RESUMO
The prefusion conformation of human metapneumovirus fusion protein (hMPV Pre-F) is critical for eliciting the most potent neutralizing antibodies and is the preferred immunogen for an efficacious vaccine against hMPV respiratory infections. Here we show that an additional cleavage event in the F protein allows closure and correct folding of the trimer. We therefore engineered the F protein to undergo double cleavage, which enabled screening for Pre-F stabilizing substitutions at the natively folded protomer interfaces. To identify these substitutions, we developed an AI convolutional classifier that successfully predicts complex polar interactions often overlooked by physics-based methods and visual inspection. The combination of additional processing, stabilization of interface regions and stabilization of the membrane-proximal stem, resulted in a Pre-F protein vaccine candidate without the need for a heterologous trimerization domain that exhibited high expression yields and thermostability. Cryo-EM analysis shows the complete ectodomain structure, including the stem, and a specific interaction of the newly identified cleaved C-terminus with the adjacent protomer. Importantly, the protein induces high and cross-neutralizing antibody responses resulting in near complete protection against hMPV challenge in cotton rats, making the highly stable, double-cleaved hMPV Pre-F trimer an attractive vaccine candidate.
Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , Metapneumovirus , Proteínas Virais de Fusão , Vacinas Virais , Metapneumovirus/imunologia , Metapneumovirus/genética , Animais , Anticorpos Neutralizantes/imunologia , Humanos , Anticorpos Antivirais/imunologia , Proteínas Virais de Fusão/imunologia , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/genética , Vacinas Virais/imunologia , Infecções por Paramyxoviridae/prevenção & controle , Infecções por Paramyxoviridae/imunologia , Microscopia Crioeletrônica , Engenharia de Proteínas/métodos , Sigmodontinae , Feminino , Multimerização Proteica , Modelos MolecularesRESUMO
Respirovirus 3 is a leading cause of severe acute respiratory infections in vulnerable human populations. Entry into host cells is facilitated by the attachment glycoprotein and the fusion glycoprotein (F). Because of its crucial role, F represents an attractive therapeutic target. Here, we identify 13 F-directed heavy-chain-only antibody fragments that neutralize recombinant respirovirus 3. High-resolution cryo-EM structures of antibody fragments bound to the prefusion conformation of F reveal three distinct, previously uncharacterized epitopes. All three antibody fragments bind quaternary epitopes on F, suggesting mechanisms for neutralization that may include stabilization of the prefusion conformation. Studies in cotton rats demonstrate the prophylactic efficacy of these antibody fragments in reducing viral load in the lungs and nasal passages. These data highlight the potential of heavy-chain-only antibody fragments as effective interventions against respirovirus 3 infection and identify neutralizing epitopes that can be targeted for therapeutic development.
Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , Microscopia Crioeletrônica , Epitopos , Animais , Anticorpos Neutralizantes/imunologia , Humanos , Anticorpos Antivirais/imunologia , Epitopos/imunologia , Sigmodontinae , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/química , Proteínas Virais de Fusão/imunologia , Proteínas Virais de Fusão/química , Feminino , Camelus/imunologia , Camelus/virologiaRESUMO
The Paramyxoviridae family encompasses medically significant RNA viruses, including human respiroviruses 1 and 3 (RV1, RV3), and zoonotic pathogens like Nipah virus (NiV). RV3, previously known as parainfluenza type 3, for which no vaccines or antivirals have been approved, causes respiratory tract infections in vulnerable populations. The RV3 fusion (F) protein is inherently metastable and will likely require prefusion (preF) stabilization for vaccine effectiveness. Here we used structure-based design to stabilize regions involved in structural transformation to generate a preF protein vaccine antigen with high expression and stability, and which, by stabilizing the coiled-coil stem region, does not require a heterologous trimerization domain. The preF candidate induces strong neutralizing antibody responses in both female naïve and pre-exposed mice and provides protection in a cotton rat challenge model (female). Despite the evolutionary distance of paramyxovirus F proteins, their structural transformation and local regions of instability are conserved, which allows successful transfer of stabilizing substitutions to the distant preF proteins of RV1 and NiV. This work presents a successful vaccine antigen design for RV3 and provides a toolbox for future paramyxovirus vaccine design and pandemic preparedness.
Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , Sigmodontinae , Proteínas Virais de Fusão , Vacinas Virais , Animais , Feminino , Proteínas Virais de Fusão/imunologia , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/química , Camundongos , Vacinas Virais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Humanos , Camundongos Endogâmicos BALB C , Infecções por Paramyxoviridae/prevenção & controle , Infecções por Paramyxoviridae/imunologia , Infecções por Paramyxoviridae/virologia , Vírus da Parainfluenza 3 Humana/imunologia , Vírus da Parainfluenza 3 Humana/genéticaRESUMO
A safe and effective vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may be required to end the coronavirus disease 2019 (COVID-19) pandemic1-8. For global deployment and pandemic control, a vaccine that requires only a single immunization would be optimal. Here we show the immunogenicity and protective efficacy of a single dose of adenovirus serotype 26 (Ad26) vector-based vaccines expressing the SARS-CoV-2 spike (S) protein in non-human primates. Fifty-two rhesus macaques (Macaca mulatta) were immunized with Ad26 vectors that encoded S variants or sham control, and then challenged with SARS-CoV-2 by the intranasal and intratracheal routes9,10. The optimal Ad26 vaccine induced robust neutralizing antibody responses and provided complete or near-complete protection in bronchoalveolar lavage and nasal swabs after SARS-CoV-2 challenge. Titres of vaccine-elicited neutralizing antibodies correlated with protective efficacy, suggesting an immune correlate of protection. These data demonstrate robust single-shot vaccine protection against SARS-CoV-2 in non-human primates. The optimal Ad26 vector-based vaccine for SARS-CoV-2, termed Ad26.COV2.S, is currently being evaluated in clinical trials.
Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Macaca mulatta , Pandemias/prevenção & controle , Pneumonia Viral/imunologia , Pneumonia Viral/prevenção & controle , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologia , Animais , COVID-19 , Vacinas contra COVID-19 , Modelos Animais de Doenças , Feminino , Imunidade Celular , Imunidade Humoral , Macaca mulatta/imunologia , Macaca mulatta/virologia , Masculino , SARS-CoV-2 , Vacinação , Carga ViralRESUMO
Endogenous retroviruses (ERVs), remnants of ancient germline infections, comprise 8% of the human genome. The most recently integrated includes human ERV-K (HERV-K) where several envelope (env) sequences remain intact. Viral pseudotypes decorated with one of those Envs are infectious. Using a recombinant vesicular stomatitis virus encoding HERV-K Env as its sole attachment and fusion protein (VSV-HERVK) we conducted a genome-wide haploid genetic screen to interrogate the host requirements for infection. This screen identified 11 genes involved in heparan sulfate biosynthesis. Genetic inhibition or chemical removal of heparan sulfate and addition of excess soluble heparan sulfate inhibit infection. Direct binding of heparin to soluble HERV-K Env and purified VSV-HERVK defines it as critical for viral attachment. Cell surface bound VSV-HERVK particles are triggered to infect on exposure to acidic pH, whereas acid pH pretreatment of virions blocks infection. Testing of additional endogenous HERV-K env sequences reveals they bind heparin and mediate acid pH triggered fusion. This work reconstructs and defines key steps in the infectious entry pathway of an extinct virus.
Assuntos
Retrovirus Endógenos/fisiologia , Heparitina Sulfato/metabolismo , Proteínas do Envelope Viral/metabolismo , Tropismo Viral/fisiologia , Internalização do Vírus , HumanosRESUMO
Human beta1-coronavirus (ß1CoV) OC43 emerged relatively recently through a single zoonotic introduction. Like related animal ß1CoVs, OC43 uses 9-O-acetylated sialic acid as receptor determinant. ß1CoV receptor binding is typically controlled by attachment/fusion spike protein S and receptor-binding/receptor-destroying hemagglutinin-esterase protein HE. We show that following OC43's introduction into humans, HE-mediated receptor binding was selected against and ultimately lost through progressive accumulation of mutations in the HE lectin domain. Consequently, virion-associated receptor-destroying activity toward multivalent glycoconjugates was reduced and altered such that some clustered receptor populations are no longer cleaved. Loss of HE lectin function was also observed for another respiratory human coronavirus, HKU1. This thus appears to be an adaptation to the sialoglycome of the human respiratory tract and for replication in human airways. The findings suggest that the dynamics of virion-glycan interactions contribute to host tropism. Our observations are relevant also to other human respiratory viruses of zoonotic origin, particularly influenza A virus.
