus Humano A/efectos de los fármacos , Infecciones por Enterovirus/tratamiento farmacológico , Formaldehído/farmacología , Vacunas de Productos Inactivados/uso terapéutico , Animales , Línea Celular , Chlorocebus aethiops , Enterovirus Humano A/patogenicidad , Infecciones por Enterovirus/inmunología , Humanos , Células VeroRESUMEN
Combination vaccines can reduce the number of injections and simplify the immunization schedule required to prevent different diseases. Here we assessed the immunogenicity in a mouse model of a vaccine composition comprising inactivated influenza viruses (H5N1/H1N1), enterovirus 71 (EV71), and/or Japanese encephalitis virus (JEV) and investigated whether the vaccine formulations can overcome the immunologic interference between the individual vaccine components. We demonstrated that the antigenic competition happens between H5N1/H1N1 or H5N1/EV71 inactivated virions when the vaccine combinations either formulated with Alum suspensions or without adjuvant. In the presence of PELC emulsified particles, EV71-specific immune responses before and after incorporating H5N1 virus into EV71 vaccine were detected of no significant difference; in addition, H5N1- and EV71-specific immune responses were found at the same level when H5N1/EV71/JEV consolidating into combination vaccine. Emulsified vaccine formulation was represented as a potential tool that is found to reduce the number of injections required to prevent multiple infectious strains causing the same disease (H5N1/H1N1) and/or that protect against different diseases (H5N1/EV71). Combination vaccines can also include a third component to protect against H5N1/EV71/JEV at the same time.
Asunto(s)
Virus de la Encefalitis Japonesa (Especie)/inmunología , Enterovirus Humano A/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H5N1 del Virus de la Influenza A/inmunología , Vacunas Virales/inmunología , Virión/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Compuestos de Alumbre/administración & dosificación , Animales , Anticuerpos Antivirales/sangre , Composición de Medicamentos , Emulsiones/administración & dosificación , Ratones Endogámicos BALB C , Vacunas Combinadas/administración & dosificación , Vacunas Combinadas/inmunología , Vacunas Virales/administración & dosificaciónRESUMEN
BACKGROUND: Large-scale outbreaks of enterovirus 71 (EV71) infections have occurred in Asia-Pacific regions. Severe complications include encephalitis and poliomyelitis-like paralysis, cardiopulmonary collapse, and death, necessitating an effective vaccine against EV71. METHODS: In this randomized Phase I study, we evaluated the safety and immunogenicity of an inactivated alum-adjuvanted EV71 whole-virus vaccine produced on Vero cell cultures. Sixty healthy volunteers aged 20-60 years received two doses of vaccine, administered 21 days apart. Each dose contained either 5 µg of EV71 antigen with 150 µg of adjuvant (Group A05) or 10 µg of EV71 antigen with 300 µg of adjuvant (Group B10). Serologic analysis was performed at baseline, day 21, and day 42. RESULTS: There were no serious adverse events. Mild injection site pain and myalgia were the most common adverse events with either vaccine formulation. The immunogenicity data showed that 90% of vaccine recipients have a 4-fold or greater increase in neutralization antibody titers (NT) after the first dose, without a further increase in NT after the second dose. The seroconversion rates on day 21 and day 42 were 86.7% and 93.1% respectively, in Group A05, and 92.9% and 96.3%, respectively, in Group B10. Thus, 5 µg and 10 µg of the EV71 vaccine can induce a remarkable immune response in healthy adults after only the first vaccination. CONCLUSION: The 5 µg and 10 µg adjuvanted EV71 vaccines are generally safe and immunogenic in healthy adults. (ClinicalTrials.gov number, NCT01268787).
