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1.
Microbes Infect ; 22(2): 74-79, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32017984

RESUMO

On 10 January 2020, a new coronavirus causing a pneumonia outbreak in Wuhan City in central China was denoted as 2019-nCoV by the World Health Organization (WHO). As of 24 January 2020, there were 887 confirmed cases of 2019-nCoV infection, including 26 deaths, reported in China and other countries. Therefore, combating this new virus and stopping the epidemic is a matter of urgency. Here, we focus on advances in research and development of fast diagnosis methods, as well as potential prophylactics and therapeutics to prevent or treat 2019-nCoV infection.


Assuntos
Betacoronavirus/patogenicidade , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/terapia , Pneumonia Viral/diagnóstico , Pneumonia Viral/terapia , Sequência de Aminoácidos , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Antivirais/uso terapêutico , Betacoronavirus/isolamento & purificação , China/epidemiologia , Humanos , Técnicas de Amplificação de Ácido Nucleico , RNA Viral/análise , Alinhamento de Sequência , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Virais/imunologia , Vacinas Virais/uso terapêutico
2.
Emerg Microbes Infect ; 9(1): 382-385, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32065055

RESUMO

The newly identified 2019 novel coronavirus (2019-nCoV) has caused more than 11,900 laboratory-confirmed human infections, including 259 deaths, posing a serious threat to human health. Currently, however, there is no specific antiviral treatment or vaccine. Considering the relatively high identity of receptor-binding domain (RBD) in 2019-nCoV and SARS-CoV, it is urgent to assess the cross-reactivity of anti-SARS CoV antibodies with 2019-nCoV spike protein, which could have important implications for rapid development of vaccines and therapeutic antibodies against 2019-nCoV. Here, we report for the first time that a SARS-CoV-specific human monoclonal antibody, CR3022, could bind potently with 2019-nCoV RBD (KD of 6.3 nM). The epitope of CR3022 does not overlap with the ACE2 binding site within 2019-nCoV RBD. These results suggest that CR3022 may have the potential to be developed as candidate therapeutics, alone or in combination with other neutralizing antibodies, for the prevention and treatment of 2019-nCoV infections. Interestingly, some of the most potent SARS-CoV-specific neutralizing antibodies (e.g. m396, CR3014) that target the ACE2 binding site of SARS-CoV failed to bind 2019-nCoV spike protein, implying that the difference in the RBD of SARS-CoV and 2019-nCoV has a critical impact for the cross-reactivity of neutralizing antibodies, and that it is still necessary to develop novel monoclonal antibodies that could bind specifically to 2019-nCoV RBD.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Reações Cruzadas , Glicoproteína da Espícula de Coronavírus/imunologia , Sequência de Aminoácidos , Infecções por Coronavirus , Humanos , Pneumonia Viral , Estrutura Terciária de Proteína , Vírus da SARS/imunologia
3.
Science ; 367(6483): 1260-1263, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32075877

RESUMO

The outbreak of a novel coronavirus (2019-nCoV) represents a pandemic threat that has been declared a public health emergency of international concern. The CoV spike (S) glycoprotein is a key target for vaccines, therapeutic antibodies, and diagnostics. To facilitate medical countermeasure development, we determined a 3.5-angstrom-resolution cryo-electron microscopy structure of the 2019-nCoV S trimer in the prefusion conformation. The predominant state of the trimer has one of the three receptor-binding domains (RBDs) rotated up in a receptor-accessible conformation. We also provide biophysical and structural evidence that the 2019-nCoV S protein binds angiotensin-converting enzyme 2 (ACE2) with higher affinity than does severe acute respiratory syndrome (SARS)-CoV S. Additionally, we tested several published SARS-CoV RBD-specific monoclonal antibodies and found that they do not have appreciable binding to 2019-nCoV S, suggesting that antibody cross-reactivity may be limited between the two RBDs. The structure of 2019-nCoV S should enable the rapid development and evaluation of medical countermeasures to address the ongoing public health crisis.


