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1.
Nature ; 586(7830): 567-571, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32756549

RESUMO

A vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is needed to control the coronavirus disease 2019 (COVID-19) global pandemic. Structural studies have led to the development of mutations that stabilize Betacoronavirus spike proteins in the prefusion state, improving their expression and increasing immunogenicity1. This principle has been applied to design mRNA-1273, an mRNA vaccine that encodes a SARS-CoV-2 spike protein that is stabilized in the prefusion conformation. Here we show that mRNA-1273 induces potent neutralizing antibody responses to both wild-type (D614) and D614G mutant2 SARS-CoV-2 as well as CD8+ T cell responses, and protects against SARS-CoV-2 infection in the lungs and noses of mice without evidence of immunopathology. mRNA-1273 is currently in a phase III trial to evaluate its efficacy.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/imunologia , Pneumonia Viral/prevenção & controle , Vacinas Virais/imunologia , Vacina de mRNA-1273 contra 2019-nCoV , Animais , Anticorpos Neutralizantes/imunologia , Betacoronavirus/genética , Linfócitos T CD8-Positivos/imunologia , COVID-19 , Vacinas contra COVID-19 , Ensaios Clínicos Fase III como Assunto , Infecções por Coronavirus/genética , Infecções por Coronavirus/virologia , Feminino , Pulmão/imunologia , Pulmão/virologia , Camundongos , Mutação , Nariz/imunologia , Nariz/virologia , Pneumonia Viral/virologia , RNA Mensageiro/genética , RNA Viral/genética , SARS-CoV-2 , Células Th1/imunologia , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/imunologia , Vacinas Virais/química , Vacinas Virais/genética
2.
Proc Natl Acad Sci U S A ; 120(29): e2305896120, 2023 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-37428933

RESUMO

Vaccines have played a fundamental role in the control of infectious diseases. We previously developed a messenger RNA (mRNA) vaccine against HIV-1 that forms virus-like particles (VLPs) through coexpression of the viral envelope with Gag. Here, we applied the same principle to the design of a VLP-forming mRNA vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To promote cognate interaction with simian immunodeficiency virus (SIV) Gag, we engineered different chimeric proteins encompassing the ectodomain and the transmembrane region of the SARS-CoV-2 Spike protein from the Wuhan-Hu-1 strain fused to the gp41 cytoplasmic tail of either HIV-1 (strain WITO) or SIV (strain mac239) with or without a partial truncation at amino acid 745 to enhance membrane expression. Upon cotransfection with SIV gag mRNA, the Spike-SIVCT.745 (SSt) chimera yielded the highest level of cell-surface expression and extracellular VLP release. Immunization of BALB/c mice with SSt+gag mRNA at 0, 4, and 16 wk induced higher titers of Spike-binding and autologous neutralizing antibodies at all time points compared to SSt mRNA alone. Furthermore, mice immunized with SSt+gag mRNA developed neutralizing antibodies effective against different variants of concern. These data demonstrate that the Gag/VLP mRNA platform can be successfully applied to vaccines against different agents for the prevention of infectious diseases of global relevance.


Assuntos
COVID-19 , Vírus da Imunodeficiência Símia , Humanos , Animais , Camundongos , Vacinas contra COVID-19/genética , Anticorpos Antivirais , SARS-CoV-2/genética , COVID-19/prevenção & controle , Anticorpos Neutralizantes , Glicoproteína da Espícula de Coronavírus/genética , Vírus da Imunodeficiência Símia/genética
4.
NPJ Vaccines ; 9(1): 103, 2024 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-38858423

RESUMO

Acellular multivalent vaccines for pertussis (DTaP and Tdap) prevent symptomatic disease and infant mortality, but immunity to Bordetella pertussis infection wanes significantly over time resulting in cyclic epidemics of pertussis. The messenger RNA (mRNA) vaccine platform provides an opportunity to address complex bacterial infections with an adaptable approach providing Th1-biased responses. In this study, immunogenicity and challenge models were used to evaluate the mRNA platform with multivalent vaccine formulations targeting both B. pertussis antigens and diphtheria and tetanus toxoids. Immunization with mRNA formulations were immunogenetic, induced antigen specific antibodies, as well as Th1 T cell responses. Upon challenge with either historical or contemporary B. pertussis strains, 6 and 10 valent mRNA DTP vaccine provided protection equal to that of 1/20th human doses of either DTaP or whole cell pertussis vaccines. mRNA DTP immunized mice were also protected from pertussis toxin challenge as measured by prevention of lymphocytosis and leukocytosis. Collectively these pre-clinical mouse studies illustrate the potential of the mRNA platform for multivalent bacterial pathogen vaccines.

