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The spike (S) glycoprotein of SARS CoV-2 is the target of neutralizing antibodies (NAbs) that are crucial for vaccine effectiveness. The S1 subunit binds ACE2 while the S2 subunit mediates virus-cell membrane fusion. S2 is a class I fusion glycoprotein subunit and contains a central coiled coil that acts as a scaffold for the conformational changes associated with fusion function. The coiled coil of S2 is unusual in that the 3-4 repeat of inward-facing positions are mostly occupied by polar residues that mediate few inter-helical contacts in the prefusion trimer. We examined how insertion of bulkier hydrophobic residues (Val, Leu, Ile, Phe) to fill a cavity next to Ala1016 and Ala1020 in the 3-4 repeat affects the stability and antigenicity of S trimers. Substitution of Ala1016 with bulkier hydrophobic residues in the context of a prefusion-stabilized S trimer, S2P-FHA, was associated with increased thermal stability. S glycoprotein membrane fusion function was retained with Ala1016/Ala1020 cavity-filling mutations associated with improved recombinant S2P-FHA thermostability, however 2 mutants, A1016L and A1016V/A1020I, lacked ability to mediate entry of S-HIV-1 pseudoparticles into 293-ACE2 cells. When assessed as immunogens, two thermostable S2P-FHA mutants derived from the ancestral isolate, A1016L (16L) and A1016V/A1020I (VI) elicited neutralizing antibody with 50%-inhibitory dilutions (ID50s) in the range 2,700-5,110 for ancestral and Delta-derived viruses, and 210-1,744 for Omicron BA.1. The antigens elicited antibody specificities directed to the receptor-binding domain (RBD), N-terminal domain (NTD), fusion peptide and stem region of S2. The VI mutation enabled the production of intrinsically stable Omicron BA.1 and Omicron BA.4/5 S2P-FHA-like ectodomain oligomers in the absence of an external trimerization motif (T4 foldon), thus representing an alternative approach for stabilizing oligomeric S glycoprotein vaccines.
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COVID-19 , Síndrome Respiratorio Agudo Grave , Humanos , Glicoproteína de la Espiga del Coronavirus , Enzima Convertidora de Angiotensina 2 , Anticuerpos NeutralizantesRESUMEN
BACKGROUND & AIMS: In individuals highly exposed to HCV, reinfection is common, suggesting that natural development of sterilising immunity is difficult. In those that are reinfected, some will develop a persistent infection, while a small proportion repeatedly clear the virus, suggesting natural protection is possible. The aim of this study was to characterise immune responses associated with rapid natural clearance of HCV reinfection. METHODS: Broad neutralising antibodies (nAbs) and Envelope 2 (E2)-specific memory B cell (MBC) responses were examined longitudinally in 15 individuals with varied reinfection outcomes. RESULTS: Broad nAb responses were associated with MBC recall, but not with clearance of reinfection. Strong evidence of antigen imprinting was found, and the B-cell receptor repertoire showed a high level of clonality with ongoing somatic hypermutation of many clones over subsequent reinfection events. Single-cell transcriptomic analyses showed that cleared reinfections featured an activated transcriptomic profile in HCV-specific B cells that rapidly expanded upon reinfection. CONCLUSIONS: MBC quality, but not necessarily breadth of nAb responses, is important for protection against antigenically diverse variants, which is encouraging for HCV vaccine development. IMPACT AND IMPLICATIONS: HCV continues to have a major health burden globally. Limitations in the health infrastructure for diagnosis and treatment, as well as high rates of reinfection, indicate that a vaccine that can protect against chronic HCV infection will greatly complement current efforts to eliminate HCV-related disease. With alternative approaches to testing vaccines, such as controlled human inoculation trials under consideration, we desperately need to identify the correlates of immune protection. In this study, in a small but rare cohort of high-risk injecting drug users who were reinfected multiple times, breadth of neutralisation was not associated with ultimate clearance of the reinfection event. Alternatively, characteristics of the HCV-specific B-cell response associated with B-cell proliferation were. This study indicates that humoral responses are important for protection and suggests that for genetically very diverse viruses, such as HCV, it may be beneficial to look beyond just antibodies as correlates of protection.