Assuntos
Betacoronavirus/química , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/ultraestrutura , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Betacoronavirus/metabolismo , Betacoronavirus/ultraestrutura , Reações Cruzadas , Microscopia Crioeletrônica , Processamento de Imagem Assistida por Computador , Modelos Moleculares , Peptidil Dipeptidase A/metabolismo , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Multimerização Proteica , Receptores Virais/metabolismo , Vírus da SARS/química , Vírus da SARS/imunologia , Vírus da SARS/ultraestrutura , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo
4.
Viruses ; 12(3)2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32106567

RESUMO

The beginning of 2020 has seen the emergence of COVID-19 outbreak caused by a novel coronavirus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). There is an imminent need to better understand this new virus and to develop ways to control its spread. In this study, we sought to gain insights for vaccine design against SARS-CoV-2 by considering the high genetic similarity between SARS-CoV-2 and SARS-CoV, which caused the outbreak in 2003, and leveraging existing immunological studies of SARS-CoV. By screening the experimentally-determined SARS-CoV-derived B cell and T cell epitopes in the immunogenic structural proteins of SARS-CoV, we identified a set of B cell and T cell epitopes derived from the spike (S) and nucleocapsid (N) proteins that map identically to SARS-CoV-2 proteins. As no mutation has been observed in these identified epitopes among the 120 available SARS-CoV-2 sequences (as of 21 February 2020), immune targeting of these epitopes may potentially offer protection against this novel virus. For the T cell epitopes, we performed a population coverage analysis of the associated MHC alleles and proposed a set of epitopes that is estimated to provide broad coverage globally, as well as in China. Our findings provide a screened set of epitopes that can help guide experimental efforts towards the development of vaccines against SARS-CoV-2.


Assuntos
Betacoronavirus/imunologia , Proteínas do Nucleocapsídeo/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Virais/imunologia , Betacoronavirus/genética , Infecções por Coronavirus/prevenção & controle , Mapeamento de Epitopos , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Genoma Viral , Humanos , Proteínas do Nucleocapsídeo/genética , Filogenia , Pneumonia Viral/prevenção & controle , Estrutura Terciária de Proteína , Vírus da SARS/genética , Vírus da SARS/imunologia , Glicoproteína da Espícula de Coronavírus/genética
5.
Emerg Microbes Infect ; 8(1): 1593-1603, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31711379

RESUMO

The ongoing Middle East respiratory syndrome coronavirus (MERS-CoV) outbreaks pose a worldwide public health threat. Blocking MERS-CoV zoonotic transmission from dromedary camels, the animal reservoir, could potentially reduce the number of primary human cases. Here we report MERS-CoV transmission from experimentally infected llamas to naïve animals. Directly inoculated llamas shed virus for at least 6 days and could infect all in-contact naïve animals 4-5 days after exposure. With the aim to block virus transmission, we examined the efficacy of a recombinant spike S1-protein vaccine. In contrast to naïve animals, in-contact vaccinated llamas did not shed infectious virus upon exposure to directly inoculated llamas, consistent with the induction of strong virus neutralizing antibody responses. Our data provide further evidence that vaccination of the reservoir host may impede MERS-CoV zoonotic transmission to humans.


Assuntos
Infecções por Coronavirus/prevenção & controle , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Glicoproteína da Espícula de Coronavírus/administração & dosagem , Vacinas Virais/administração & dosagem , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Camelídeos Americanos , Camelus/virologia , Infecções por Coronavirus/virologia , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinação , Vacinas Virais/genética , Vacinas Virais/imunologia , Zoonoses/imunologia , Zoonoses/prevenção & controle , Zoonoses/transmissão , Zoonoses/virologia
6.
Emerg Microbes Infect ; 8(1): 1528-1534, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31645223