Asunto(s)
Enterovirus/inmunología , Vacunación/efectos adversos , Vacunas de Productos Inactivados/efectos adversos , Vacunas de Productos Inactivados/inmunología , Vacunas Virales/efectos adversos , Vacunas Virales/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Adulto , Compuestos de Alumbre , Animales , Formación de Anticuerpos/inmunología , Chlorocebus aethiops , Infecciones por Enterovirus/inmunología , Infecciones por Enterovirus/prevención & control , Femenino , Voluntarios Sanos , Humanos , Masculino , Persona de Mediana Edad , Vacunas de Productos Inactivados/administración & dosificación , Células Vero , Vacunas Virales/administración & dosificación , Adulto JovenRESUMEN
BACKGROUND: Avian H5N1 influenza has caused human infections globally and has a high mortality rate. Rapid production of effective vaccines is needed. METHODS: A phase 1, randomized, observer-blinded clinical trial was conducted to examine the safety and immunogenicity of an inactivated whole virion vaccine against the influenza A/H5N1 virus produced from the Madin-Darby canine kidney (MDCK) cell line. Participants were randomized to four groups and administered two intramuscular doses of vaccine containing 3 µg hemagglutinin (HA), 3 µg HA with 300 µg aluminum phosphate (AlPO4), 6 µg HA, and 6 µg HA with 300 µg AlPO4, respectively, at two visits, 21 days apart. Serum hemagglutination inhibition (HAI) and neutralizing antibody levels were determined at baseline and on Days 21 and 42. RESULTS: Sixty healthy individuals were enrolled. The neutralization assay showed a significant immune response in the 6 µg with ALPO4 group on Day 42 compared to pre-vaccination levels (11.32±9.77 vs. 4.00±0, p=0.02). The adjuvant effect in neutralization assay was also significant on Day 42 in the 6 µg group (4.52±1.94 without adjuvant vs. 11.32±9.77 with adjuvant, p=0.02). HAI assay also showed an aluminum adjuvant-induced increasing trend in HAI geometric mean titer on Day 42 in the 3 µg and 6 µg groups (6.02 versus 8.20, p=0.05 and 5.74 versus 8.21, p=0.14). The most frequent adverse event was local pain (20% to 60%). There were no vaccine-related severe adverse effects. CONCLUSION: MDCK cell line-derived H5N1 vaccine was well tolerated. It is necessary to investigate further the immunogenicity of higher antigen doses and the role of aluminum adjuvant in augmenting the effect of the vaccine.
Asunto(s)
Subtipo H5N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/efectos adversos , Vacunas contra la Influenza/inmunología , Gripe Humana/prevención & control , Adyuvantes Inmunológicos/administración & dosificación , Adulto , Compuestos de Aluminio/administración & dosificación , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Perros , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/epidemiología , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/patología , Femenino , Voluntarios Sanos , Pruebas de Inhibición de Hemaglutinación , Humanos , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/aislamiento & purificación , Inyecciones Intramusculares , Células de Riñón Canino Madin Darby , Masculino , Persona de Mediana Edad , Pruebas de Neutralización , Fosfatos/administración & dosificación , Estudios Prospectivos , Método Simple Ciego , Taiwán , Vacunación/métodos , Vacunas de Productos Inactivados/administración & dosificación , Vacunas de Productos Inactivados/efectos adversos , Vacunas de Productos Inactivados/inmunología , Vacunas de Productos Inactivados/aislamiento & purificación , Cultivo de Virus/métodos , Adulto JovenRESUMEN
BACKGROUND: Coxsackie virus A16 (CVA16) infections have become a serious public health problem in the Asia-Pacific region. It manifests most often in childhood exanthema, commonly known as hand-foot-and-mouth disease (HFMD). There are currently no vaccine or effective medical treatments available. PRINCIPAL FINDING: In this study, we describe the production, purification and characterization of CVA16 virus produced from Vero cells grown on 5 g/L Cytodex 1 microcarrier beads in a five-liter serum-free bioreactor system. The viral titer was found to be >10(6) the tissue culture's infectious dose (TCID(50)) per mL within 7 days post-infection when a multiplicity of infection (MOI) of 10(-5) was used for initial infection. Two CVA16 virus fractions were separated and detected when the harvested CVA16 viral concentrate was purified by a sucrose gradient zonal ultracentrifugation. The viral particles detected in the 24-28% sucrose fractions had low viral infectivity and RNA content. The viral particles obtained from 35-38% sucrose fractions were found to have high viral infectivity and RNA content, and composed of four viral proteins (VP1, VP2, VP3 and VP4), as shown by SDS-PAGE analyses. These two virus fractions were formalin-inactivated and only the infectious particle fraction was found to be capable of inducing CVA16-specific neutralizing antibody responses in both mouse and rabbit immunogenicity studies. But these antisera failed to neutralize enterovirus 71. In addition, rabbit antisera did not react with any peptides derived from CVA16 capsid proteins. Mouse antisera recognized a single linear immunodominant epitope of VP3 corresponding to residues 176-190. CONCLUSION: These results provide important information for cell-based CVA16 vaccine development. To eliminate HFMD, a bivalent EV71/CVA16 vaccine formulation is necessary.