Assuntos
Adaptação Biológica , Coronavirus Humano OC43/genética , Hemaglutininas Virais/genética , Hemaglutininas Virais/metabolismo , Lectinas/genética , Lectinas/metabolismo , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo , Ligação Viral , Animais , Coronavirus Humano OC43/fisiologia , Humanos , Mutação , Ligação Proteica , Receptores Virais/metabolismoRESUMO
Hemagglutinin-esterases (HEs) are bimodular envelope proteins of orthomyxoviruses, toroviruses, and coronaviruses with a carbohydrate-binding "lectin" domain appended to a receptor-destroying sialate-O-acetylesterase ("esterase"). In concert, these domains facilitate dynamic virion attachment to cell-surface sialoglycans. Most HEs (type I) target 9-O-acetylated sialic acids (9-O-Ac-Sias), but one group of coronaviruses switched to using 4-O-Ac-Sias instead (type II). This specificity shift required quasisynchronous adaptations in the Sia-binding sites of both lectin and esterase domains. Previously, a partially disordered crystal structure of a type II HE revealed how the shift in lectin ligand specificity was achieved. How the switch in esterase substrate specificity was realized remained unresolved, however. Here, we present a complete structure of a type II HE with a receptor analog in the catalytic site and identify the mutations underlying the 9-O- to 4-O-Ac-Sia substrate switch. We show that (i) common principles pertaining to the stereochemistry of protein-carbohydrate interactions were at the core of the transition in lectin ligand and esterase substrate specificity; (ii) in consequence, the switch in O-Ac-Sia specificity could be readily accomplished via convergent intramolecular coevolution with only modest architectural changes in lectin and esterase domains; and (iii) a single, inconspicuous Ala-to-Ser substitution in the catalytic site was key to the emergence of the type II HEs. Our findings provide fundamental insights into how proteins "see" sugars and how this affects protein and virus evolution.
Assuntos
Coronavirus/enzimologia , Hemaglutininas Virais/metabolismo , Lectinas/metabolismo , Mutação/genética , Receptores Virais/metabolismo , Ácidos Siálicos/metabolismo , Proteínas Virais de Fusão/metabolismo , Animais , Sítios de Ligação , Domínio Catalítico , Colo/metabolismo , Cristalografia por Raios X , Hemaglutininas Virais/química , Hemaglutininas Virais/genética , Humanos , Lectinas/química , Camundongos , Simulação de Acoplamento Molecular , Receptores de Coronavírus , Receptores Virais/química , Receptores Virais/genética , Ácidos Siálicos/química , Estereoisomerismo , Especificidade por Substrato , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/genéticaRESUMO
Sialic acids, terminal sugars of glycoproteins and glycolipids, play important roles in development, cellular recognition processes and host-pathogen interactions. A common modification of sialic acids is 9-O-acetylation, which has been implicated in sialoglycan recognition, ganglioside biology, and the survival and drug resistance of acute lymphoblastic leukaemia cells. Despite many functional implications, the molecular basis of 9-O-acetylation has remained elusive thus far. Following cellular approaches, including selective gene knockout by CRISPR/Cas genome editing, we here show that CASD1--a previously identified human candidate gene--is essential for sialic acid 9-O-acetylation. In vitro assays with the purified N-terminal luminal domain of CASD1 demonstrate transfer of acetyl groups from acetyl-coenzyme A to CMP-activated sialic acid and formation of a covalent acetyl-enzyme intermediate. Our study provides direct evidence that CASD1 is a sialate O-acetyltransferase and serves as key enzyme in the biosynthesis of 9-O-acetylated sialoglycans.
Assuntos
Acetiltransferases/genética , Ácidos Siálicos/metabolismo , Sialiltransferases/metabolismo , Acetilação , Acetiltransferases/metabolismo , Animais , Células CHO , Sistemas CRISPR-Cas , Catálise , Domínio Catalítico , Linhagem Celular , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Cricetulus , Cães , Eletroforese em Gel de Poliacrilamida , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Técnicas In Vitro , Células Madin Darby de Rim Canino , Espectrometria de Massas , Camundongos , Mutagênese Sítio-Dirigida , Organismos Geneticamente Modificados , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae , Células Sf9 , SpodopteraRESUMO
Sialic acids (Sias), 9-carbon-backbone sugars, are among the most complex and versatile molecules of life. As terminal residues of glycans on proteins and lipids, Sias are key elements of glycotopes of both cellular and microbial lectins and thus act as important molecular tags in cell recognition and signaling events. Their functions in such interactions can be regulated by post-synthetic modifications, the most common of which is differential Sia-O-acetylation (O-Ac-Sias). The biology of O-Ac-Sias remains mostly unexplored, largely because of limitations associated with their specific in situ detection. Here, we show that dual-function hemagglutinin-esterase envelope proteins of nidoviruses distinguish between a variety of closely related O-Ac-Sias. By using soluble forms of hemagglutinin-esterases as lectins and sialate-O-acetylesterases, we demonstrate differential expression of distinct O-Ac-sialoglycan populations in an organ-, tissue- and cell-specific fashion. Our findings indicate that programmed Sia-O-acetylation/de-O-acetylation may be critical to key aspects of cell development, homeostasis, and/or function.