RESUMO

Dromedary camels are important reservoir hosts of various coronaviruses, including Middle East respiratory syndrome coronavirus (MERS-CoV) that cause human infections. CoV genomes regularly undergo recombination during infection as observed in bat SARS-related CoVs. Here we report for the first time that only a small proportion of MERS-CoV receptor-binding domain positive (RBD) of spike protein positive camel sera in Kenya were also seropositive to MERS-CoV nucleocapsid (NP). In contrast, many of them contain antibodies against bat HKU8-related (HKU8r)-CoVs. Among 584 camel samples that were positive against MERS-CoV RBD, we found only 0.48 (8.22%) samples were also positive for NP. Furthermore, we found bat HKU8r-CoV NP antibody in 73 (12.5%) of the MERS-CoV RBD positive and NP negative samples, yet found only 3 (0.43%) of the HKU8r-CoV S1 antibody in the same samples. These findings may indicate co-infection with MERS-CoV and a HKU8r-CoV in camels. It may also raise the possibility of the circulation of a recombinant coronavirus virus with the spike of MERS-CoV and the NP of a HKU8r-CoV in Kenya. We failed to find molecular evidence of an HKU8r-CoV or a putative recombinant virus. Our findings should alert other investigators to look for molecular evidence of HKU8r-CoV or recombinants.


Assuntos
Camelus/virologia , Infecções por Coronavirus/veterinária , Coronavirus/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Animais , Anticorpos Antivirais/sangue , Camelus/sangue , Quirópteros/virologia , Coronavirus/genética , Coronavirus/isolamento & purificação , Infecções por Coronavirus/sangue , Infecções por Coronavirus/virologia , Quênia , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/isolamento & purificação , Proteínas do Nucleocapsídeo/imunologia , Recombinação Genética , Glicoproteína da Espícula de Coronavírus/imunologia
7.
J Vet Med Sci ; 81(10): 1438-1444, 2019 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-31474664

RESUMO

Transmissible gastroenteritis (TGE), caused by transmissible gastroenteritis virus (TGEV), is a highly infectious disease in pigs. Vaccination is an effective approach to prevent TGEV infection. Here, we evaluated the potential of TGEV S1 as a DNA vaccine and porcine interleukin (pIL)-12 as an adjuvant in a mouse model. A DNA vaccine was constructed with the TGEV S1 gene to induce immune response in an experimental mouse model; pIL-12 was chosen as the immunological adjuvant within this DNA vaccine. The pVAX1-(TGEV-S1) and pVAX1-(pIL-12) vectors were transfected into BHK-21 cells and expressed in vitro. Experimental mice were separately immunized with each of the recombinant plasmids and controls through the intramuscular route. The lymphocytes isolated from the blood and spleen were analyzed for proliferation, cytotoxic activities, and populations of CD4+ and CD8+ cells. The titers of TGEV S1 in an enzyme-linked immunosorbent assay (ELISA) and TGEV neutralizing antibodies and the concentrations of interferon (IFN)-γ and IL-4 were also analyzed in the serum. The plasmids pVAX1-(TGEV-S1) and pVAX1-(pIL-12) could be expressed in BHK-21 cells, and the combination of pVAX1-(TGEV-S1) and pVAX1-(pIL-12) could induce a significant increase in all markers. pIL-12 could act as an immunological adjuvant in the DNA vaccine for TGEV-S1. Furthermore, the DNA vaccine prepared using TGEV-S1 and porcine IL-12 could induce excellent humoral and cellular immune responses.


Assuntos
Interleucina-12/imunologia , Plasmídeos/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas de DNA/virologia , Adjuvantes Imunológicos , Animais , Gastroenterite Suína Transmissível/prevenção & controle , Imunidade Celular , Imunidade Humoral , Interleucina-12/genética , Camundongos , Modelos Animais , Glicoproteína da Espícula de Coronavírus/genética , Suínos , Vírus da Gastroenterite Transmissível/genética , Vírus da Gastroenterite Transmissível/imunologia , Vacinas de DNA/imunologia , Vacinas Virais/imunologia
8.
PLoS One ; 14(7): e0220196, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31329652