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Following the COVID-19 pandemic, novel vaccines have successfully reduced severe disease and death. Despite eliciting lower antibody responses, adenoviral vector vaccines are nearly as effective as mRNA vaccines. Therefore, protection against severe disease may be mediated by immune memory cells. We here evaluated plasma antibody and memory B cells (Bmem) targeting the SARS-CoV-2 Spike receptor-binding domain (RBD) elicited by the adenoviral vector vaccine ChAdOx1 (AstraZeneca), their capacity to bind Omicron subvariants, and compared this to the response to mRNA BNT162b2 (Pfizer-BioNTech) vaccination. Whole blood was sampled from 31 healthy adults pre-vaccination and 4 weeks after dose one and dose two of ChAdOx1. Neutralizing antibodies (NAb) against SARS-CoV-2 were quantified at each time point. Recombinant RBDs of the Wuhan-Hu-1 (WH1), Delta, BA.2, and BA.5 variants were produced for ELISA-based quantification of plasma IgG and incorporated separately into fluorescent tetramers for flow cytometric identification of RBD-specific Bmem. NAb and RBD-specific IgG levels were over eight times lower following ChAdOx1 vaccination than BNT162b2. In ChAdOx1-vaccinated individuals, median plasma IgG recognition of BA.2 and BA.5 as a proportion of WH1-specific IgG was 26% and 17%, respectively. All donors generated resting RBD-specific Bmem, which were boosted after the second dose of ChAdOx1 and were similar in number to those produced by BNT162b2. The second dose of ChAdOx1 boosted Bmem that recognized VoC, and 37% and 39% of WH1-specific Bmem recognized BA.2 and BA.5, respectively. These data uncover mechanisms by which ChAdOx1 elicits immune memory to confer effective protection against severe COVID-19.
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Vacuna BNT162 , COVID-19 , Adulto , Humanos , Células B de Memoria , Pandemias , COVID-19/prevención & control , SARS-CoV-2 , Vacunación , Adenoviridae , Anticuerpos Neutralizantes , Inmunoglobulina G , Anticuerpos AntiviralesRESUMEN
Current serological tests cannot differentiate between total immunoglobulin A (IgA) and dimeric IgA (dIgA) associated with mucosal immunity. Here, we describe two new assays, dIgA-ELISA and dIgA-multiplex bead assay (MBA), that utilize the preferential binding of dIgA to a chimeric form of secretory component, allowing the differentiation between dIgA and monomeric IgA. dIgA responses elicited through severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection were measured in (i) a longitudinal panel, consisting of 74 samples (n = 20 individuals) from hospitalized cases of coronavirus disease 2019 (COVID-19); (ii) a longitudinal panel, consisting of 96 samples (n = 10 individuals) from individuals with mild COVID-19; (iii) a cross-sectional panel with PCR-confirmed SARS-CoV-2 infection with mild COVID-19 (n = 199) and (iv) pre-COVID-19 samples (n = 200). The dIgA-ELISA and dIgA-MBA demonstrated a specificity for dIgA of 99% and 98.5%, respectively. Analysis of dIgA responses in the longitudinal panels revealed that 70% (ELISA) and 50% (MBA) of patients elicited a dIgA response by day 20 after PCR diagnosis with a SARS-CoV-2 infection. Individuals with mild COVID-19 displayed increased levels of dIgA within the first 3 weeks after diagnosis but responses appeared to be short lived, compared with sustained IgA levels. However, in samples from hospitalized patients with COVID-19 we observed high and sustained levels of dIgA, up to 245 days after PCR diagnosis. Our results suggest that severe COVID-19 infections are associated with sustained levels of plasma dIgA compared with mild cases.
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COVID-19 , Humanos , COVID-19/diagnóstico , SARS-CoV-2/metabolismo , Estudios Transversales , Inmunoglobulina A , Anticuerpos Antivirales , Inmunoglobulina MRESUMEN
A vaccine to prevent hepatitis C virus (HCV) infection is urgently needed for use alongside direct-acting antiviral drugs to achieve elimination targets. We have previously shown that a soluble recombinant form of the glycoprotein E2 ectodomain (residues 384 to 661) that lacks three variable regions (Δ123) is able to elicit a higher titer of broadly neutralizing antibodies (bNAbs) than the parental form (receptor-binding domain [RBD]). In this study, we engineered a viral nanoparticle that displays HCV glycoprotein E2 on a duck hepatitis B virus (DHBV) small surface antigen (S) scaffold. Four variants of E2-S virus-like particles (VLPs) were constructed: Δ123-S, RBD-S, Δ123A7-S, and RBDA7-S; in the last two, 7 cysteines were replaced with alanines. While all four E2-S variant VLPs display E2 as a surface antigen, the Δ123A7-S and RBDA7-S VLPs were the most efficiently secreted from transfected mammalian cells and displayed epitopes recognized by cross-genotype broadly neutralizing monoclonal antibodies (bNMAbs). Both Δ123A7-S and RBDA7-S VLPs were immunogenic in guinea pigs, generating high titers of antibodies reactive to native E2 and able to prevent the interaction between E2 and the cellular receptor CD81. Four out of eight animals immunized with Δ123A7-S elicited neutralizing antibodies (NAbs), with three of those animals generating bNAbs against 7 genotypes. Immune serum generated by animals with NAbs mapped to major neutralization epitopes located at residues 412 to 420 (epitope I) and antigenic region 3. VLPs that display E2 glycoproteins represent a promising vaccine platform for HCV and could be adapted to large-scale manufacturing in yeast systems. IMPORTANCE There is currently no vaccine to prevent hepatitis C virus infection, which affects more than 71 million people globally and is a leading cause of progressive liver disease, including cirrhosis and cancer. Broadly neutralizing antibodies that recognize the E2 envelope glycoprotein can protect against heterologous viral infection and correlate with viral clearance in humans. However, broadly neutralizing antibodies are difficult to generate due to conformational flexibility of the E2 protein and epitope occlusion. Here, we show that a VLP vaccine using the duck hepatitis B virus S antigen fused to HCV glycoprotein E2 assembles into virus-like particles that display epitopes recognized by broadly neutralizing antibodies and elicit such antibodies in guinea pigs. This platform represents a novel HCV vaccine candidate amenable to large-scale manufacture at low cost.
