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1.
Proc Natl Acad Sci U S A ; 118(45)2021 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-34670842

RESUMO

Vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other pathogens with pandemic potential requires safe, protective, inexpensive, and easily accessible vaccines that can be developed and manufactured rapidly at a large scale. DNA vaccines can achieve these criteria, but induction of strong immune responses has often required bulky, expensive electroporation devices. Here, we report an ultra-low-cost (<1 USD), handheld (<50 g) electroporation system utilizing a microneedle electrode array ("ePatch") for DNA vaccination against SARS-CoV-2. The low cost and small size are achieved by combining a thumb-operated piezoelectric pulser derived from a common household stove lighter that emits microsecond, bipolar, oscillatory electric pulses and a microneedle electrode array that targets delivery of high electric field strength pulses to the skin's epidermis. Antibody responses against SARS-CoV-2 induced by this electroporation system in mice were strong and enabled at least 10-fold dose sparing compared to conventional intramuscular or intradermal injection of the DNA vaccine. Vaccination was well tolerated with mild, transient effects on the skin. This ePatch system is easily portable, without any battery or other power source supply, offering an attractive, inexpensive approach for rapid and accessible DNA vaccination to combat COVID-19, as well as other epidemics.


Assuntos
Vacinas contra COVID-19/administração & dosagem , COVID-19/imunologia , COVID-19/prevenção & controle , Eletroporação/instrumentação , SARS-CoV-2 , Vacinas de DNA/administração & dosagem , Animais , Vacinas contra COVID-19/genética , Vacinas contra COVID-19/imunologia , Custos e Análise de Custo , Eletroporação/economia , Eletroporação/métodos , Desenho de Equipamento , Feminino , Genes Reporter , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Microeletrodos , Agulhas , Pandemias/prevenção & controle , Estudo de Prova de Conceito , Ratos , Ratos Wistar , Pele/imunologia , Pele/metabolismo , Transfecção , Vacinação/economia , Vacinação/instrumentação , Vacinação/métodos , Vacinas de DNA/genética , Vacinas de DNA/imunologia
2.
J Infect Dis ; 218(suppl_5): S545-S552, 2018 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-29893888

RESUMO

In this study, we investigated immune responses induced by purified Ebola virus (EBOV) soluble glycoprotein (sGP) subunit vaccines via intradermal immunization with microneedle (MN) patches in comparison with intramuscular (IM) injection in mice. Our results showed that MN delivery of EBOV sGP was superior to IM injection in eliciting higher levels and longer lasting antibody responses against EBOV sGP and GP antigens. Moreover, sGP-specific immune responses induced by MN or IM immunizations were effectively augmented by formulating sGP with a saponin-based adjuvant, and they were shown to confer complete protection of mice against lethal mouse-adapted EBOV (MA-EBOV) challenge. In comparison, mice that received sGP without adjuvant by MN or IM immunizations succumbed to lethal MA-EBOV challenge. These results show that immunization with EBOV sGP subunit vaccines with adjuvant by MN patches, which have been shown to provide improved safety and thermal stability, is a promising approach to protect against EBOV infection.


Assuntos
Vacinas contra Ebola/imunologia , Vacinação , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Antivirais/biossíntese , Formação de Anticorpos , Vacinas contra Ebola/administração & dosagem , Feminino , Células HEK293 , Células HeLa , Doença pelo Vírus Ebola/prevenção & controle , Humanos , Injeções Intramusculares , Camundongos , Camundongos Endogâmicos BALB C , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologia
3.
J Virol ; 89(2): 1205-17, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25392212