5.
Acta Crystallogr D Biol Crystallogr ; 69(Pt 11): 2174-85, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24189228

RESUMO

Structured RNA molecules are key players in ensuring cellular viability. It is now emerging that, like proteins, the functions of many nucleic acids are dictated by their tertiary folds. At the same time, the number of known crystal structures of nucleic acids is also increasing rapidly. In this context, molecular replacement will become an increasingly useful technique for phasing nucleic acid crystallographic data in the near future. Here, strategies to select, create and refine molecular-replacement search models for nucleic acids are discussed. Using examples taken primarily from research on group II introns, it is shown that nucleic acids are amenable to different and potentially more flexible and sophisticated molecular-replacement searches than proteins. These observations specifically aim to encourage future crystallographic studies on the newly discovered repertoire of noncoding transcripts.


Assuntos
Substituição de Aminoácidos/genética , Íntrons , RNA Longo não Codificante/química , Sequência Conservada/genética , Cristalografia por Raios X/métodos , Previsões/métodos , Modelos Moleculares , RNA Longo não Codificante/genética , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica/genética
6.
PLoS Comput Biol ; 8(8): e1002639, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22927804

RESUMO

Predicting which mutations proteins tolerate while maintaining their structure and function has important applications for modeling fundamental properties of proteins and their evolution; it also drives progress in protein design. Here we develop a computational model to predict the tolerated sequence space of HIV-1 protease reachable by single mutations. We assess the model by comparison to the observed variability in more than 50,000 HIV-1 protease sequences, one of the most comprehensive datasets on tolerated sequence space. We then extend the model to a second protein, reverse transcriptase. The model integrates multiple structural and functional constraints acting on a protein and uses ensembles of protein conformations. We find the model correctly captures a considerable fraction of protease and reverse-transcriptase mutational tolerance and shows comparable accuracy using either experimentally determined or computationally generated structural ensembles. Predictions of tolerated sequence space afforded by the model provide insights into stability-function tradeoffs in the emergence of resistance mutations and into strengths and limitations of the computational model.


Assuntos
Protease de HIV/genética , Transcriptase Reversa do HIV/genética , Mutação , Protease de HIV/química , Transcriptase Reversa do HIV/química , Modelos Moleculares , Conformação Proteica
7.
Cell Rep Med ; 4(11): 101253, 2023 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-37918405

RESUMO

Colonization of the gut and airways by pathogenic bacteria can lead to local tissue destruction and life-threatening systemic infections, especially in immunologically compromised individuals. Here, we describe an mRNA-based platform enabling delivery of pathogen-specific immunoglobulin A (IgA) monoclonal antibodies into mucosal secretions. The platform consists of synthetic mRNA encoding IgA heavy, light, and joining (J) chains, packaged in lipid nanoparticles (LNPs) that express glycosylated, dimeric IgA with functional activity in vitro and in vivo. Importantly, mRNA-derived IgA had a significantly greater serum half-life and a more native glycosylation profile in mice than did a recombinantly produced IgA. Expression of an mRNA encoded Salmonella-specific IgA in mice resulted in intestinal localization and limited Peyer's patch invasion. The same mRNA-LNP technology was used to express a Pseudomonas-specific IgA that protected from a lung challenge. Leveraging the mRNA antibody technology as a means to intercept bacterial pathogens at mucosal surfaces opens up avenues for prophylactic and therapeutic interventions.