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Hepacivirus , Hepatitis C , Proteínas del Envoltorio Viral , Vacunas contra Hepatitis Viral , Animales , Antígenos de Superficie/inmunología , Anticuerpos ampliamente neutralizantes/inmunología , Epítopos/inmunología , Cobayas , Hepacivirus/genética , Hepacivirus/inmunología , Antígenos de Superficie de la Hepatitis B/química , Hepatitis C/inmunología , Anticuerpos contra la Hepatitis C/inmunología , Humanos , Proteínas del Envoltorio Viral/inmunología , Vacunas contra Hepatitis Viral/inmunologíaRESUMEN
The E2 glycoprotein of hepatitis C virus (HCV) is the major target of broadly neutralizing antibodies (bNAbs) that are critical for the efficacy of a prophylactic HCV vaccine. We previously showed that a cell culture-derived, disulfide-linked high-molecular-weight (HMW) form of the E2 receptor-binding domain lacking three variable regions, Δ123-HMW, elicits broad neutralizing activity against the seven major genotypes of HCV. A limitation to the use of this antigen is that it is produced only at low yields and does not have a homogeneous composition. Here, we employed a sequential reduction and oxidation strategy to efficiently refold two high-yielding monomeric E2 species, D123 and a disulfide-minimized version (D123A7), into disulfide-linked HMW-like species (Δ123r and Δ123A7r). These proteins exhibited normal reactivity to bNAbs with continuous epitopes on the neutralizing face of E2, but reduced reactivity to conformation-dependent bNAbs and nonneutralizing antibodies (non-NAbs) compared with the corresponding monomeric species. Δ123r and Δ123A7r recapitulated the immunogenic properties of cell culture-derived D123-HMW in guinea pigs. The refolded antigens elicited antibodies that neutralized homologous and heterologous HCV genotypes, blocked the interaction between E2 and its cellular receptor CD81, and targeted the AS412, AS434, and AR3 domains. Of note, antibodies directed to epitopes overlapping with those of non-NAbs were absent. The approach to E2 antigen engineering outlined here provides an avenue for the development of preventive HCV vaccine candidates that induce bNAbs at higher yield and lower cost.
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Glicoproteínas/inmunología , Hepacivirus/inmunología , Antígenos de la Hepatitis/inmunología , Inmunogenicidad Vacunal , Mutación Missense , Vacunas contra Hepatitis Viral/inmunología , Proteínas Virales/inmunología , Sustitución de Aminoácidos , Animales , Anticuerpos Neutralizantes/inmunología , Glicoproteínas/genética , Cobayas , Hepacivirus/genética , Anticuerpos Antihepatitis/inmunología , Antígenos de la Hepatitis/genética , Humanos , Vacunas contra Hepatitis Viral/genética , Proteínas Virales/genéticaRESUMEN
BACKGROUND & AIMS: Neutralising antibodies (NAbs) play a key role in clearance of HCV. NAbs have been isolated and mapped to several domains on the HCV envelope proteins. However, the immunodominance of these epitopes in HCV infection remains unknown, hindering efforts to elicit optimal epitope-specific responses. Furthermore, it remains unclear which epitope-specific responses are associated with broad NAb (bNAb) activity in primary HCV infection. The aim of this study was to define B cell immunodominance in primary HCV, and its implications on neutralisation breadth and clearance. METHODS: Using samples from 168 patients with primary HCV infection, the antibody responses targeted 2 immunodominant domains, termed domains B and C. Genotype 1 and 3 infections were associated with responses targeted towards different bNAb domains. RESULTS: No epitopes were uniquely targeted by clearers compared to those who developed chronic infection. Samples with bNAb activity were enriched for multi-specific responses directed towards the epitopes antigenic region 3, antigenic region 4, and domain D, and did not target non-neutralising domains. CONCLUSIONS: This study outlines for the first time a clear NAb immunodominance profile in primary HCV infection, and indicates that it is influenced by the infecting virus. It also highlights the need for a vaccination strategy to induce multi-specific responses that do not target non-neutralising domains. LAY SUMMARY: Neutralising antibodies will likely form a key component of a protective hepatitis C virus vaccine. In this work we characterise the predominant neutralising and non-neutralising antibody (epitope) targets in acute hepatitis C virus infection. We have defined the natural hierarchy of epitope immunodominance, and demonstrated that viral genotype can impact on this hierarchy. Our findings highlight key epitopes that are associated with broadly neutralising antibodies, and the deleterious impact of mounting a response towards some of these domains on neutralising breadth. These findings should guide future efforts to design immunogens aimed at generating neutralising antibodies with a vaccine candidate.