RESUMO

UNLABELLED: The Ebola virus (EBOV) surface glycoprotein (GP1,2) mediates host cell attachment and fusion and is the primary target for host neutralizing antibodies. Expression of GP1,2 at high levels disrupts normal cell physiology, and EBOV uses an RNA-editing mechanism to regulate expression of the GP gene. In this study, we demonstrate that high levels of GP1,2 expression impair production and release of EBOV virus-like particles (VLPs) as well as infectivity of GP1,2-pseudotyped viruses. We further show that this effect is mediated through two mechanisms. First, high levels of GP1,2 expression reduce synthesis of other proteins needed for virus assembly. Second, viruses containing high levels of GP1,2 are intrinsically less infectious, possibly due to impaired receptor binding or endosomal processing. Importantly, proteolysis can rescue the infectivity of high-GP1,2-containing viruses. Taken together, our findings indicate that GP1,2 expression levels have a profound effect on factors that contribute to virus fitness and that RNA editing may be an important mechanism employed by EBOV to regulate GP1,2 expression in order to optimize virus production and infectivity. IMPORTANCE: The Ebola virus (EBOV), as well as other members of the Filoviridae family, causes severe hemorrhagic fever that is highly lethal, with up to 90% mortality. The EBOV surface glycoprotein (GP1,2) plays important roles in virus infection and pathogenesis, and its expression is tightly regulated by an RNA-editing mechanism during virus replication. Our study demonstrates that the level of GP1,2 expression profoundly affects virus particle production and release and uncovers a new mechanism by which Ebola virus infectivity is regulated by the level of GP1,2 expression. These findings extend our understanding of EBOV infection and replication in adaptation of host environments, which will aid the development of countermeasures against EBOV infection.


Assuntos
Ebolavirus/fisiologia , Regulação Viral da Expressão Gênica , Glicoproteínas de Membrana/biossíntese , Internalização do Vírus , Liberação de Vírus , Replicação Viral , Linhagem Celular , Humanos , Edição de RNA
4.
Pharm Res ; 33(4): 868-78, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26620313

RESUMO

PURPOSE: This study tested the hypothesis that encapsulation of influenza vaccine in microneedle patches increases vaccine stability during storage at elevated temperature. METHODS: Whole inactivated influenza virus vaccine (A/Puerto Rico/8/34) was formulated into dissolving microneedle patches and vaccine stability was evaluated by in vitro and in vivo assays of antigenicity and immunogenicity after storage for up to 3 months at 4, 25, 37 and 45°C. RESULTS: While liquid vaccine completely lost potency as determined by hemagglutination (HA) activity within 1-2 weeks outside of refrigeration, vaccine in microneedle patches lost 40-50% HA activity during or shortly after fabrication, but then had no significant additional loss of activity over 3 months of storage, independent of temperature. This level of stability required reduced humidity by packaging with desiccant, but was not affected by presence of oxygen. This finding was consistent with additional stability assays, including antigenicity of the vaccine measured by ELISA, virus particle morphological structure captured by transmission electron microscopy and protective immune responses by immunization of mice in vivo. CONCLUSIONS: These data show that inactivated influenza vaccine encapsulated in dissolving microneedle patches has enhanced stability during extended storage at elevated temperatures.


Assuntos
Sistemas de Liberação de Medicamentos , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/administração & dosagem , Agulhas , Infecções por Orthomyxoviridae/prevenção & controle , Vacinas de Produtos Inativados/administração & dosagem , Animais , Armazenamento de Medicamentos , Temperatura Alta , Humanos , Imunização , Vacinas contra Influenza/imunologia , Vacinas contra Influenza/uso terapêutico , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Camundongos , Infecções por Orthomyxoviridae/imunologia , Adesivo Transdérmico , Vacinas de Produtos Inativados/imunologia , Vacinas de Produtos Inativados/uso terapêutico
5.
J Infect Dis ; 212 Suppl 2: S398-403, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-25877553

