Lasting immunity following SARS-CoV-2 infection is questioned because serum antibodies decline in convalescence. However, functional immunity is mediated by long-lived memory T and B (Bmem) cells. Therefore, we generated fluorescently-labeled tetramers of the spike receptor binding domain (RBD) and nucleocapsid protein (NCP) to determine the longevity and immunophenotype of SARS-CoV-2-specific Bmem cells in COVID-19 patients. A total of 36 blood samples were obtained from 25 COVID-19 patients between 4 and 242 days post-symptom onset including 11 paired samples. While serum IgG to RBD and NCP was identified in all patients, antibody levels began declining at 20 days post-symptom onset. RBD- and NCP-specific Bmem cells predominantly expressed IgM+ or IgG1+ and continued to rise until 150 days. RBD-specific IgG+ Bmem were predominantly CD27+, and numbers significantly correlated with circulating follicular helper T cell numbers. Thus, the SARS-CoV-2 antibody response contracts in convalescence with persistence of RBD- and NCP-specific Bmem cells. Flow cytometric detection of SARS-CoV-2-specific Bmem cells enables detection of long-term immune memory following infection or vaccination for COVID-19.

The hepatitis C virus (HCV) glycoproteins E1 and E2 form a heterodimer that mediates CD81 receptor binding and viral entry. In this study, we used site-directed mutagenesis to examine the functional role of a conserved G 436 WLAGLFY motif of E2. The mutants could be placed into two groups based on the ability of mature virion-incorporated E1E2 to bind the large extracellular loop (LEL) of CD81 versus the ability to mediate cellular entry of pseudotyped retroviral particles. Group 1 comprised E2 mutants where LEL binding ability largely correlated with viral entry ability, with conservative and nonconservative substitutions (W437 L/A, L438A, L441V/F, and F442A) inhibiting both functions. These data suggest that Trp-437, Leu-438, Leu-441, and Phe-442 directly interact with the LEL. Group 2 comprised E2 glycoproteins with more conservative substitutions that lacked LEL binding but retained between 20% and 60% of wild-type viral entry competence. The viral entry competence displayed by group 2 mutants was explained by residual binding by the E2 receptor binding domain to cellular full-length CD81. A subset of mutants maintained LEL binding ability in the context of intracellular E1E2 forms, but this function was largely lost in virion-incorporated glycoproteins. These data suggest that the CD81 binding site undergoes a conformational transition during glycoprotein maturation through the secretory pathway. The G436P mutant was an outlier, retaining near-wild-type levels of CD81 binding but lacking significant viral entry ability. These findings indicate that the G 436 WLA GLFY motif of E2 functions in CD81 binding and in pre-or post-CD81-dependent stages of viral entry.Hepatitis C virus (HCV) is a member of the Flaviviridae family of small, enveloped plus-strand RNA viruses that has infected over 3% of the global human population, causing significant morbidity and mortality. Hepatitis C virus encodes two type I transmembrane glycoproteins, E1 and E2, which are cleaved from the viral polyprotein precursor by signal peptidases in the endoplasmic reticulum (ER). E1 and E2 form heterogeneous mixtures of covalently and noncovalently associated heterodimers that are largely retained in the ER via retention sequences located in their transmembrane domains (5,6,14). However, a small proportion of E1E2 heterodimer escapes the ER and matures through the secretory pathway (11). Retroviruses such as human immunodeficiency virus type 1 (HIV-1) can be pseudotyped with cell surface-expressed E1E2 (E1E2-pseudotyped particles [E1E2-pps]). E1E2-pps contain noncovalently associated E1E2 heterodimers and are capable of infecting primary human hepatocytes and various human liver cell lines including Huh7 cells (2,11,23). Viral entry of E1E2-pps and cell culture-grown HCV occurs via receptor-mediated endocytosis, the E1E2 glycoproteins mediating low-pH-dependent fusion (1, 2, 23, 24, 40).The E2 glycoprotein mediates binding to cellular receptors, including the tetraspanin CD81 (34) and the high-density lipoprotein receptor scavenger recep...

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).