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Linfocitos B/inmunología , Epítopos de Linfocito B/inmunología , Hepacivirus/inmunología , Hepatitis C/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Australia/epidemiología , Femenino , Genotipo , Hepacivirus/genética , Hepatitis C/epidemiología , Hepatitis C/virología , Anticuerpos contra la Hepatitis C/inmunología , Humanos , Masculino , Estudios Prospectivos , Seroconversión , Proteínas del Envoltorio Viral/inmunología , Vacunas contra Hepatitis Viral/inmunologíaRESUMEN
The hepatitis C virus (HCV) E2 glycoprotein is a major target of the neutralizing antibody (nAb) response, with multiple type-specific and broadly neutralizing antibody (bnAb) epitopes identified. The 412-to-423 region can generate bnAbs that block interaction with the cell surface receptor CD81, with activity toward multiple HCV genotypes. In this study, we reveal the structure of rodent monoclonal antibody 24 (MAb24) with an extensive contact area toward a peptide spanning the 412-to-423 region. The crystal structure of the MAb24-peptide 412-to-423 complex reveals the paratope bound to a peptide hairpin highly similar to that observed with human MAb HCV1 and rodent MAb AP33, but with a different angle of approach. In viral outgrowth experiments, we demonstrated three distinct genotype 2a viral populations that acquired resistance to MAb24 via N415D, N417S, and N415D/H386R mutations. Importantly, the MAb24-resistant viruses exhibited significant increases in sensitivity to the majority of bnAbs directed to epitopes within the 412-to-423 region and in additional antigenic determinants located within E2 and the E1E2 complex. This study suggests that modification of N415 causes a global change in glycoprotein structure that increases its vulnerability to neutralization by other antibodies. This finding suggests that in the context of an antibody response to viral infection, acquisition of escape mutations in the 412-to-423 region renders the virus more susceptible to neutralization by other specificities of nAbs, effectively reducing the immunological fitness of the virus. A vaccine for HCV that generates polyspecific humoral immunity with specificity for the 412-to-423 region and at least one other region of E2 is desirable.IMPORTANCE Understanding how antibodies neutralize hepatitis C virus (HCV) is essential for vaccine development. This study reveals for the first time that when HCV develops resistance to a major class of bnAbs targeting the 412-to-423 region of E2, this results in a concomitant increase in sensitivity to neutralization by a majority of other bnAb specificities. Vaccines for the prevention of HCV infection should therefore generate bnAbs directed toward the 412-to-423 region of E2 and additional bnAb epitopes within the viral glycoproteins.
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Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Complejo Antígeno-Anticuerpo/metabolismo , Epítopos/metabolismo , Hepacivirus/inmunología , Anticuerpos contra la Hepatitis C/inmunología , Proteínas del Envoltorio Viral/inmunología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Neutralizantes/metabolismo , Complejo Antígeno-Anticuerpo/inmunología , Carcinoma Hepatocelular , Línea Celular Tumoral , Epítopos/inmunología , Hepacivirus/genética , Anticuerpos contra la Hepatitis C/metabolismo , Humanos , Neoplasias Hepáticas , Estructura Secundaria de Proteína , Tetraspanina 28/inmunología , Vacunas contra Hepatitis Viral/inmunologíaRESUMEN
The hepatitis C virus (HCV) envelope glycoprotein E2 is the major target of broadly neutralizing antibodies in vivo and is the focus of efforts in the rational design of a universal B cell vaccine against HCV. The E2 glycoprotein exhibits a high degree of amino acid variability which localizes to three discrete regions: hypervariable region 1 (HVR1), hypervariable region 2 (HVR2), and the intergenotypic variable region (igVR). All three variable regions contribute to immune evasion and/or isolate-specific structural variations, both important considerations for vaccine design. A high-resolution structural definition of the intact HCV envelope glycoprotein complex containing E1 and E2 remains to be elucidated, while crystallographic structures of a recombinant E2 ectodomain failed to resolve HVR1, HVR2, and a major neutralization determinant adjacent to HVR1. To obtain further information on E2, we characterized the role of all three variable regions in E2 ectodomain folding and function in the context of a recombinant ectodomain fragment (rE2). We report that removal of the variable regions accelerates binding to the major host cell receptor CD81 and that simultaneous deletion of HVR2 and the igVR is required to maintain wild-type CD81-binding characteristics. The removal of the variable regions also rescued the ability of rE2 to form a functional homodimer. We propose that the rE2 core provides novel insights into the role of the variable motifs in the higher-order assembly of the E2 ectodomain and may have implications for E1E2 structure on the virion surface. IMPORTANCE Hepatitis C virus (HCV) infection affects â¼2% of the population globally, and no vaccine is available. HCV is a highly variable virus, and understanding the presentation of key antigenic sites at the virion surface is important for the design of a universal vaccine. This study investigates the role of three surface-exposed variable regions in E2 glycoprotein folding and function in the context of a recombinant soluble ectodomain. Our data demonstrate the variable motifs modulate binding of the E2 ectodomain to the major host cell receptor CD81 and have an impact on the formation of an E2 homodimer with high-affinity binding to CD81.