RESUMO

In addition to its surface glycoprotein (GP), Ebola virus directs the production of large quantities of a truncated glycoprotein isoform (sGP) that is secreted into the extracellular space. We recently reported that sGP actively diverts host antibody responses against the epitopes that it shares with GP and thereby allows itself to absorb anti-GP antibodies, a phenomenon we termed "antigenic subversion." To investigate the effect of antigenic subversion by sGP on protection against virus infection, we compared immune responses induced by different prime-boost immunization regimens with GP and sGP DNA vaccines in mice and their efficacy against lethal Ebola virus challenge. Similar levels of anti-GP antibodies were induced by 2 immunizations with sGP and GP DNA vaccines. However, 2 immunizations with GP but not sGP DNA vaccine fully protected mice from lethal challenge. Boosting with sGP or GP DNA vaccine in mice that had been primed by GP or sGP DNA vaccine augmented the levels of anti-GP antibody responses and further improved protective efficacy against Ebola virus infection. These results show that both the quality and the levels of anti-GP antibody responses affect the efficacy of protection against Ebola virus infection.


Assuntos
Vacinas contra Ebola/imunologia , Ebolavirus/imunologia , Glicoproteínas/imunologia , Doença pelo Vírus Ebola/imunologia , Isoformas de Proteínas/imunologia , Vacinas de DNA/imunologia , Proteínas Virais/imunologia , Animais , Anticorpos Antivirais/imunologia , Formação de Anticorpos/imunologia , Feminino , Células HEK293 , Doença pelo Vírus Ebola/virologia , Humanos , Imunização Secundária/métodos , Camundongos , Camundongos Endogâmicos BALB C , Vacinação/métodos
6.
J Virol ; 88(10): 5677-86, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24623422

RESUMO

UNLABELLED: Sporadic activity by H5N2 influenza viruses has been observed in chickens in Taiwan from 2003 to 2012. The available information suggests that these viruses were generated by reassortment between a Mexican-like H5N2 virus and a local enzootic H6N1 virus. Yet the origin, prevalence, and pathogenicity of these H5N2 viruses have not been fully defined. Following the 2012 highly pathogenic avian influenza (HPAI) outbreaks, surveillance was conducted from December 2012 to July 2013 at a live-poultry wholesale market in Taipei. Our findings showed that H5N2 and H6N1 viruses cocirculated at low levels in chickens in Taiwan. Phylogenetic analyses revealed that all H5N2 viruses had hemagglutinin (HA) and neuraminidase (NA) genes derived from a 1994 Mexican-like virus, while their internal gene complexes were incorporated from the enzootic H6N1 virus lineage by multiple reassortment events. Pathogenicity studies demonstrated heterogeneous results even though all tested viruses had motifs (R-X-K/R-R) supportive of high pathogenicity. Serological surveys for common subtypes of avian viruses confirmed the prevalence of the H5N2 and H6N1 viruses in chickens and revealed an extraordinarily high seroconversion rate to an H9N2 virus, a subtype that is not found in Taiwan but is prevalent in mainland China. These findings suggest that reassortant H5N2 viruses, together with H6N1 viruses, have become established and enzootic in chickens throughout Taiwan and that a large-scale vaccination program might have been conducted locally that likely led to the introduction of the 1994 Mexican-like virus to Taiwan in 2003. IMPORTANCE: H5N2 avian influenza viruses first appeared in chickens in Taiwan in 2003 and caused a series of outbreaks afterwards. Phylogenetic analyses show that the chicken H5N2 viruses have H5 and N2 genes that are closely related to those of a vaccine strain originating from Mexico in 1994, while the contemporary duck H5N2 viruses in Taiwan belong to the Eurasian gene pool. The unusually high similarity of the chicken H5N2 viruses to the Mexican vaccine strain suggests that these viruses might have been introduced to Taiwan by using inadequately inactivated or attenuated vaccines. These chicken H5N2 viruses are developing varying levels of pathogenicity that could lead to significant consequences for the local poultry industry. These findings emphasize the need for strict quality control and competent oversight in the manufacture and usage of avian influenza virus vaccines and indicate that alternatives to widespread vaccination may be desirable.