Asunto(s)
Anticuerpos Antivirales/biosíntesis , Proteínas de la Cápside/inmunología , Enterovirus Humano A/aislamiento & purificación , Virión/aislamiento & purificación , Animales , Anticuerpos Antivirales/inmunología , Reactores Biológicos , Proteínas de la Cápside/genética , Centrifugación por Gradiente de Densidad , Chlorocebus aethiops , Electroforesis en Gel de Poliacrilamida , Enterovirus Humano A/crecimiento & desarrollo , Enterovirus Humano A/inmunología , Enterovirus Humano A/ultraestructura , Epítopos/inmunología , Enfermedad de Boca, Mano y Pie/prevención & control , Humanos , Ratones , Microscopía Electrónica de Transmisión , Pruebas de Neutralización , Conejos , Células Vero , Virión/crecimiento & desarrollo , Virión/inmunología , Virión/ultraestructuraRESUMEN
Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are major causative agents of hand, foot, and mouth diseases (HFMDs), and EV71 is now recognized as an emerging neurotropic virus in Asia. Effective medications and/or prophylactic vaccines against HFMD are not available. The current results from mouse immunogenicity studies using in-house standardized RD cell virus neutralization assays indicate that (1) VP1 peptide (residues 211-225) formulated with Freund's adjuvant (CFA/IFA) elicited low virus neutralizing antibody response (1/32 titer); (2) recombinant virus-like particles produced from baculovirus formulated with CFA/IFA could elicit good virus neutralization titer (1/160); (3) individual recombinant EV71 antigens (VP1, VP2, and VP3) formulated with CFA/IFA, only VP1 elicited antibody response with 1/128 virus neutralization titer; and (4) the formalin-inactivated EV71 formulated in alum elicited antibodies that cross-neutralized different EV71 genotypes (1/640), but failed to neutralize CVA16. In contrast, rabbits antisera could cross-neutralize strongly against different genotypes of EV71 but weakly against CVA16, with average titers 1/6400 and 1/32, respectively. The VP1 amino acid sequence dissimilarity between CVA16 and EV71 could partially explain why mouse antibodies failed to cross-neutralize CVA16. Therefore, the best formulation for producing cost-effective HFMD vaccine is a combination of formalin-inactivated EV71 and CAV16 virions.
Asunto(s)
Anticuerpos Antivirales/inmunología , Proteínas de la Cápside/inmunología , Enterovirus Humano A/inmunología , Infecciones por Enterovirus/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Proteínas de la Cápside/química , Línea Celular , Línea Celular Tumoral , Chlorocebus aethiops , Infecciones por Enterovirus/prevención & control , Infecciones por Enterovirus/virología , Femenino , Ratones , Ratones Endogámicos BALB C , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Conejos , Rabdomiosarcoma/inmunología , Rabdomiosarcoma/virología , Vacunas Sintéticas , Células Vero , Carga Viral/inmunología , Proteínas Estructurales Virales/inmunología , Vacunas Virales/farmacología , Virión/inmunologíaRESUMEN
Enterovirus 71 (EV71) is now recognized as an emerging neurotropic virus in Asia and with Coxsackie virus (CV) it is the other major causative agent of hand-foot-mouth diseases (HFMD). Effective medications and/or prophylactic vaccines against HFMD are urgently needed. From a scientific (the feasibility of bioprocess, immunological responses and potency in animal challenge model) and business development (cost of goods) points of view, we in this review address and discuss the pros and cons of different EV71 vaccine candidates that have been produced and evaluated in animal models. Epitope-based synthetic peptide vaccine candidates containing residues 211-225 of VP1 formulated with Freund's adjuvant (CFA/IFA) elicited low EV71 virus neutralizing antibody responses, but were protective in the suckling mouse challenge model. Among recombinant EV71 subunits (rVP1, rVP2 and rVP3) expressed in E. coli, purified and formulated with CFA/IFA, only VP1 elicited mouse antibody responses with measurable EV71-specific virus neutralization titers. Immunization of mice with either a DNA plasmid containing VP1 gene or VP1 expressed in Salmonella typhimurium also generated neutralizing antibody responses and protected animals against a live EV71 challenge. Recombinant EV71 virus-like particles (rVLP) produced from baculovirus formulated either with CFA/IFA or alum elicited good virus neutralization titers in both mice and non-human primates, and were found to be protective in the suckling mouse EV71 challenge model. Synthetic peptides or recombinant EV71 subunit vaccines (rVP1 and rVLP) formulated in alum were found to be poorly immunogenic in rabbits. Only formalin-inactivated (FI) EV71 virions formulated in alum elicited cross-neutralizing antibodies against different EV71 genotypes in mice, rabbits and non-human primates but induced weak neutralizing responses against CAV16. From a regulatory, economic and market acceptability standpoint, FI-EV71 virion vaccines are the most promising candidates and are currently being evaluated in human clinical trials. We further describe and analyze some new bioprocesses technologies that have great potential applications in EV71 vaccine development. This review also demonstrates the opportunities and challenges that the Asian vaccine industry faces today.