RESUMO

Middle East respiratory syndrome coronavirus (MERS-CoV) causes an acute and severe lower respiratory illness as well as vomiting, diarrhea, and renal failure. Because no licensed MERS-CoV vaccines are currently available, preventive and therapeutic measures are urgently needed. The surface spike (S) glycoprotein of MERS-CoV, which binds to the cellular receptor dipeptidyl peptidase 4 (DPP4), is considered as a major target for MERS-CoV vaccine development. Here, we designed recombinant replication-deficient adenovirus-based vaccines expressing the N-terminal domain (rAd/NTD) and receptor-binding domain (rAd/RBD) of the MERS-CoV S1 subunit and full-length Spike protein (rAd/Spike). We found that immunization with candidate vaccines via intranasal route induced S1-specific IgG antibodies and neutralizing antibodies against MERS spike pseudotyped virus. Especially, rAd/Spike induced the highest neutralizing antibody titer and the strongest cytokine-induced T cell responses among the three candidate vaccines. To compare the immune responses induced by different administration routes, rAd/Spike was administered via intranasal, sublingual, or intramuscular route. All these administration routes exhibited neutralizing effects in the serum. MERS-CoV-specific neutralizing IgA antibodies in the bronchoalveolar lavage fluid were only induced by intranasal and sublingual administration but not by intramuscular administration. Intranasal administration with rAd/Spike also created resident memory CD8 T cells in the airway and lung parenchyma. Taken together, our results showed that both the humoral and cellular immune responses are highly induced by rAd/Spike administration, suggesting that rAd/Spike may confer protection against MERS-CoV infection.


Assuntos
Infecções por Coronavirus/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinação/métodos , Adenoviridae/genética , Adenoviridae/imunologia , Administração Intranasal , Animais , Anticorpos Neutralizantes/imunologia , Células Cultivadas , Infecções por Coronavirus/prevenção & controle , Feminino , Células HEK293 , Humanos , Imunoglobulina A/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Glicoproteína da Espícula de Coronavírus/genética , Spodoptera , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia
9.
Nat Commun ; 10(1): 3068, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31296843

RESUMO

Most neutralizing antibodies against Middle East respiratory syndrome coronavirus (MERS-CoV) target the receptor-binding domain (RBD) of the spike glycoprotein and block its binding to the cellular receptor dipeptidyl peptidase 4 (DPP4). The epitopes and mechanisms of mAbs targeting non-RBD regions have not been well characterized yet. Here we report the monoclonal antibody 7D10 that binds to the N-terminal domain (NTD) of the spike glycoprotein and inhibits the cell entry of MERS-CoV with high potency. Structure determination and mutagenesis experiments reveal the epitope and critical residues on the NTD for 7D10 binding and neutralization. Further experiments indicate that the neutralization by 7D10 is not solely dependent on the inhibition of DPP4 binding, but also acts after viral cell attachment, inhibiting the pre-fusion to post-fusion conformational change of the spike. These properties give 7D10 a wide neutralization breadth and help explain its synergistic effects with several RBD-targeting antibodies.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por Coronavirus/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/metabolismo , Anticorpos Neutralizantes/ultraestrutura , Anticorpos Antivirais/sangue , Anticorpos Antivirais/metabolismo , Anticorpos Antivirais/ultraestrutura , Linhagem Celular Tumoral , Infecções por Coronavirus/sangue , Infecções por Coronavirus/virologia , Cristalografia por Raios X , Dipeptidil Peptidase 4/metabolismo , Modelos Animais de Doenças , Mapeamento de Epitopos , Epitopos/imunologia , Feminino , Células HEK293 , Humanos , Camundongos , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Testes de Neutralização , Ligação Proteica/imunologia , Domínios Proteicos/imunologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Glicoproteína da Espícula de Coronavírus/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/ultraestrutura , Células Vero , Internalização do Vírus
10.
Emerg Microbes Infect ; 8(1): 841-856, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31169078

RESUMO

The Middle East respiratory syndrome coronavirus (MERS-CoV) has spread through 27 countries and infected more than 2,200 people since its first outbreak in Saudi Arabia in 2012. The high fatality rate (35.4%) of this novel coronavirus and its persistent wide spread infectiousness in animal reservoirs have generated tremendous global public health concern. However, no licensed therapeutic agents or vaccines against MERS-CoV are currently available and only a limited few have entered clinical trials. Among all the potential targets of MERS-CoV, the spike glycoprotein (S) has been the most well-studied due to its critical role in mediating viral entry and in inducing a protective antibody response in infected individuals. The most notable studies include the recent discoveries of monoclonal antibodies and development of candidate vaccines against the S glycoprotein. Structural characterization of MERS-CoV S protein bound with these monoclonal antibodies has provided insights into the mechanisms of humoral immune responses against MERS-CoV infection. The current review aims to highlight these developments and discuss possible hurdles and strategies to translate these discoveries into ultimate medical interventions against MERS-CoV infection.