Assuntos
Evolução Molecular , Vírus da Influenza A Subtipo H5N2/classificação , Vírus da Influenza A Subtipo H5N2/genética , Influenza Aviária/virologia , Animais , Galinhas , Análise por Conglomerados , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H5N2/isolamento & purificação , Dados de Sequência Molecular , Neuraminidase/genética , Filogenia , Vírus Reordenados/classificação , Vírus Reordenados/genética , Vírus Reordenados/isolamento & purificação , Análise de Sequência de DNA , Taiwan , Proteínas Virais/genética
7.
PLoS Pathog ; 8(12): e1003065, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23271969

RESUMO

In addition to its surface glycoprotein (GP(1,2)), Ebola virus (EBOV) directs the production of large quantities of a truncated glycoprotein isoform (sGP) that is secreted into the extracellular space. The generation of secreted antigens has been studied in several viruses and suggested as a mechanism of host immune evasion through absorption of antibodies and interference with antibody-mediated clearance. However such a role has not been conclusively determined for the Ebola virus sGP. In this study, we immunized mice with DNA constructs expressing GP(1,2) and/or sGP, and demonstrate that sGP can efficiently compete for anti-GP(12) antibodies, but only from mice that have been immunized by sGP. We term this phenomenon "antigenic subversion", and propose a model whereby sGP redirects the host antibody response to focus on epitopes which it shares with membrane-bound GP(1,2), thereby allowing it to absorb anti-GP(1,2) antibodies. Unexpectedly, we found that sGP can also subvert a previously immunized host's anti-GP(1,2) response resulting in strong cross-reactivity with sGP. This finding is particularly relevant to EBOV vaccinology since it underscores the importance of eliciting robust immunity that is sufficient to rapidly clear an infection before antigenic subversion can occur. Antigenic subversion represents a novel virus escape strategy that likely helps EBOV evade host immunity, and may represent an important obstacle to EBOV vaccine design.


Assuntos
Anticorpos Antivirais/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Evasão da Resposta Imune/imunologia , Animais , Reações Cruzadas/efeitos dos fármacos , Reações Cruzadas/genética , Vacinas contra Ebola/imunologia , Vacinas contra Ebola/farmacologia , Ebolavirus/genética , Feminino , Células HeLa , Doença pelo Vírus Ebola/genética , Doença pelo Vírus Ebola/prevenção & controle , Humanos , Evasão da Resposta Imune/genética , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Vacinas de DNA/imunologia , Vacinas de DNA/farmacologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia
8.
J Virol ; 85(16): 8241-52, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21632762

RESUMO

Effective, safe, and affordable rabies vaccines are still being sought. Newcastle disease virus (NDV), an avian paramyxovirus, has shown promise as a vaccine vector for mammals. Here, we generated a recombinant avirulent NDV La Sota strain expressing the rabies virus glycoprotein (RVG) and evaluated its potential to serve as a vaccine against rabies. The recombinant virus, rL-RVG, retained its high-growth property in chicken eggs, with titers of up to 109·8 50% egg infective doses (EID50)/ml of allantoic fluid. RVG expression enabled rL-RVG to spread from cell to cell in a rabies virus-like manner, and RVG was incorporated on the surface of the rL-RVG viral particle. RVG incorporation did not alter the trypsin-dependent infectivity of the NDV vector in mammalian cells. rL-RVG and La Sota NDV showed similar levels of sensitivity to a neutralization antibody against NDV and similar levels of resistance to a neutralization antibody against rabies virus. Animal studies demonstrated that rL-RVG is safe in several species, including cats and dogs, when administered as multiple high doses of recombinant vaccine. Intramuscular vaccination with rL-RVG induced a substantial rabies virus neutralization antibody response and provided complete protection from challenge with circulating rabies virus strains. Most importantly, rL-RVG induced strong and long-lasting protective neutralization antibody responses to rabies virus in dogs and cats. A low vaccine dose of 108·³ EID50 completely protected dogs from challenge with a circulating strain of rabies virus for more than a year. This is the first study to demonstrate that immunization with an NDV-vectored vaccine can induce long-lasting, systemic protective immunity against rabies.