Assuntos
Mucosa , Nódulos Linfáticos Agregados , Camundongos , Animais , Imunoglobulina A , Anticorpos Monoclonais
8.
Antibodies (Basel) ; 11(4)2022 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-36412833

RESUMO

Monoclonal antibodies have been used successfully as recombinant protein therapy; however, for HIV, multiple broadly neutralizing antibodies may be necessary. We used the mRNA-LNP platform for in vivo co-expression of 3 broadly neutralizing antibodies, PGDM1400, PGT121, and N6, directed against the HIV-1 envelope protein. mRNA-encoded HIV-1 antibodies were engineered as single-chain Fc (scFv-Fc) to overcome heavy- and light-chain mismatch. In vitro neutralization breadth and potency of the constructs were compared to their parental IgG form. We assessed the ability of these scFv-Fcs to be expressed individually and in combination in vivo, and neutralization and pharmacokinetics were compared to the corresponding full-length IgGs. Single-chain PGDM1400 and PGT121 exhibited neutralization potency comparable to parental IgG, achieving peak systemic concentrations ≥ 30.81 µg/mL in mice; full-length N6 IgG achieved a peak concentration of 974 µg/mL, but did not tolerate single-chain conversion. The mRNA combination encoding full-length N6 IgG and single-chain PGDM1400 and PGT121 was efficiently expressed in mice, achieving high systemic concentration and desired neutralization potency. Analysis of mice sera demonstrated each antibody contributed towards neutralization of multiple HIV-1 pseudoviruses. Together, these data show that the mRNA-LNP platform provides a promising approach for antibody-based HIV treatment and is well-suited for development of combination therapeutics.

9.
bioRxiv ; 2021 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-33880468

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of a global pandemic. Safe and effective COVID-19 vaccines are now available, including mRNA-1273, which has shown 94% efficacy in prevention of symptomatic COVID-19 disease. However, the emergence of SARS-CoV-2 variants has led to concerns of viral escape from vaccine-induced immunity. Several variants have shown decreased susceptibility to neutralization by vaccine-induced immunity, most notably B.1.351 (Beta), although the overall impact on vaccine efficacy remains to be determined. Here, we present the initial evaluation in mice of 2 updated mRNA vaccines designed to target SARS-CoV-2 variants: (1) monovalent mRNA-1273.351 encodes for the spike protein found in B.1.351 and (2) mRNA-1273.211 comprising a 1:1 mix of mRNA-1273 and mRNA-1273.351. Both vaccines were evaluated as a 2-dose primary series in mice; mRNA-1273.351 was also evaluated as a booster dose in animals previously vaccinated with mRNA-1273. The results demonstrated that a primary vaccination series of mRNA-1273.351 was effective at increasing neutralizing antibody titers against B.1.351, while mRNA-1273.211 was effective at providing broad cross-variant neutralization. A third (booster) dose of mRNA-1273.351 significantly increased both wild-type and B.1.351-specific neutralization titers. Both mRNA-1273.351 and mRNA-1273.211 are being evaluated in pre-clinical challenge and clinical studies.

10.
Front Immunol ; 12: 772864, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34956199

RESUMO

Nipah virus (NiV) represents a significant pandemic threat with zoonotic transmission from bats-to-humans with almost annual regional outbreaks characterized by documented human-to-human transmission and high fatality rates. Currently, no vaccine against NiV has been approved. Structure-based design and protein engineering principles were applied to stabilize the fusion (F) protein in its prefusion trimeric conformation (pre-F) to improve expression and increase immunogenicity. We covalently linked the stabilized pre-F through trimerization domains at the C-terminus to three attachment protein (G) monomers, forming a chimeric design. These studies detailed here focus on mRNA delivery of NiV immunogens in mice, assessment of mRNA immunogen-specific design elements and their effects on humoral and cellular immunogenicity. The pre-F/G chimera elicited a strong neutralizing antibody response and a superior NiV-specific Tfh and other effector T cell response compared to G alone across both the mRNA and protein platforms. These findings enabled final candidate selection of pre-F/G Fd for clinical development.