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Hepacivirus/fisiología , Proteínas del Envoltorio Viral/química , Internalización del Virus , Regulación Alostérica , Anticuerpos Neutralizantes/química , Anticuerpos Antivirales/química , Línea Celular Tumoral , Epítopos/química , Epítopos/inmunología , Células HEK293 , Hepatocitos/virología , Humanos , Cinética , Unión Proteica , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Tetraspanina 28/química , Proteínas del Envoltorio Viral/fisiologíaRESUMEN
A vaccine that prevents hepatitis C virus (HCV) infection is urgently needed to support an emerging global elimination program. However, vaccine development has been confounded because of HCV's high degree of antigenic variability and the preferential induction of type-specific immune responses with limited potency against heterologous viral strains and genotypes. We showed previously that deletion of the three variable regions from the E2 receptor-binding domain (Δ123) increases the ability of human broadly neutralizing antibodies (bNAbs) to inhibit E2-CD81 receptor interactions, suggesting improved bNAb epitope exposure. In this study, the immunogenicity of Δ123 was examined. We show that high-molecular-weight forms of Δ123 elicit distinct antibody specificities with potent and broad neutralizing activity against all seven HCV genotypes. Antibody competition studies revealed that immune sera raised to high-molecular-weight Δ123 was poly specific, given that it inhibited the binding of human bNAbs directed to three major neutralization epitopes on E2. By contrast, the immune sera raised to monomeric Δ123 predominantly blocked the binding of a non-neutralizing antibody to Δ123, while having reduced ability to block bNAb binding to E2, and neutralization was largely toward the homologous genotype. This increased ability of oligomeric Δ123 to generate bNAbs correlates with occlusion of the non-neutralizing face of E2 in this glycoprotein form. CONCLUSION: The results from this study reveal new information on the antigenic and immunogenic potential of E2-based immunogens and provide a pathway for the development of a simple, recombinant protein-based prophylactic vaccine for HCV with potential for universal protection. (Hepatology 2017;65:1117-1131).
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Hepacivirus/genética , Hepatitis C/genética , Proteínas del Envoltorio Viral/genética , Vacunas contra Hepatitis Viral/farmacología , Animales , Anticuerpos Neutralizantes/inmunología , Especificidad de Anticuerpos/genética , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Epítopos/genética , Genotipo , Cobayas , Hepacivirus/inmunología , Hepatitis C/inmunología , Anticuerpos contra la Hepatitis C/inmunología , Distribución Aleatoria , Estadísticas no Paramétricas , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
The hepatitis C virus (HCV) RNA genome of 9.6 kb encodes only 10 proteins, and so is highly dependent on host hepatocyte factors to facilitate replication. We aimed to identify host factors involved in the egress of viral particles. By screening the supernatant of HCV-infected Huh7 cells using SILAC-based proteomics, we identified the transmembrane protein calsyntenin-1 as a factor specifically secreted by infected cells. Calsyntenin-1 has previously been shown to mediate transport of endosomes along microtubules in neurons, through interactions with kinesin light chain-1. Here we demonstrate for the first time, we believe, a similar role for calsyntenin-1 in Huh7 cells, mediating intracellular transport of endosomes. In HCV-infected cells we show that calsyntenin-1 contributes to the early stages of the viral replication cycle and the formation of the replication complex. Importantly, we demonstrate in our model that silencing calsyntenin-1 disrupts the viral replication cycle, confirming the reliance of HCV on this protein as a host factor. Characterizing the function of calsyntenin-1 will increase our understanding of the HCV replication cycle and pathogenesis, with potential application to other viruses sharing common pathways.
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Proteínas de Unión al Calcio/metabolismo , Hepacivirus/fisiología , Interacciones Huésped-Patógeno , Replicación Viral , Línea Celular , Hepatocitos/virología , HumanosRESUMEN
UNLABELLED: Hepatitis C virus (HCV) envelope glycoproteins E1 and E2 form a heterodimer and mediate receptor interactions and viral fusion. Both E1 and E2 are targets of the neutralizing antibody (NAb) response and are candidates for the production of vaccines that generate humoral immunity. Previous studies demonstrated that N-terminal hypervariable region 1 (HVR1) can modulate the neutralization potential of monoclonal antibodies (MAbs), but no information is available on the influence of HVR2 or the intergenotypic variable region (igVR) on antigenicity. In this study, we examined how the variable regions influence the antigenicity of the receptor binding domain of E2 spanning HCV polyprotein residues 384 to 661 (E2661) using a panel of MAbs raised against E2661 and E2661 lacking HVR1, HVR2, and the igVR (Δ123) and well-characterized MAbs isolated from infected humans. We show for a subset of both neutralizing and nonneutralizing MAbs that all three variable regions decrease the ability of MAbs to bind E2661 and reduce the ability of MAbs to inhibit E2-CD81 interactions. In addition, we describe a new MAb directed toward the region spanning residues 411 to 428 of E2 (MAb24) that demonstrates broad neutralization against all 7 genotypes of HCV. The ability of MAb24 to inhibit E2-CD81 interactions is strongly influenced by the three variable regions. Our data suggest that HVR1, HVR2, and the igVR modulate exposure of epitopes on the core domain of E2 and their ability to prevent E2-CD81 interactions. These studies suggest that the function of HVR2 and the igVR is to modulate antibody recognition of glycoprotein E2 and may contribute to immune evasion. IMPORTANCE: This study reveals conformational and antigenic differences between the Δ123 and intact E2661 glycoproteins and provides new structural and functional data about the three variable regions and their role in occluding neutralizing and nonneutralizing epitopes on the E2 core domain. The variable regions may therefore function to reduce the ability of HCV to elicit NAbs directed toward the conserved core domain. Future studies aimed at generating a three-dimensional structure for intact E2 containing HVR1, and the adjoining NAb epitope at residues 412 to 428, together with HVR2, will reveal how the variable regions modulate antigenic structure.