Asunto(s)
Enterovirus Humano A/inmunología , Enfermedad de Boca, Mano y Pie/prevención & control , Vacunas Virales/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Antígenos Virales/genética , Antígenos Virales/inmunología , Ensayos Clínicos como Asunto , Modelos Animales de Enfermedad , Enterovirus Humano A/genética , Femenino , Enfermedad de Boca, Mano y Pie/epidemiología , Humanos , Masculino , Ratones , Primates , Conejos , Análisis de Supervivencia , Vacunas de Productos Inactivados/administración & dosificación , Vacunas de Productos Inactivados/genética , Vacunas de Productos Inactivados/inmunología , Vacunas de Productos Inactivados/aislamiento & purificación , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/aislamiento & purificación , Proteínas Estructurales Virales/genética , Proteínas Estructurales Virales/inmunología , Vacunas Virales/administración & dosificación , Vacunas Virales/genética , Vacunas Virales/aislamiento & purificaciónRESUMEN
BACKGROUND: Enterovirus 71 (EV71) has caused several epidemics of hand, foot and mouth diseases (HFMD) in Asia and now is being recognized as an important neurotropic virus. Effective medications and prophylactic vaccine against EV71 infection are urgently needed. Based on the success of inactivated poliovirus vaccine, a prototype chemically inactivated EV71 vaccine candidate has been developed and currently in human phase 1 clinical trial. PRINCIPAL FINDING: In this report, we present the development of a serum-free cell-based EV71 vaccine. The optimization at each step of the manufacturing process was investigated, characterized and quantified. In the up-stream process development, different commercially available cell culture media either containing serum or serum-free was screened for cell growth and virus yield using the roller-bottle technology. VP-SFM serum-free medium was selected based on the Vero cell growth profile and EV71 virus production. After the up-stream processes (virus harvest, diafiltration and concentration), a combination of gel-filtration liquid chromatography and/or sucrose-gradient ultracentrifugation down-stream purification processes were investigated at a pilot scale of 40 liters each. Although the combination of chromatography and sucrose-gradient ultracentrifugation produced extremely pure EV71 infectious virus particles, the overall yield of vaccine was 7-10% as determined by a VP2-based quantitative ELISA. Using chromatography as the downstream purification, the virus yield was 30-43%. To retain the integrity of virus neutralization epitopes and the stability of the vaccine product, the best virus inactivation was found to be 0.025% formalin-treatment at 37 °C for 3 to 6 days. Furthermore, the formalin-inactivated virion vaccine candidate was found to be stable for >18 months at 4 °C and a microgram of viral proteins formulated with alum adjuvant could induce strong virus-neutralizing antibody responses in mice, rats, rabbits, and non-human primates. CONCLUSION: These results provide valuable information supporting the current cell-based serum-free EV71 vaccine candidate going into human Phase I clinical trials.