Assuntos
Antígenos Virais/genética , Lipossomos/administração & dosagem , Nanopartículas/administração & dosagem , Vírus Nipah/imunologia , Proteínas do Envelope Viral/genética , Proteínas Virais de Fusão/genética , Vacinas Virais/administração & dosagem , Vacinas de mRNA/administração & dosagem , Animais , Antígenos Virais/imunologia , Feminino , Imunoglobulina G/sangue , Camundongos , Parcerias Público-Privadas , RNA Mensageiro/administração & dosagem , Linfócitos T/imunologia , Proteínas do Envelope Viral/imunologia , Proteínas Virais de Fusão/imunologia
11.
Vaccine ; 39(51): 7394-7400, 2021 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-34815117

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of a global pandemic. Safe and effective COVID-19 vaccines are now available, including mRNA-1273, which has shown 94% efficacy in prevention of symptomatic COVID-19 disease. However, the emergence of SARS-CoV-2 variants has led to concerns of viral escape from vaccine-induced immunity. Several variants have shown decreased susceptibility to neutralization by vaccine-induced immunity, most notably B.1.351 (Beta), although the overall impact on vaccine efficacy remains to be determined. Here, we present the initial evaluation in mice of 2 updated mRNA vaccines designed to target SARS-CoV-2 variants: (1) monovalent mRNA-1273.351 encodes for the spike protein found in B.1.351 and (2) mRNA-1273.211 comprising a 1:1 mix of mRNA-1273 and mRNA-1273.351. Both vaccines were evaluated as a 2-dose primary series in mice; mRNA-1273.351 was also evaluated as a booster dose in animals previously vaccinated with mRNA-1273. The results demonstrated that a primary vaccination series of mRNA-1273.351 was effective at increasing neutralizing antibody titers against B.1.351, while mRNA-1273.211 was effective at providing broad cross-variant neutralization. A third (booster) dose of mRNA-1273.351 significantly increased both wild-type and B.1.351-specific neutralization titers. Both mRNA-1273.351 and mRNA-1273.211 are being evaluated in pre-clinical challenge and clinical studies.


Assuntos
Vacinas contra COVID-19 , COVID-19 , Vacina de mRNA-1273 contra 2019-nCoV , Animais , Anticorpos Antivirais , Humanos , Camundongos , SARS-CoV-2 , Vacinação , Eficácia de Vacinas , Vacinas Sintéticas , Vacinas de mRNA
12.
Nat Med ; 27(12): 2234-2245, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34887575

RESUMO

The development of a protective vaccine remains a top priority for the control of the HIV/AIDS pandemic. Here, we show that a messenger RNA (mRNA) vaccine co-expressing membrane-anchored HIV-1 envelope (Env) and simian immunodeficiency virus (SIV) Gag proteins to generate virus-like particles (VLPs) induces antibodies capable of broad neutralization and reduces the risk of infection in rhesus macaques. In mice, immunization with co-formulated env and gag mRNAs was superior to env mRNA alone in inducing neutralizing antibodies. Macaques were primed with a transmitted-founder clade-B env mRNA lacking the N276 glycan, followed by multiple booster immunizations with glycan-repaired autologous and subsequently bivalent heterologous envs (clades A and C). This regimen was highly immunogenic and elicited neutralizing antibodies against the most prevalent (tier-2) HIV-1 strains accompanied by robust anti-Env CD4+ T cell responses. Vaccinated animals had a 79% per-exposure risk reduction upon repeated low-dose mucosal challenges with heterologous tier-2 simian-human immunodeficiency virus (SHIV AD8). Thus, the multiclade env-gag VLP mRNA platform represents a promising approach for the development of an HIV-1 vaccine.