Assuntos
Anticorpos Neutralizantes/biossíntese , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/imunologia , Vacina Antirrábica , Vírus da Raiva/imunologia , Raiva/prevenção & controle , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/imunologia , Western Blotting , Gatos , Linhagem Celular Tumoral , Chlorocebus aethiops , Cricetinae , Cães , Imunofluorescência , Regulação Viral da Expressão Gênica , Glicoproteínas/biossíntese , Glicoproteínas/genética , Glicoproteínas/imunologia , Humanos , Camundongos , Vírus da Doença de Newcastle/fisiologia , Raiva/imunologia , Vacina Antirrábica/administração & dosagem , Vacina Antirrábica/genética , Vacina Antirrábica/imunologia , Vírus da Raiva/genética , Vacinação , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia , Células Vero , Proteínas do Envelope Viral/biossíntese
9.
Proc Natl Acad Sci U S A ; 106(19): 7968-73, 2009 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-19416832

RESUMO

Influenza prophylaxis would benefit from a simple method to administer influenza vaccine into skin without the need for hypodermic needles. In this study, solid metal microneedle arrays (MNs) were investigated as a system for cutaneous vaccine delivery using influenza virus antigen. The MNs with 5 monument-shaped microneedles per array were produced and coated with inactivated influenza virus A/PR/8/34 (IIV). As much as 10 microg of viral proteins could be coated onto an array of 5 microneedles, and the coated IIV was delivered into skin at high efficiency within minutes. The coated MNs were used to immunize mice in comparison with conventional intramuscular injection at the same dose. Analysis of immune responses showed that a single immunization with IIV-coated MNs induced strong antibody responses against influenza virus, with significant levels of hemagglutination inhibition activities (>1:40), which were comparable to those induced by conventional intramuscular immunization. Moreover, mice immunized by a single dose of IIV coated on MNs were effectively protected against lethal challenge by a high dose of mouse-adapted influenza virus A/PR/8/34. These results show that MNs are highly effective as a simple method of vaccine delivery to elicit protective immune responses against virus infection.


Assuntos
Imunização/métodos , Vacinas contra Influenza/administração & dosagem , Orthomyxoviridae/imunologia , Administração Cutânea , Animais , Anticorpos/química , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Hemaglutinação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Vírus da Influenza A/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Vacinas/química
10.
J Virol ; 84(17): 8926-36, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20538851

RESUMO

The molecular mechanisms associated with rabies virus (RV) virulence are not fully understood. In this study, the RV Flury low-egg-passage (LEP) and high-egg-passage (HEP) strains were used as models to explore the attenuation mechanism of RV. The results of our studies confirmed that the R333Q mutation in the glycoprotein (G(R333Q)) is crucial for the attenuation of Flury RV in mice. The R333Q mutation is stably maintained in the HEP genome background but not in the LEP genome background during replication in mouse brain tissue or cell culture. Further investigation using chimeric viruses revealed that the polymerase L gene determines the genetic stability of the G(R333Q) mutation during replication. Moreover, a recombinant RV containing the LEP G protein with the R333Q mutation and the HEP L gene showed significant attenuation, genetic stability, enhancement of apoptosis, and immunogenicity. These results indicate that attenuation of the RV Flury strain results from the coevolution of G and L elements and provide important information for the generation of safer and more effective modified live rabies vaccine.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Doenças do Cão/virologia , Glicoproteínas/genética , Mutação de Sentido Incorreto , Vacina Antirrábica/genética , Vírus da Raiva/genética , Raiva/veterinária , Proteínas Virais/metabolismo , Animais , Sequência de Bases , Encéfalo/imunologia , Encéfalo/virologia , Linhagem Celular , Cricetinae , RNA Polimerases Dirigidas por DNA/genética , Doenças do Cão/imunologia , Cães , Feminino , Glicoproteínas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Raiva/imunologia , Raiva/virologia , Vacina Antirrábica/imunologia , Vírus da Raiva/enzimologia , Vírus da Raiva/patogenicidade , Vírus da Raiva/fisiologia , Proteínas Virais/genética , Virulência , Replicação Viral
11.
Virol J ; 8: 454, 2011 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-21943337