Asunto(s)
Enterovirus Humano A/inmunología , Infecciones por Enterovirus/prevención & control , Vacunas Virales , Compuestos de Aluminio , Animales , Técnicas de Cultivo Celular por Lotes , Reactores Biológicos , Chlorocebus aethiops , Medio de Cultivo Libre de Suero , Enterovirus Humano A/crecimiento & desarrollo , Humanos , Macaca , Fosfatos , Vacunas de Productos Inactivados/inmunología , Vacunas de Productos Inactivados/aislamiento & purificación , Células Vero , Vacunas Virales/inmunología , Vacunas Virales/aislamiento & purificación , Inactivación de VirusRESUMEN
Enterovirus 71 (EV71) has recently emerged as an important neurotropic virus in Asia because effective medications and prophylactic vaccine against EV71 infection are not available. Based on the success of inactivated poliovirus vaccine, the Vero cell-based chemically inactivated EV71 vaccine candidate could be developed. Identification of EV71 vaccine strain which can grow to high titer in Vero cell and induce cross-genotype virus neutralizing antibody responses represents the first step in vaccine development. In this report we describe the characterization and validation of a clinical isolate E59 belonging to B4 sub-genotype based on VP1 genetic analysis. Before selected as the vaccine strain, the genetic stability of E59 in passage had been analyzed based on the nucleotide sequences obtained from the Master Virus Seed, Working Seed banks and the virus harvested from the production lots, and found to be identical to those found in the original isolate. These results indicate that E59 vaccine strain has strong genetic stability in passage. Using this vaccine strain the prototype EV71 vaccine candidate was produced from 20L of Vero cell grown in serum-containing medium. The production processes were investigated, characterized and quantified to establish the potential vaccine manufacturing process including the time for virus harvest, the membrane for diafiltration and concentration, the gel-filtration chromatography for the down-stream virus purification, and the methods for viral inactivation. Finally, the inactivated virion vaccine candidate containing sub-microgram of viral proteins formulated with alum adjuvant was found to induce strong virus neutralizing antibody responses in mice and rabbits. Therefore, these results provide valuable information for cell-based EV71 vaccine development.
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Enterovirus Humano A/inmunología , Enterovirus Humano A/aislamiento & purificación , Vacunas Virales/inmunología , Vacunas Virales/aislamiento & purificación , Adyuvantes Inmunológicos/administración & dosificación , Compuestos de Alumbre/administración & dosificación , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Chlorocebus aethiops , Enterovirus Humano A/genética , Enterovirus Humano A/crecimiento & desarrollo , Femenino , Inestabilidad Genómica , Ratones , Ratones Endogámicos BALB C , ARN Viral/genética , Análisis de Secuencia de ADN , Pase Seriado , Vacunas de Productos Inactivados/administración & dosificación , Vacunas de Productos Inactivados/genética , Vacunas de Productos Inactivados/inmunología , Vacunas de Productos Inactivados/aislamiento & purificación , Células Vero , Vacunas Virales/administración & dosificación , Vacunas Virales/genética , Inactivación de VirusRESUMEN
Enterovirus 71 (EV71), the etiologic agent causes outbreaks with significant mortality in young children in Asia and currently there is no vaccine available. In this study, we report a quantitative enzyme linked immunosorbent assay (Q-ELISA) to determine the concentration of the EV71 VP2 antigen. EV71 virus-like particles (VLPs) were produced in the baculovirus expression system and used as the EV71 antigen reference standard. Antisera from both EV71-immunized chickens and rabbits were very efficient and useful as capture antibodies to bind various forms of EV71 antigens, whereas a commercial VP2-specific virus neutralizing monoclonal antibody MAB979 was found to be suitable for quantifying the amount of VP2 antigen. This Q-ELISA was used successfully to determine VP2 content at each stage of EV71 vaccine manufacturing process, particularly during the upstream harvest, downstream purification and viral inactivation steps. The amount of VP2 antigen and the magnitude of neutralizing titers were found to be dose-dependent in mice immunized with vaccine candidates. These results indicate that Q-ELISA could provide off-line timely quantitative measurements of VP2 antigen throughout the production cycle to evaluate critical attributes and conditions that may affect virus yields in culture media, the quality of purification methods, the stability and potency of final vaccine formulations.
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Anticuerpos Antivirales/inmunología , Antígenos Virales/análisis , Proteínas de la Cápside/análisis , Enterovirus/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Vacunas Virales , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Antígenos Virales/inmunología , Biotecnología/métodos , Proteínas de la Cápside/inmunología , Chlorocebus aethiops , Enterovirus/crecimiento & desarrollo , Infecciones por Enterovirus/inmunología , Infecciones por Enterovirus/prevención & control , Femenino , Humanos , Inmunización , Ratones , Ratones Endogámicos BALB C , Conejos , Estándares de Referencia , Sensibilidad y Especificidad , Vacunación , Células Vero , Vacunas Virales/normasRESUMEN
BACKGROUND: Enterovirus 71 (EV71) infections manifest most commonly as a childhood exanthema known as hand-foot-and-mouth disease (HFMD) and can cause neurological disease during acute infection. PRINCIPAL FINDING: In this study, we describe the production, purification and characterization of EV71 virus produced from Vero cells grown in a five-liter serum-free bioreactor system containing 5 g/L Cytodex 1 microcarrier. The viral titer was >10(6) TCID(50)/mL by 6 days post infection when a MOI of 10(-5) was used at the initial infection. Two EV71 virus fractions were separated and detected when the harvested EV71 virus concentrate was purified by sucrose gradient zonal ultracentrifugation. The EV71 viral particles detected in the 24-28% sucrose fractions had an icosahedral structure 30-31 nm in diameter and had low viral infectivity and RNA content. Three major viral proteins (VP0, VP1 and VP3) were observed by SDS-PAGE. The EV71 viral particles detected in the fractions containing 35-38% sucrose were 33-35 nm in size, had high viral infectivity and RNA content, and were composed of four viral proteins (VP1, VP2, VP3 and VP4), as shown by SDS-PAGE analyses. The two virus fractions were formalin-inactivated and induced high virus neutralizing antibody responses in mouse immunogenicity studies. Both mouse antisera recognized the immunodominant linear neutralization epitope of VP1 (residues 211-225). CONCLUSION: These results provide important information for cell-based EV71 vaccine development, particularly for the preparation of working standards for viral antigen quantification.