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Anticuerpos Monoclonales de Origen Murino/química , Anticuerpos Neutralizantes/química , Hepacivirus/química , Anticuerpos contra la Hepatitis C/química , Proteínas del Envoltorio Viral/química , Animales , Anticuerpos Monoclonales de Origen Murino/inmunología , Anticuerpos Neutralizantes/inmunología , Línea Celular , Hepacivirus/genética , Hepacivirus/inmunología , Anticuerpos contra la Hepatitis C/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Tetraspanina 28/química , Tetraspanina 28/genética , Tetraspanina 28/inmunología , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
Booster vaccinations are recommended to improve protection against severe disease from SARS-CoV-2 infection. With primary vaccinations involving various adenoviral vector and mRNA-based formulations, it remains unclear if these differentially affect the immune response to booster doses. We examined the effects of homologous (mRNA/mRNA) and heterologous (adenoviral vector/mRNA) vaccination on antibody and memory B cell (Bmem) responses against ancestral and Omicron subvariants. Healthy adults who received primary BNT162b2 (mRNA) or ChAdOx1 (vector) vaccination were sampled 1-month and 6-months after their 2nd and 3rd dose (homologous or heterologous) vaccination. Recombinant spike receptor-binding domain (RBD) proteins from ancestral, Omicron BA.2 and BA.5 variants were produced for ELISA-based serology, and tetramerized for immunophenotyping of RBD-specific Bmem. Dose 3 boosters significantly increased ancestral RBD-specific plasma IgG and Bmem in both cohorts. Up to 80% of ancestral RBD-specific Bmem expressed IgG1+. IgG4+ Bmem were detectable after primary mRNA vaccination, and expanded significantly to 5-20% after dose 3, whereas heterologous boosting did not elicit IgG4+ Bmem. Recognition of Omicron BA.2 and BA.5 by ancestral RBD-specific plasma IgG increased from 20% to 60% after the 3rd dose in both cohorts. Reactivity of ancestral RBD-specific Bmem to Omicron BA.2 and BA.5 increased following a homologous booster from 40% to 60%, but not after a heterologous booster. A 3rd mRNA dose generates similarly robust serological and Bmem responses in homologous and heterologous vaccination groups. The expansion of IgG4+ Bmem after mRNA priming might result from the unique vaccine formulation or dosing schedule affecting the Bmem response duration and antibody maturation.
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Objectives: Despite vaccination strategies, people with chronic kidney disease, particularly kidney transplant recipients (KTRs), remained at high risk of poor COVID-19 outcomes. We assessed serological responses to the three-dose COVID-19 vaccine schedule in KTRs and people on dialysis, as well as seroresponse predictors and the relationship between responses and breakthrough infection. Methods: Plasma from 30 KTRs and 17 people receiving dialysis was tested for anti-Spike receptor binding domain (RBD) IgG and neutralising antibodies (NAb) to the ancestral and Omicron BA.2 variant after Doses 2 and 3 of vaccination. Results: After three doses, KTRs achieved lower anti-Spike RBD IgG levels (P < 0.001) and NAb titres than people receiving dialysis (P = 0.002). Seropositive cross-reactive Omicron neutralisation levels were achieved in 11/27 (40.7%) KTRs and 11/14 (78.6%) dialysis recipients. ChAdOx1/viral-vector vaccine type, higher mycophenolate dose (> 1 g per day) and lower absolute B-cell counts predicted poor serological responses in KTRs. ChAdOx-1 vaccine type and higher monocyte counts were negative predictors in dialysis recipients. Among ancestral NAb seroresponders, higher NAb levels positively correlated with higher Omicron neutralisation (R = 0.9, P < 0.001). More KTRs contracted SARS-CoV-2 infection (14/30; 47%) than dialysis recipients (5/17; 29%) and had more severe disease. Those with breakthrough infections had significantly lower median interdose incremental change in anti-Spike RBD IgG and ancestral NAb titres. Conclusion: Serological responses to COVID-19 vaccines in KTRs lag behind their dialysis counterparts. KTRs remained at high risk of breakthrough infection after their primary vaccination schedule underlining their need for booster doses, strict infection prevention measures and close surveillance.