RESUMO

The rabies Flury Low Egg Passage virus (LEP) has been widely used as a seed virus to generate inactive vaccine. Here, we established a reverse genetic system for LEP and generated a recombinant LEP virus (rLEP-G) that carries two identical G genes. This recombinant virus showed similar properties to those of LEP with respect to in vitro growth, neurotropism index, and virulence in mice. rLEP-G produced 4.3-fold more G protein than did LEP in BHK-21 cells. The inactivated vaccine generated from rLEP-G induced significantly higher virus neutralization titers in mice and dogs than those produced in response to LEP-derived vaccine. Our results suggest that rLEP-G is an improved seed virus candidate for inactivated rabies virus vaccine manufacture.


Assuntos
Anticorpos Antivirais/biossíntese , Antígenos Virais/imunologia , Glicoproteínas/imunologia , Imunização , Vacina Antirrábica/administração & dosagem , Vírus da Raiva/imunologia , Raiva/prevenção & controle , Vacinas de Produtos Inativados/administração & dosagem , Vacinas Sintéticas/administração & dosagem , Proteínas do Envelope Viral/imunologia , Animais , Antígenos Virais/química , Antígenos Virais/genética , Western Blotting , Linhagem Celular , Cricetinae , Cães , Feminino , Glicoproteínas/química , Glicoproteínas/genética , Injeções Intramusculares , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos , Reação em Cadeia da Polimerase , Raiva/imunologia , Raiva/veterinária , Raiva/virologia , Vacina Antirrábica/genética , Vacina Antirrábica/imunologia , Vírus da Raiva/química , Vírus da Raiva/genética , Vírus da Raiva/patogenicidade , Transdução Genética , Vacinas de Produtos Inativados/genética , Vacinas de Produtos Inativados/imunologia , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Virulência/genética , Virulência/imunologia
12.
J Biomed Biotechnol ; 2010: 497219, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20508832

RESUMO

The immune response induced by immunization with HIV Env DNA and virus-like particle (VLP) vaccines was investigated. Immunization with the HIV Env DNA vaccine induced a strong CD8 T cell response but relatively weak antibody response against the HIV Env whereas immunization with VLPs induced higher levels of antibody responses but little CD8 T cell response. Interestingly, immunization with a mixture the HIV Env DNA and VLP vaccines induced enhanced CD8 T cell and antibody responses. Further, it was observed that the mixing of DNA and VLP vaccines during immunization is necessary for augmenting induction of CD8 T cell responses and such augmentation of CD8 T cell responses was also observed by mixing the HIV Env DNA vaccine with control VLPs. These results show that immunization with a mixture of DNA and VLP vaccines combines advantages of both vaccine platforms for eliciting high levels of both antibody and CD8 T cell responses.

13.
Front Microbiol ; 11: 304, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32174901

RESUMO

Ebolavirus (EBOV) infection in humans causes severe hemorrhagic fevers with high mortality rates that range from 30 to 80% as shown in different outbreaks. Thus the development of safe and efficacious EBOV vaccines remains an important goal for biomedical research. We have shown in early studies that immunization with insect cell-produced EBOV virus-like particles (VLPs) is able to induce protect vaccinated mice against lethal EBOV challenge. In the present study, we investigated immune responses induced by Ebola VLPs via two different routes, intramuscular and intradermal immunizations, in guinea pigs. Analyses of antibody responses revealed that similar levels of total IgG antibodies against the EBOV glycoprotein (GP) were induced by the two different immunization methods. However, further characterization showed that the EBOV GP-specific antibodies induced by intramuscular immunization were mainly of the IgG2 subtype whereas both IgG1 and IgG2 antibodies against EBOV GP were induced by intradermal immunization. In contrast, antibody responses against the EBOV matrix protein VP40 induced by intramuscular or intradermal immunizations exhibited similar IgG1 and IgG2 profiles. More interestingly, we found that the sites that the IgG1 antibodies induced by intradermal immunizations bind to in GP are different from those that bind to the IgG2 antibodies induced by intramuscular immunization. Further analyses revealed that sera from all vaccinated guinea pigs exhibited neutralizing activity against Ebola GP-mediated HIV pseudovirion infection at high levels. Moreover, all EBOV VLP-vaccinated guinea pigs survived the challenge by a high dose (1000 pfu) of guinea pig-adapted EBOV, while all control guinea pigs immunized with irrelevant VLPs succumbed to the challenge. The induction of both IgG1 and IgG2 antibody responses that recognized broader sites in GP by intradermal immunization of EBOV VLPs indicates that this approach may represent a more advantageous route of vaccination against virus infection.