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Reactores Biológicos/microbiología , Enterovirus Humano A/aislamiento & purificación , Enterovirus/aislamiento & purificación , Virión/aislamiento & purificación , Animales , Centrifugación por Gradiente de Densidad , Chlorocebus aethiops , Electroforesis en Gel de Poliacrilamida , Enterovirus/crecimiento & desarrollo , Enterovirus Humano A/crecimiento & desarrollo , Infecciones por Enterovirus/virología , Enfermedad de Boca, Mano y Pie/virología , Ratones , Vacunas , Células Vero , Virión/crecimiento & desarrolloRESUMEN
Cross-presentation by DCs is the major mechanism by which exogenous antigens activate CTLs. However, the mechanisms of entry and presentation of vaccine peptides by DCs remain unclear. In this study, we determined that the mechanisms of antigen presentation differed between nonlipidated and monopalmitoylated peptide antigens. We found that a nonlipidated long peptide could be taken up by DCs and that the peptide could be colocalized with early endosomes. The uptake of nonlipidated peptides by DCs was inhibited at low temperatures or by the depolymerization of actin filaments or microtubules. In contrast, lipidated peptides were internalized by DCs at low temperatures, and internalization was not inhibited when actin filaments or microtubules were depolymerized. Moreover, lipidated peptide, but not nonlipidated peptide, was internalized by nonphagocytic Jurkat cells. The endosomal/lysosomal and proteasomal degradation pathways were necessary for nonlipidated presentation leading to the activation of CD8(+) T cells, but the proteasomal degradation pathway alone was sufficient to process lipidated peptides for MHC class I presentation. We further found that lipidated peptides could enhance peptide-specific T cell responses in vitro and in vivo and induced stronger antitumor responses than nonlipidated peptides. Taken together, our results demonstrate that DCs present lipidated peptides through an endocytosis-independent pathway to promote strong anti-tumor effects in vivo.
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Presentación de Antígeno/inmunología , Células Dendríticas/inmunología , Lipopéptidos/inmunología , Neoplasias/inmunología , Linfocitos T CD8-positivos/inmunología , Endocitosis , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Células Jurkat , Activación de Linfocitos/inmunología , Complejo de la Endopetidasa Proteasomal/inmunología , Complejo de la Endopetidasa Proteasomal/metabolismoRESUMEN
Enterovirus 71 (EV71) infections in children manifest as exanthema and are most commonly known as hand-foot-and-mouth disease (HFMD). Because it can cause severe neurological complications like poliomyelitis, EV71 has now emerged as an important neurotropic virus in Asia. EV71 virus has been shown to consist of 3 (A, B and C) genotypes and many subgenotypes. Although EV71 vaccine development has recently yielded promising preclinical results, yet the correlation between the content of antigen(s) in vaccine candidates and the level of protective antibody responses is not established. The neutralization epitope(s) of EV71 antigens could be used as the surrogate biomarker of vaccine potency. Using peptide ELISA, antisera generated from animals immunized with formalin-inactivated EV71 virion vaccine formulated in alum, EV71-specific neutralizing monoclonal antibody (nMAb) and a panel of 153 overlapping synthetic peptides covering the entire sequences of VP1, VP2 and VP3 of EV71, we screened for immunodominant linear neutralization epitope(s). Synthetic peptide VP2-28, corresponding to residues 136-150 of VP2, was found to bind to and inhibit the binding to EV71 of nMAb MAB979 that was found to have cross-neutralizing activity against different genotypes of EV71 virus. In addition, VP2-28 was found to be recognized only by neutralizing antisera generated from rabbits immunized with the formalin-inactivated whole EV71 virion vaccine but not by antisera from immunized mice and rats. During the epitope mapping, a murine EV71 genotype- and strain-specific linear neutralization epitope VP1-43 was identified within residues 211-220 of VP1. Furthermore, based on sequence alignment and structure prediction analysis using poliovirus as the template for molecular modeling, the VP1-43 and VP2-28 epitopes were shown to run in parallel within 0.1 nm and form a rim of the canyon at the junction site of VP1 and VP2 in the viral capsid. In mouse, rat and rabbit immunogenicity studies, a dose-dependent relationship between the number of VP2-28 epitope units measured by a quantitative assay in vaccine preparations and the magnitude of neutralizing titers was demonstrated. VP2-28 has amino acid sequences that are highly conserved among EV71 genotypes, is not affected by formalin-treatment and long-term storage. Thus, VP2-28 could be used as the surrogate biomarker in the potency testing of candidate EV71 vaccines.