Assuntos
Anticorpos Antivirais/imunologia , Infecções por Coronavirus/prevenção & controle , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Vacinas Virais/imunologia , Animais , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Glicoproteína da Espícula de Coronavírus/administração & dosagem , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genética
11.
Virology ; 533: 77-85, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31128495

RESUMO

Emerging porcine epidemic diarrhea viruses (PEDVs) have caused large economic losses since 2010, and G2b is the prevalent globally epidemic genotype. Given the fastidious isolation of emerging PEDV in cell culture and difficulties in retaining the isolate infectivity upon further in vitro passage, highly attenuated recombinant vesicular stomatitis virus (rVSVMT) was used as a vector to express the PEDV spike (S) protein, aiming to develop a subunit vaccine against G2b viruses. An S protein with 19 of its cytoplasmic domain amino acids deleted could be incorporated into VSV particles, generating rVSVMT (VSVMT-SΔ19) with high efficiency. Our results suggest that VSVMT-SΔ19 could effectively induce PEDV-specific immunity in pigs via intramuscular, but not intranasal, immunization. Notably, immunizations of sows with VSV MT-SΔ19 provided protective lactogenic immunity against a virulent G2b PEDV challenge in piglets. Consequently, recombinant VSVMT may be a promising platform for preparing a subunit vaccine against PEDV.


Assuntos
Infecções por Coronavirus/veterinária , Vírus da Diarreia Epidêmica Suína/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Doenças dos Suínos/prevenção & controle , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/virologia , Feminino , Expressão Gênica , Camundongos , Camundongos Endogâmicos BALB C , Vírus da Diarreia Epidêmica Suína/classificação , Vírus da Diarreia Epidêmica Suína/genética , Glicoproteína da Espícula de Coronavírus/administração & dosagem , Glicoproteína da Espícula de Coronavírus/genética , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/virologia , Vírus da Estomatite Vesicular Indiana/genética , Vírus da Estomatite Vesicular Indiana/metabolismo , Vacinas Virais/administração & dosagem , Vacinas Virais/genética
12.
Emerg Microbes Infect ; 8(1): 760-772, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31130102

RESUMO

The recently identified Middle East Respiratory Syndrome Coronavirus (MERS-CoV) causes severe and fatal acute respiratory illness in humans. However, no approved prophylactic and therapeutic interventions are currently available. The MERS-CoV envelope spike protein serves as a crucial target for neutralizing antibodies and vaccine development, as it plays a critical role in mediating viral entry through interactions with the cellular receptor, dipeptidyl peptidase 4 (DPP4). Here, we constructed a recombinant rare serotype of the chimpanzee adenovirus 68 (AdC68) that expresses full-length MERS-CoV S protein (AdC68-S). Single intranasal immunization with AdC68-S induced robust and sustained neutralizing antibody and T cell responses in BALB/c mice. In a human DPP4 knock-in (hDPP4-KI) mouse model, it completely protected against lethal challenge with a mouse-adapted MERS-CoV (MERS-CoV-MA). Passive transfer of immune sera to naïve hDPP4-KI mice also provided survival advantages from lethal MERS-CoV-MA challenge. Analysis of sera absorption and isolated monoclonal antibodies from immunized mice demonstrated that the potent and broad neutralizing activity was largely attributed to antibodies targeting the receptor binding domain (RBD) of the S protein. These results show that AdC68-S can induce protective immune responses in mice and represent a promising candidate for further development against MERS-CoV infection in both dromedaries and humans.


Assuntos
Infecções por Coronavirus/prevenção & controle , Portadores de Fármacos/administração & dosagem , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Virais/imunologia , Adenoviridae/genética , Administração Intranasal , Animais , Animais Geneticamente Modificados , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Humanos , Imunização Passiva , Camundongos Endogâmicos BALB C , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Glicoproteína da Espícula de Coronavírus/genética , Análise de Sobrevida , Linfócitos T/imunologia , Resultado do Tratamento , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genética
13.
J Microbiol Biotechnol ; 29(5): 813-819, 2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-30982320