The E1E2 glycoprotein heterodimer of Hepatitis C virus mediates viral entry. E2 attaches the virus to cellular receptors; however, the function of E1 is unknown. We tested the hypothesis that E1 is a truncated class II fusion protein. We mutated amino acids within a predicted fusion peptide (residues 276-286) and a truncated C-terminal stem-like motif, containing a membrane-proximal heptad-repeat sequence (residues 330-347). The fusion peptide mutation F285A abolished viral entry, while mutation of other hydrophobic residues had no effect. Alanine replacement of heptad-repeat residues blocked entry in three of five cases, whereas substitution with the helix breaker, Pro, led to loss of entry function in all cases. The mutations did not affect glycoprotein expression, heterodimerization with E2 or global folding, in contrast to the effects of mutations in the fusion motifs of prototypical class II fusion proteins. Our data suggest that E1 is unlikely to function in an analogous manner to other class II fusion glycoproteins.Virus replication is initiated when viral attachment proteins bind to cellular receptors. In the case of enveloped viruses, water molecules bound to the polar headgroups of viral and cellular membranes exert strong repulsive forces when brought into close proximity. Viral fusion protein(s) overcome this hydration barrier by catalysing membrane fusion. Fusion proteins characteristically comprise two hydrophobic membrane-interactive regions: a C-terminal transmembrane domain (TMD) and a fusion peptide that is located either at the N terminus or internally (Kielian & Rey, 2006). X-ray crystallography has thus far revealed two structural classes of fusion glycoprotein (Kielian, 2006;Kielian & Rey, 2006;Skehel & Wiley, 2000;Stiasny & Heinz, 2006). Class I fusion proteins [e.g. human immunodeficiency virus 1 (HIV-1) gp41, influenza virus HA2] are helical, trimeric rods that project as spikes from the viral envelope. In the fusion-activated state, their N (fusion peptide-proximal) and C (TMD-proximal) termini become juxtaposed at one end of a helical hairpin core domain. Class II fusion glycoproteins (e.g. flavivirus E, alpha virus E1) comprise three domains rich in b-strands that lie roughly parallel to the viral membrane. At neutral pH, the metastable state of E, which has dual receptor-binding and fusion functions, is maintained in a homodimer by monomer-monomer interactions that sequester the fusion loop. In the case of alphaviruses, glycoprotein E2 mediates receptor binding, whereas the associated E1 trimer mediates fusion. E1 metastability is maintained through E1-E2 interactions. At low fusion pH, E and E1 have almost identical trimeric structures where membrane-inserted fusion loops are atop three uptilted protomers. Trimerization creates three surface-exposed hydrophobic grooves along the trimer axis for the antiparallel packing of the TMD-proximal amphipathic a-helical stem to form a hairpin. Thus, hairpin formation is employed by both classes of fusion glycoprotein to appose membrane-associ...

The hepatitis C virus glycoprotein E2 receptor-binding domain is encompassed by amino acids 384 to 661 (E2 661 ) and contains two hypervariable sequences, HVR1 and HVR2. E2 sequence comparisons revealed a third variable region, located between residues 570 and 580, that varies widely between genotypes, designated here as igVR, the intergenotypic variable region. A secreted E2 661 glycoprotein with simultaneous deletions of the three variable sequences retained its ability to bind CD81 and conformation-dependent monoclonal antibodies (MAbs) and displayed enhanced binding to a neutralizing MAb directed to E2 immunogenic domain B. Our data provide insights into the E2 structure by suggesting that the three variable regions reside outside a conserved E2 core.Hepatitis C virus (HCV) is a member of the Flaviviridae family and is classified into six major genotypes and numerous subtypes that differ in nucleotide sequence by up to 35% and 25%, respectively (37). The virus encodes two envelope glycoproteins, E1 (polyprotein residues 191 to 383 [H77c numbering is used throughout this study]) and E2 (residues 384 to 746) that function in viral entry as noncovalently associated heterodimers (1) (Fig. 1A). Glycoprotein E2 attaches the virus to host cell receptors that include the tetraspanin CD81 (33), claudin-1 (14), and the high-density lipoprotein receptor scavenger receptor, class B type I (SR-B1) (35), while E1 contains an internal fusion peptide-like sequence and membrane-proximal heptad repeat, both containing residues essential for viral entry function (9, 16).The receptor-binding domain (RBD) of E2 is encompassed by polyprotein residues 384 to 661 (E2 661 ) (Fig. 1A). Recombinant forms of E2 661 RBD are efficiently secreted from transfected cells and are able to interact with CD81, SR-B1, and other cell surface molecules (4,33,35). The E2 RBD contains two hypervariable regions, HVR1 (residues 384 to 410) and HVR2 (residues 474 to 482) (21, 42). Hypervariable region 1, located at the N terminus of E2, is the most variable region in the HCV genome, is highly immunogenic, and rapidly accumulates neutralization escape mutations (15). Despite the high level of amino acid variability in HVR1, there is an overall conservation of basic residues that are important for viral entry (3,32). HVR1 also appears to play a role in the enhancement of viral entry via high-density lipoproteins present in human serum, which upregulate the SR-B1-mediated endocytosis of virions (2,7,26,29,40).Hypervariable region 2 is located within the region flanked by Cys-459 to Cys-486 (21). Although originally described as a 7-residue sequence, comparison of E2 sequences from different HCV genotypes suggests it may extend from residue 461 to 481 (Fig. 1B and data not shown). The degree of sequence identity across the Cys-459 to Cys-486 region ranges from 39% (genotypes 1a and b) to 93% (genotype 5a), and the region is 28 to 30 residues in length (data not shown). In comparison to the HVR1 sequence, the sequence of HVR2 is relatively stable within HCV...