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Background: Hepatitis C virus (HCV) infections are more prevalent in people who inject drugs (PWID) who often experience additional health risks. HCV induces inflammation and immune alterations that contribute to hepatic and non-hepatic morbidities. It remains unclear whether curative direct acting antiviral (DAA) therapy completely reverses immune alterations in PWID. Methods: Plasma biomarkers of immune activation associated with chronic disease risk were measured in HCV-seronegative (n=24) and HCV RNA+ (n=32) PWID at baseline and longitudinally after DAA therapy. Adjusted generalised estimating equations were used to assess longitudinal changes in biomarker levels. Comparisons between community controls (n=29) and HCV-seronegative PWID were made using adjusted multiple regression modelling. Results: HCV-seronegative PWID exhibited significantly increased levels of inflammatory biomarkers including soluble (s) TNF-RII, IL-6, sCD14 and sCD163 and the diabetes index HbA1c as compared to community controls. CXCL10, sTNF-RII, vascular cell adhesion molecule-1 and lipopolysaccharide binding protein (LBP) were additionally elevated in PWID with viremic HCV infection as compared to HCV- PWID. Whilst curative DAA therapy reversed some biomarkers, others including LBP and sTNF-RII remained elevated 48 weeks after HCV cure. Conclusion: Elevated levels of inflammatory and chronic disease biomarkers in PWID suggest an increased risk of chronic morbidities such as diabetes and cardiovascular disease. HCV infection in PWID poses an additional disease burden, amplified by the incomplete reversal of immune dysfunction following DAA therapy. These findings highlight the need for heightened clinical surveillance of PWID for chronic inflammatory diseases, particularly those with a history of HCV infection.
Asunto(s)
Diabetes Mellitus , Hepatitis C Crónica , Hepatitis C , Abuso de Sustancias por Vía Intravenosa , Humanos , Hepacivirus , Antivirales/uso terapéutico , Abuso de Sustancias por Vía Intravenosa/complicaciones , Abuso de Sustancias por Vía Intravenosa/tratamiento farmacológico , Abuso de Sustancias por Vía Intravenosa/epidemiología , Hepatitis C Crónica/complicaciones , Hepatitis C Crónica/tratamiento farmacológico , Hepatitis C/complicaciones , Hepatitis C/tratamiento farmacológico , Biomarcadores , Diabetes Mellitus/tratamiento farmacológicoRESUMEN
Hepatitis C virus glycoprotein E2 contains 18 conserved cysteines predicted to form nine disulfide pairs. In this study, a comprehensive cysteine-alanine mutagenesis scan of all 18 cysteine residues was performed in E1E2-pseudotyped retroviruses (HCVpp) and recombinant E2 receptor-binding domain (E2 residues 384 to 661 [E2(661)]). All 18 cysteine residues were absolutely required for HCVpp entry competence. The phenotypes of individual cysteines and pairwise mutation of disulfides were largely the same for retrovirion-incorporated E2 and E2(661), suggesting their disulfide arrangements are similar. However, the contributions of each cysteine residue and the nine disulfides to E2 structure and function varied. Individual Cys-to-Ala mutations revealed discordant effects, where removal of one Cys within a pair had minimal effect on H53 recognition and CD81 binding (C486 and C569) while mutation of its partner abolished these functions (C494 and C564). Removal of disulfides at C581-C585 and C452-C459 significantly reduced the amount of E1 coprecipitated with E2, while all other disulfides were absolutely required for E1E2 heterodimerization. Remarkably, E2(661) tolerates the presence of four free cysteines, as simultaneous mutation of C452A, C486A, C569A, C581A, C585A, C597A, and C652A (M+C597A) retained wild-type CD81 binding. Thus, only one disulfide from each of the three predicted domains, C429-C552 (DI), C503-C508 (DII), and C607-C644 (DIII), is essential for the assembly of the E2(661) CD81-binding site. Furthermore, the yield of total monomeric E2 increased to 70% in M+C597A. These studies reveal the contribution of each cysteine residue and the nine disulfide pairs to E2 structure and function.