RESUMO

Middle East respiratory syndrome coronavirus (MERS-CoV) induces severe respiratory impairment with a reported mortality rate of ~36% in humans. The absence of clinically available MERS-CoV vaccines and treatments to date has resulted in uncontrolled incidence and propagation of the virus. In vaccine design, fusion with the IgG Fc domain is reported to increase the immunogenicity of various vaccine antigens. However, limited reports have documented the potential negative effects of Fc fusion on vaccine antigens. To determine whether Fc fusion affects the immunogenicity of MERS-CoV antigen, we constructed a Fcassociated MERS-CoV spike protein (eS770-Fc, 110 kDa), whereby human IgG4 Fc domain was fused to MERS-CoV spike protein (eS770) via a Gly/Pro linker using baculovirus as the expression system. For comparative analyses, two eS770 proteins lacking the IgG4 Fc domain were generated using the IdeS protease (eS770-ΔFc) or His tag attachment (eS770-His) and the immunogenicity of the above constructs were examined following intramuscular immunization in mice. Contrary to expectations, non-Fc spike proteins (eS770-ΔFc, eS770-His; 90 kDa) showed higher immunogenicity than the Fc fusion protein (eS770-Fc). Moreover, unlike non- Fc spike proteins, eS770-Fc immunization did not elicit neutralizing antibodies against MERSCoV. The lower immunogenicity of Fc-fused eS770 was related to alterations in the structural conformation of the spike protein. Taken together, our results indicate that IgG Fc fusion reduces the immunogenicity of eS770 by interfering with the proper folding structure.


Assuntos
Infecções por Coronavirus/prevenção & controle , Imunogenicidade da Vacina , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Dobramento de Proteína , Proteínas Recombinantes de Fusão/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , Antígenos Virais/genética , Feminino , Imunização , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G , Camundongos , Camundongos Endogâmicos BALB C , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/patogenicidade , Testes de Neutralização , Células Sf9 , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Vacinação , Vacinas de Subunidades/imunologia , Vacinas Virais/genética
14.
Emerg Microbes Infect ; 8(1): 516-530, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30938227

RESUMO

The Middle-East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic virus that causes severe and often fatal respiratory disease in humans. Efforts to develop antibody-based therapies have focused on neutralizing antibodies that target the receptor binding domain of the viral spike protein thereby blocking receptor binding. Here, we developed a set of human monoclonal antibodies that target functionally distinct domains of the MERS-CoV spike protein. These antibodies belong to six distinct epitope groups and interfere with the three critical entry functions of the MERS-CoV spike protein: sialic acid binding, receptor binding and membrane fusion. Passive immunization with potently as well as with poorly neutralizing antibodies protected mice from lethal MERS-CoV challenge. Collectively, these antibodies offer new ways to gain humoral protection in humans against the emerging MERS-CoV by targeting different spike protein epitopes and functions.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Infecções por Coronavirus/prevenção & controle , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Infecções por Coronavirus/genética , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Humanos , Imunização Passiva , Camundongos , Coronavírus da Síndrome Respiratória do Oriente Médio/química , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Domínios Proteicos , Receptores Virais/genética , Receptores Virais/imunologia , Glicoproteína da Espícula de Coronavírus/genética
15.
Virus Res ; 266: 34-42, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30965063

RESUMO

The porcine epidemic diarrhea virus (PEDV) collagenase equivalent domain (COE, residues 499-638), a crucial antigenic region within the viral spike (S) glycoprotein, has been widely utilized for the development of subunit vaccines to prevent viral infection. In the current study, we immunized BALB/c mice with recombinant truncated PEDV COE protein and obtained 14 COE-specific monoclonal antibodies (mAbs). Based on the reactivity analysis of the mAbs with two prevalent PEDV strains in G2 type and the attenuated CV777 strain in G1 type, 6 mAbs were selected for subsequent identification of COE mAb-binding epitopes. Dot-blot hybridization and enzyme-linked immunosorbent assays (ELISAs) identified the peptide 592TSLLASACTIDLFGYP607 as a novel linear B-cell epitope involved in binding of mAbs 4D8F10 and 6F3E3. Subsequently, alanine (A)-scanning mutagenesis demonstrated that residues 606Y, 605G and 604F were core residues involved in recognition. Importantly, this novel COE epitope, including core residues, is conserved among G1 and G2 type PEDV strains. Further experiment indicates that the mAbs 4D8F10 and 6F3E3 were suitable for PEDV detection via mAb binding to the conserved epitope. The current work actually provides potential uses for the development of diagnostic methods to detect PEDV.