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Anticuerpos Antivirales/inmunología , Proteínas de la Cápside/inmunología , Enterovirus/inmunología , Mapeo Epitopo , Epítopos Inmunodominantes/química , Epítopos Inmunodominantes/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales/sangre , Proteínas de la Cápside/química , Línea Celular Tumoral , Chlorocebus aethiops , Reacciones Cruzadas , Enterovirus/clasificación , Enterovirus/genética , Infecciones por Enterovirus/prevención & control , Infecciones por Enterovirus/virología , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Pruebas de Neutralización , Péptidos/síntesis química , Péptidos/inmunología , Poliovirus/química , Poliovirus/genética , Conejos , Ratas , Vacunas de Productos Inactivados/inmunología , Células Vero , Vacunas Virales/inmunologíaRESUMEN
BACKGROUND: Highly pathogenic influenza viruses pose a constant threat which could lead to a global pandemic. Vaccination remains the principal measure to reduce morbidity and mortality from such pandemics. The availability and surging demand for pandemic vaccines needs to be addressed in the preparedness plans. This study presents an improved high-yield manufacturing process for the inactivated influenza H5N1 vaccines using Madin-Darby canine kidney (MDCK) cells grown in a serum-free (SF) medium microcarrier cell culture system. PRINCIPAL FINDING: The current study has evaluated the performance of cell adaptation switched from serum-containing (SC) medium to several commercial SF media. The selected SF medium was further evaluated in various bioreactor culture systems for process scale-up evaluation. No significant difference was found in the cell growth in different sizes of bioreactors studied. In the 7.5 L bioreactor runs, the cell concentration reached to 2.3 × 10(6) cells/mL after 5 days. The maximum virus titers of 1024 Hemagglutinin (HA) units/50 µL and 7.1 ± 0.3 × 10(8) pfu/mL were obtained after 3 days infection. The concentration of HA antigen as determined by SRID was found to be 14.1 µg/mL which was higher than those obtained from the SC medium. A mouse immunogenicity study showed that the formalin-inactivated purified SF vaccine candidate formulated with alum adjuvant could induce protective level of virus neutralization titers similar to those obtained from the SC medium. In addition, the H5N1 viruses produced from either SC or SF media showed the same antigenic reactivity with the NIBRG14 standard antisera. CONCLUSIONS: The advantages of this SF cell-based manufacturing process could reduce the animal serum contamination, the cost and lot-to-lot variation of SC medium production. This study provides useful information to manufacturers that are planning to use SF medium for cell-based influenza vaccine production.