14.
J Virol ; 82(1): 220-8, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17942562

RESUMO

In 2001 and 2003, we isolated two H5N1 viruses, A/swine/Fujian/1/01 (SW/FJ/01) and A/swine/Fujian/1/03 (SW/FJ/03), from pigs in Fujian Province, southern China. Genetically, these two viruses are similar, although the NS gene of the SW/FJ/03 virus has a 15-nucleotide deletion at coding positions 612 to 626. The SW/FJ/01 virus is highly lethal for chickens, whereas the SW/FJ/03 virus is nonpathogenic for chickens when administrated intravenously or intranasally. To understand the molecular basis for the difference in virulence, we used reverse genetics to create a series of single-gene recombinants of both viruses. We found that a recombinant virus containing the mutated NS gene from the SW/FJ/03 virus in the SW/FJ/01 virus background was completely attenuated in chickens. We also found that viruses expressing the mutant NS1 protein of SW/FJ/03 did not antagonize the induction of interferon (IFN) protein. Conversely, only the recombinant virus containing the wild-type SW/FJ/01 NS gene in the SW/FJ/03 background was lethal in chickens and antagonized IFN protein levels. Further, we proved that the NS1 genes of the two viruses differ in their stabilities in the host cells and in their abilities to interact with the chicken cleavage and polyadenylation specificity factor. These results indicate that the deletion of amino acids 191 to 195 of the NS1 protein is critical for the attenuation of the SW/FJ/03 virus in chickens and that this deletion affects the ability of the virus to antagonize IFN induction in host cells.


Assuntos
Virus da Influenza A Subtipo H5N1/patogenicidade , Vírus da Influenza A/patogenicidade , Influenza Aviária/virologia , Deleção de Sequência , Proteínas não Estruturais Virais/genética , Animais , Encéfalo/virologia , Embrião de Galinha , Galinhas , Feminino , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/imunologia , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Vírus da Influenza A/genética , Vírus da Influenza A/imunologia , Vírus da Influenza A/isolamento & purificação , Interferons/antagonistas & inibidores , Interferons/imunologia , Rim/virologia , Dose Letal Mediana , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , Baço/virologia , Proteínas não Estruturais Virais/imunologia , Virulência/genética
15.
Sci Rep ; 8(1): 11193, 2018 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-30046140

RESUMO

Development of a safe and efficacious filovirus vaccine is of high importance to public health. In this study, we compared immune responses induced by Ebola virus (EBOV) glycoprotein (GP) subunit vaccines via intradermal immunization with microneedle (MN) patches and the conventional intramuscular (IM) injection in mice, which showed that MN delivery of GP induced higher levels and longer lasting antibody responses against GP than IM injection. Further, we found that EBOV GP in formulation with a saponin-based adjuvant, Matrix-M, can be efficiently loaded onto MN patches. Co-delivery of Matrix-M with GP significantly enhanced induction of antibody responses by MN delivery, as also observed for IM injection. Results from challenge studies showed that all mice that received the GP/adjuvant formulation by MN or IM immunizations were protected from lethal EBOV challenge. Further, 4 out of 5 mice vaccinated by MN delivery of unadjuvanted GP also survived the challenge, whereas only 1 out of 5 mice vaccinated by IM injection of unadjuvanted GP survived the challenge. These results demonstrate that MN patch delivery of EBOV GP subunit vaccines, which is expected to enable improved safety and thermal stability, can confer effective protection against EBOV infection that is superior to IM vaccination.