Assuntos
Epitopos de Linfócito B/imunologia , Vírus da Diarreia Epidêmica Suína/imunologia , Glicoproteína da Espícula de Coronavírus/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Sequência Conservada , Infecções por Coronavirus/imunologia , Mapeamento de Epitopos , Epitopos de Linfócito B/química , Epitopos de Linfócito B/genética , Feminino , Células HEK293 , Humanos , Imunização , Camundongos Endogâmicos BALB C , Filogenia , Vírus da Diarreia Epidêmica Suína/classificação , Vírus da Diarreia Epidêmica Suína/genética , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Deleção de Sequência , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Células Vero
16.
Viruses ; 11(3)2019 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-30832356

RESUMO

MERS-CoV is present in dromedary camels throughout the Middle East and Africa. Dromedary camels are the primary zoonotic reservoir for human infections. Interruption of the zoonotic transmission chain from camels to humans, therefore, may be an effective strategy to control the ongoing MERS-CoV outbreak. Here we show that vaccination with an adjuvanted MERS-CoV Spike protein subunit vaccine confers complete protection from MERS-CoV disease in alpaca and results in reduced and delayed viral shedding in the upper airways of dromedary camels. Protection in alpaca correlates with high serum neutralizing antibody titers. Lower titers of serum neutralizing antibodies correlate with delayed and significantly reduced shedding in the nasal turbinates of dromedary camels. Together, these data indicate that induction of robust neutralizing humoral immune responses by vaccination of naïve animals reduces shedding that potentially could diminish the risk of zoonotic transmission.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Anticorpos Antivirais/sangue , Infecções por Coronavirus/veterinária , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/sangue , Camelídeos Americanos/imunologia , Camelus/imunologia , Infecções por Coronavirus/prevenção & controle , Feminino , Imunidade Humoral , Masculino , Coronavírus da Síndrome Respiratória do Oriente Médio/isolamento & purificação , Eliminação de Partículas Virais
17.
Emerg Infect Dis ; 25(6): 1161-1168, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30900977

RESUMO

The unexpectedly large outbreak of Middle East respiratory syndrome in South Korea in 2015 was initiated by an infected traveler and amplified by several "superspreading" events. Previously, we reported the emergence and spread of mutant Middle East respiratory syndrome coronavirus bearing spike mutations (I529T or D510G) with reduced affinity to human receptor CD26 during the outbreak. To assess the potential association of spike mutations with superspreading events, we collected virus genetic information reported during the outbreak and systemically analyzed the relationship of spike sequences and epidemiology. We found sequential emergence of the spike mutations in 2 superspreaders. In vivo virulence of the mutant viruses seems to decline in human patients, as assessed by fever duration in affected persons. In addition, neutralizing activity against these 2 mutant viruses in serum samples from mice immunized with wild-type spike antigen were gradually reduced, suggesting emergence and wide spread of neutralization escapers during the outbreak.


Assuntos
Doenças Transmissíveis Emergentes/epidemiologia , Doenças Transmissíveis Emergentes/virologia , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Mutação , Adulto , Idoso , Anticorpos Neutralizantes/imunologia , Doenças Transmissíveis Emergentes/história , Doenças Transmissíveis Emergentes/imunologia , Infecções por Coronavirus/história , Infecções por Coronavirus/imunologia , Surtos de Doenças , Feminino , Genótipo , História do Século XXI , Humanos , Masculino , Pessoa de Meia-Idade , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Testes de Neutralização , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia
18.
Viral Immunol ; 32(3): 144-150, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30855219