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Subtipo H5N1 del Virus de la Influenza A/crecimiento & desarrollo , Vacunas contra la Influenza/biosíntesis , Gripe Aviar/prevención & control , Vacunas de Productos Inactivados/biosíntesis , Animales , Reactores Biológicos , Aves , Técnicas de Cultivo de Célula/métodos , Línea Celular , Proliferación Celular , Medio de Cultivo Libre de Suero , Perros , PandemiasRESUMEN
Polysaccharide-based vaccines against Neisseria meningitidis (Nm) serogroups A, C, Y and W135 have been available since 1970, but similar vaccine candidates developed for Nm group B (NmB) have not been successful due to both poor immunogenicity and their potential immunological cross-reactivity with human neurological tissue. In previous reports, a protective antigen and vaccine candidate, Ag473, was identified using proteomics and NmB-specific bactericidal monoclonal antibody. To initiate human phase one clinical trials, antigen production and characterization, pre-clinical toxicology and animal studies are required. In the present study, we report the biochemical characterization of Escherichia coli-expressed recombinant Ag473 (rAg473). Using MALDI-TOF mass analysis, chromatographically purified rAg473 was found to have two major isoforms that have molecular masses of 11,306 and 11,544amu, respectively. The isoforms were separated using RP-HPLC and pooled into two fractions. Based on the chromatogram, the ratio of lipoproteins in fractions #1 and #2 was found to be 1-2. GC-MS analysis of lipoproteins was performed, and the acylated fatty acids were identified. The results indicated that the first lipoproteins in fraction #1 contained the lipids palmitic acid (C16:0), cyclopropaneoctanoic acid (C17:1) and, predominately, stearic acid (C18:0). A different lipid composition of cyclopropaneoctanoic acid (C17:1), oleic acid (C18:1) and, predominately, palmitic acid (C16:0) was found in the second lipoprotein fraction. Both lipoprotein isoforms were tested and found to have Toll-like receptor (TLR) agonist activity in stimulating cytokine secretion from THP-1 cells. Circular dichroism (CD) analysis showed the secondary structure of rAg473 to be dominated by α-helices (48%), and the overall protein structure was stable up to 60°C and could refold after having been exposed to a temperature cycle from 20 to 90°C. In addition, the solubility of rAg473 (5mg/mL) was not affected after several freeze-thaw cycles. These biophysical and immunological properties make rAg473 a good vaccine candidate against NmB.
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Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Neisseria meningitidis Serogrupo B/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/química , Antígenos Bacterianos/genética , Antígenos Bacterianos/aislamiento & purificación , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Cromatografía , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Ácidos Grasos/análisis , Cromatografía de Gases y Espectrometría de Masas , Expresión Génica , Calor , Inmunoglobulina G/sangre , Lipoproteínas/química , Lipoproteínas/genética , Lipoproteínas/inmunología , Lipoproteínas/aislamiento & purificación , Ratones , Ratones Endogámicos BALB C , Peso Molecular , Neisseria meningitidis Serogrupo B/genética , Conformación Proteica , Isoformas de Proteínas , Estabilidad Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Receptores Toll-Like/agonistasRESUMEN
BACKGROUND: Antigen sparing and cross-protective immunity are regarded as crucial in pandemic influenza vaccine development. Both targets can be achieved by adjuvantation strategy to elicit a robust and broadened immune response. We assessed the immunogenicity of an inactivated H5N1 whole-virion vaccine (A/Vietnam/1194/2004 NIBRG-14, clade 1) formulated with emulsified nanoparticles and investigated whether it can induce cross-clade protecting immunity. METHODOLOGY/PRINCIPAL FINDINGS: After formulation with PELC, a proprietary water-in-oil-in-water nanoemulsion comprising of bioresorbable polymer/Span(R)85/squalene, inactivated virus was intramuscularly administered to mice in either one-dose or two-dose schedule. We found that the antigen-specific serum antibody responses elicited after two doses of non-adjuvanted vaccine were lower than those observed after a single dose of adjuvanted vaccine, PELC and the conventional alum adjuvant as well. Moreover, 5 microg HA of PELC-formulated inactivated virus were capable of inducing higher antibodies than those obtained from alum-adjuvanted vaccine. In single-dose study, we found that encapsulating inactivated virus into emulsified PELC nanoparticles could induce better antibody responses than those formulated with PELC-adsorbed vaccine. However, the potency was rather reduced when the inactivated virus and CpG (an immunostimulatory oligodeoxynucleotide containing unmethylated cytosine-guanosine motifs) were co-encapsulated within the emulsion. Finally, the mice who received PELC/CpG(adsorption)-vaccine could easily and quickly reach 100% of seroprotection against a homologous virus strain and effective cross-protection against a heterologous virus strain (A/Whooper swan/Mongolia/244/2005, clade 2.2). CONCLUSIONS/SIGNIFICANCE: Encapsulating inactivated H5N1 influenza virus and CpG into emulsified nanoparticles critically influences the humoral responses against pandemic influenza. These results demonstrated that the use of PELC could be as antigen-sparing in preparation for a potential shortage of prophylactic vaccines against local infectious diseases, in particular pandemic influenza. Moreover, the cross-clade neutralizing antibody responses data verify the potential of such adjuvanted H5N1 candidate vaccine as an effective tool in pre-pandemic preparedness.