Assuntos
Anticorpos Antivirais/imunologia , Vacinas contra Ebola/imunologia , Glicoproteínas/administração & dosagem , Doença pelo Vírus Ebola/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Antivirais/genética , Formação de Anticorpos/imunologia , Ebolavirus/imunologia , Ebolavirus/patogenicidade , Glicoproteínas/imunologia , Doença pelo Vírus Ebola/genética , Doença pelo Vírus Ebola/prevenção & controle , Humanos , Imunização , Injeções Intradérmicas , Camundongos , Vacinação , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/uso terapêutico
17.
Microbes Infect ; 19(12): 626-634, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28899815

RESUMO

Elderly humans over 65 years old are at great risk to pathogenesis by influenza virus infection. However, although influenza vaccines provide effective protection in healthy young adults, protection of elderly adults is substantially lower even with a good match between the vaccine and the circulating influenza virus. To gain insight of the underlying mechanism for the reduced immunogenicity of influenza vaccines in the aged population, we investigated immunogenicity of influenza virus-like particle vaccines in aged mice, which represent a useful model for studying aging associated impairment in immune responses. Specifically, we investigated the effect of inhibiting regulatory T cells in aged mice on induction of protective immune responses by influenza vaccines. Our results showed that injecting anti-CD25 antibodies could down-regulate CD25 on the surface of regulatory T cells and significantly increase the levels of antibody responses induced by VLP immunization in aged mice. Further, the profiles of antibody responses were also changed towards Th1 type by regulatory T cell blockage in aged mice. Moreover, aged mice that were treated by anti-CD25 antibodies prior to vaccination were more effectively protected against lethal influenza virus challenge.


Assuntos
Envelhecimento/imunologia , Anticorpos Antivirais/sangue , Vírus da Influenza A Subtipo H1N1/imunologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Linfócitos T Reguladores/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Formação de Anticorpos/imunologia , Feminino , Imunogenicidade da Vacina/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Vacinas contra Influenza/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/virologia , Células Th1/imunologia , Vacinação
20.
Emerg Microbes Infect ; 5: e65, 2016 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-27329850

RESUMO

After three decades of intensive research efforts, an effective vaccine against HIV-1 remains to be developed. Several broadly neutralizing antibodies to HIV-1, such as 10E8, recognize the membrane proximal external region (MPER) of the HIV-1 gp41 protein. Thus, the MPER is considered to be a very important target for vaccine design. However, the MPER segment has very weak immunogenicity and tends to insert its epitope residues into the cell membrane, thereby avoiding antibody binding. To address this complication in vaccine development, we herein designed a peptide, designated 10E8-4P, containing four copies of the 10E8 epitope as an immunogen. As predicted by structural simulation, 10E8-4P exhibits a well-arranged tandem helical conformation, with the key residues in the 10E8 epitope oriented at different angles, thus suggesting that some of these key residues may be exposed outside of the lipid membrane. Compared with a peptide containing a single 10E8 epitope (10E8-1P), 10E8-4P not only exhibited better antigenicity but also elicited neutralizing antibody response against HIV-1 pseudoviruses, whereas 10E8-1P could not induce detectable neutralizing antibody response. Importantly, antibodies elicited by 10E8-4P also possessed a strong ability to activate an antibody-dependent cell-mediated cytotoxicity (ADCC) reporter gene, thus suggesting that they may have ADCC activity. Therefore, this strategy shows promise for further optimization and application in future HIV-1 vaccine design.


Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/biossíntese , Citotoxicidade Celular Dependente de Anticorpos/genética , Epitopos/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Vacinas contra a AIDS/química , Animais , Dicroísmo Circular , Ensaio de Imunoadsorção Enzimática , Epitopos/química , Genes Reporter , Anticorpos Anti-HIV/biossíntese , Humanos , Testes de Neutralização , Ligação Proteica , Coelhos
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