Asunto(s)
Secuencia Conservada , Cisteína/química , Hepacivirus/fisiología , Hepatitis C/virología , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/metabolismo , Secuencia de Aminoácidos , Línea Celular , Cisteína/genética , Cisteína/metabolismo , Hepacivirus/química , Hepacivirus/genética , Humanos , Datos de Secuencia Molecular , Unión Proteica , Pliegue de Proteína , Proteínas del Envoltorio Viral/genética , Internalización del VirusRESUMEN
The protonation of histidine in acidic environments underpins its role in regulating the function of pH-sensitive proteins. For pH-sensitive viral fusion proteins, histidine protonation in the endosome leads to the activation of their membrane fusion function. The HCV (hepatitis C virus) glycoprotein E1-E2 heterodimer mediates membrane fusion within the endosome, but the roles of conserved histidine residues in the formation of a functional heterodimer and in sensing pH changes is unknown. We examined the functional roles of conserved histidine residues located within E1 and E2. The E1 mutations, H222A/R, H298R and H352A, disrupted E1-E2 heterodimerization and reduced virus entry. A total of five out of six histidine residues located within the E2 RBD (receptor-binding domain) were important for the E2 fold, and their substitution with arginine or alanine caused aberrant heterodimerization and/or CD81 binding. Distinct roles in E1-E2 heterodimerization and in virus entry were identified for His691 and His693 respectively within the membrane-proximal stem region. Viral entry and cell-cell fusion at neutral and low pH values were enhanced with H445R, indicating that the protonation state of His445 is a key regulator of HCV fusion. However, H445R did not overcome the block to virus entry induced by bafilomycin A1, indicating a requirement for an endosomal activation trigger in addition to acidic pH.
Asunto(s)
Hepacivirus/fisiología , Tetraspanina 28/química , Proteínas del Envoltorio Viral/biosíntesis , Internalización del Virus , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secuencia Conservada , Células HEK293 , Hepacivirus/patogenicidad , Histidina/genética , Humanos , Concentración de Iones de Hidrógeno , Mutagénesis Sitio-Dirigida , Unión Proteica , Pliegue de Proteína , Multimerización de Proteína , Estabilidad Proteica , Estructura Terciaria de Proteína , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , ViriónRESUMEN
The HCV envelope glycoproteins E1 and E2 contain eight and 18 highly conserved cysteine residues, respectively. Here, we examined the oxidation state of E1E2 heterodimers incorporated into retroviral pseudotyped particles (HCVpp) and investigated the significance of free sulfhydryl groups in cell culture-derived HCV (HCVcc) and HCVpp entry. Alkylation of free sulfhydryl groups on HCVcc/pp with a membrane-impermeable sulfhydryl-alkylating reagent 4-(N-maleimido)benzyl-α-trimethylammonium iodide (M135) prior to virus attachment to cells abolished infectivity in a dose-dependent manner. Labeling of HCVpp envelope proteins with EZ-Link maleimide-PEG2-biotin (maleimide-biotin) detected free thiol groups in both E1 and E2. Unlike retroviruses that employ disulfide reduction to facilitate virus entry, the infectivity of alkylated HCVcc could not be rescued by addition of exogenous reducing agents. Furthermore, the infectivity of HCVcc bound to target cells was not affected by addition of M135 indicative of a change in glycoprotein oxidation state from reduced to oxidized following virus attachment to cells. By contrast, HCVpp entry was reduced by 61% when treated with M135 immediately following attachment to cells, suggesting that the two model systems might demonstrate variations in oxidation kinetics. Glycoprotein oxidation was not altered following binding of HCVpp incorporated E1E2 to soluble heparin or recombinant CD81. These results suggest that HCV entry is dependent on the presence of free thiol groups in E1 and E2 prior to cellular attachment and reveals a new essential component of the HCV entry process.
Asunto(s)
Cisteína/metabolismo , Hepacivirus/metabolismo , Multimerización de Proteína/fisiología , Proteínas del Envoltorio Viral/metabolismo , Internalización del Virus , Alquilantes/farmacología , Alquilación/efectos de los fármacos , Células HEK293 , Humanos , Oxidación-Reducción/efectos de los fármacos , Compuestos de Amonio Cuaternario/farmacología , Proteínas Recombinantes/metabolismo , Tetraspanina 28/metabolismoRESUMEN
The three variable regions of hepatitis C virus (HCV) glycoprotein E2 can be removed simultaneously from the E2 ectodomain (residues 384-661) without affecting folding or CD81 binding. In this study, we show that deletion of hypervariable region (HVR) 2 or the intergenotypic variable region (igVR) in the context of the E1E2 polyprotein eliminates formation of heterodimers, reduces CD81 binding and abolishes virus entry. The replication competence of genomic RNA transcribed from the JFH1 infectious HCV clone was not affected by the HVR1, HVR2 or igVR deletions in transfected Huh7.5 cells. However, infectivity of the resultant cell-culture-derived HCV (HCVcc) was abolished by HVR2 or igVR deletions, while deletion of HVR1 led to a 5- to 10-fold reduction in infectivity. Serial passage of cells transfected with genomes lacking HVR1 generated reverted viruses with wild-type levels of infectivity. Sequencing of viral cDNA obtained after full reversion revealed mutations in E1 (I262L) and E2 (N415D) that were present in 35 and 27â% of clones, respectively. Insertion of N415D into HVR1-deleted HCV genomes conferred wild-type levels of infectivity, while I262L increased infectivity by 2.5-fold. These results suggest that HVR2 and the igVR, but not HVR1, are essential for structural integrity and function of the HCV glycoprotein heterodimer.