Assuntos
Anticorpos Neutralizantes/imunologia , Genes env , Genes gag , Anticorpos Anti-HIV/biossíntese , HIV-1/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vacinas Sintéticas/imunologia , Vacinas de mRNA/imunologia , Animais , Anticorpos Anti-HIV/imunologia , Imunização Secundária , Macaca mulatta , Fatores de Risco , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas de mRNA/administração & dosagem
13.
bioRxiv ; 2020 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-32577634

RESUMO

A SARS-CoV-2 vaccine is needed to control the global COVID-19 public health crisis. Atomic-level structures directed the application of prefusion-stabilizing mutations that improved expression and immunogenicity of betacoronavirus spike proteins. Using this established immunogen design, the release of SARS-CoV-2 sequences triggered immediate rapid manufacturing of an mRNA vaccine expressing the prefusion-stabilized SARS-CoV-2 spike trimer (mRNA-1273). Here, we show that mRNA-1273 induces both potent neutralizing antibody and CD8 T cell responses and protects against SARS-CoV-2 infection in lungs and noses of mice without evidence of immunopathology. mRNA-1273 is currently in a Phase 2 clinical trial with a trajectory towards Phase 3 efficacy evaluation.

14.
Sci Immunol ; 4(35)2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101672

RESUMO

Infection with chikungunya virus (CHIKV) causes an acute illness characterized by fever, rash, and arthralgia. However, CHIKV infection can sometimes progress to chronic arthritis or even lethal disease. CHIKV continues to cause substantial morbidity worldwide as its vector mosquitoes expand and spread. There are currently no approved vaccines or antiviral drugs available for the prevention or treatment of CHIKV. Although antibody therapy has shown promise in the prevention or treatment of CHIKV disease in preclinical models, challenges remain for implementing such therapies. Here, from the B cells of a survivor of natural CHIKV infection, we isolated ultrapotent neutralizing human monoclonal antibodies (mAbs) and encoded their sequences into mRNA molecules delivered by infusion. One human mAb, CHKV-24, was expressed to biologically significant levels in vivo after infusion of mRNAs in lipid nanoparticles in mice. We evaluated the protective capacity of CHKV-24 mAb immunoglobulin G protein or mRNA in mouse models of CHIKV infection. Treatment with CHKV-24 mRNA protected mice from arthritis, musculoskeletal tissue infection, and lethality and reduced viremia to undetectable levels at 2 days after inoculation. Infusion of macaques with CHKV-24 mRNA achieved a mean maximal mAb concentration of 10.1 to 35.9 micrograms per milliliter, with a half-life of 23 days, a level well above what is needed for protection in mice. Studies with CHKV-24 mRNA in macaques demonstrated a dose-response effect after the first dose of mRNA and maintained levels after second dose. These preclinical data with CHKV-24 mRNA suggest that it might be useful to prevent human disease.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Febre de Chikungunya/imunologia , Vírus Chikungunya/imunologia , Nanocápsulas/química , RNA Mensageiro/farmacologia , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/uso terapêutico , Linfócitos B , Linhagem Celular , Febre de Chikungunya/terapia , Febre de Chikungunya/virologia , Cricetinae , Modelos Animais de Doenças , Feminino , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/uso terapêutico , Lipídeos/química , Macaca , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/química , RNA Mensageiro/uso terapêutico
15.
J Mol Biol ; 421(1): 6-26, 2012 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-22425640

RESUMO

The discovery that RNA molecules can fold into complex structures and carry out diverse cellular roles has led to interest in developing tools for modeling RNA tertiary structure. While significant progress has been made in establishing that the RNA backbone is rotameric, few libraries of discrete conformations specifically for use in RNA modeling have been validated. Here, we present six libraries of discrete RNA conformations based on a simplified pseudo-torsional notation of the RNA backbone, comparable to phi and psi in the protein backbone. We evaluate the ability of each library to represent single nucleotide backbone conformations, and we show how individual library fragments can be assembled into dinucleotides that are consistent with established RNA backbone descriptors spanning from sugar to sugar. We then use each library to build all-atom models of 20 test folds, and we show how the composition of a fragment library can limit model quality. Despite the limitations inherent in using discretized libraries, we find that several hundred discrete fragments can rebuild RNA folds up to 174 nucleotides in length with atomic-level accuracy (<1.5 Å RMSD). We anticipate that the libraries presented here could easily be incorporated into RNA structural modeling, analysis, or refinement tools.


Assuntos
Biblioteca Gênica , Modelos Moleculares , Conformação de Ácido Nucleico , Nucleotídeos/química , Dobramento de RNA
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