RESUMO

Porcine epidemic diarrhea caused by porcine epidemic diarrhea virus (PEDV) is a highly contagious disease in newborn piglets. The spike (S) protein is the surface glycoprotein of PEDV, which can induce specific neutralization antibodies and is a candidate antigen for vaccination attempts. In our study, the S1 region of PEDV strain CH/JLDH/2016 spike gene was inserted into the Lactococcus lactis expression vector, pNZ8149, resulting in recombinant plasmid pNZ8149-S1, and the immunogenicity of recombinant L. lactis pNZ8149-S1/NZ3900 was evaluated in mice. After immunization, significantly higher levels of anti-PEDV serum IgG antibodies and mucosal sIgA antibodies were detected in mice orally administered with pNZ8149-S1/NZ3900, compared with control groups pNZ8149/NZ3900, NZ3900, and phosphate buffered saline (p < 0.01). Lymphocyte proliferation assay results showed that the recombinant L. lactis pNZ8149-S1/NZ3900 significantly stimulated the proliferation of splenic lymphocytes (p < 0.01). In addition, the recombinant L. lactis vaccine could induce high levels of IL-4 and IFN-γ in immunized mice (p < 0.01). The results of our study suggest that the recombinant L. lactis pNZ8149-S1/NZ3900 can provide a promising vaccine strategy against PEDV infection.


Assuntos
Infecções por Coronavirus/imunologia , Imunogenicidade da Vacina , Lactococcus lactis/genética , Vírus da Diarreia Epidêmica Suína/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Proliferação de Células , Feminino , Imunoglobulina G/sangue , Interferon gama/imunologia , Interleucina-4/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Vírus da Diarreia Epidêmica Suína/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Vacinas Virais/genética
19.
Vet Res ; 50(1): 12, 2019 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-30744668

RESUMO

Infectious bronchitis virus (IBV) causes a major disease problem for the poultry industry worldwide. The currently used live-attenuated vaccines have the tendency to mutate and/or recombine with circulating field strains resulting in the emergence of vaccine-derived variant viruses. In order to circumvent these issues, and to develop a vaccine that is more relevant to Egypt and its neighboring countries, a recombinant avirulent Newcastle disease virus (rNDV) strain LaSota was constructed to express the codon-optimized S glycoprotein of the Egyptian IBV variant strain IBV/Ck/EG/CU/4/2014 belonging to GI-23 lineage, that is prevalent in Egypt and in the Middle East. A wild type and two modified versions of the IBV S protein were expressed individually by rNDV. A high level of S protein expression was detected in vitro by Western blot and immunofluorescence analyses. All rNDV-vectored IBV vaccine candidates were genetically stable, slightly attenuated and showed growth patterns comparable to that of parental rLaSota virus. Single-dose vaccination of 1-day-old SPF White Leghorn chicks with the rNDVs expressing IBV S protein provided significant protection against clinical disease after IBV challenge but did not show reduction in tracheal viral shedding. Single-dose vaccination also provided complete protection against virulent NDV challenge. However, prime-boost vaccination using rNDV expressing the wild type IBV S protein provided better protection, after IBV challenge, against clinical signs and significantly reduced tracheal viral shedding. These results indicate that the NDV-vectored IBV vaccines are promising bivalent vaccine candidates to control both infectious bronchitis and Newcastle disease in Egypt.


Assuntos
Galinhas , Infecções por Coronavirus/veterinária , Vírus da Bronquite Infecciosa/imunologia , Vírus da Doença de Newcastle/imunologia , Doenças das Aves Domésticas/prevenção & controle , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Virais/imunologia , Animais , Linhagem Celular , Infecções por Coronavirus/prevenção & controle , Egito , Vetores Genéticos/imunologia , Vírus da Bronquite Infecciosa/genética , Vírus da Doença de Newcastle/genética , Glicoproteína da Espícula de Coronavírus/genética , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genética
20.
Viruses ; 11(1)2019 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-30658390

RESUMO

Efforts towards developing a vaccine for Middle East respiratory syndrome coronavirus (MERS-CoV) have yielded promising results. Utilizing a variety of platforms, several vaccine approaches have shown efficacy in animal models and begun to enter clinical trials. In this review, we summarize the current progress towards a MERS-CoV vaccine and highlight potential roadblocks identified from previous attempts to generate coronavirus vaccines.


Assuntos
Infecções por Coronavirus/prevenção & controle , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por Coronavirus/imunologia , Modelos Animais de Doenças , Humanos , Camundongos , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinação , Vacinas Atenuadas/imunologia
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