Reduced Sensitivity of Commercial Spike-Specific Antibody Assays after Primary Infection with the SARS-CoV-2 Omicron Variant.

The SARS-CoV-2 Omicron variant is characterized by substantial changes in the antigenic structure of the Spike (S) protein. Therefore, antibodies induced by primary Omicron infection lack neutralizing activity against earlier variants. In this study, we analyzed whether these antigenic changes impact the sensitivity of commercial anti-SARS-CoV-2 antibody assays. Sera from 37 unvaccinated, convalescent individuals after putative primary Omicron infection were tested with a panel of 20 commercial anti-SARS-CoV-2 immunoassays. As controls, we used samples from 43 individuals after primary infection with the SARS-CoV-2 ancestral wild-type strain. In addition, variant-specific live-virus neutralization assays were used as a reference for the presence of SARS-CoV-2-specific antibodies in the samples. Notably, in Omicron convalescents, there was a statistically significant reduction in the sensitivity of all antibody assays containing S or its receptor-binding-domain (RBD) as antigens. Furthermore, antibody levels quantified by these assays displayed a weaker correlation with Omicron-specific neutralizing antibody titers than with those against the wild type. In contrast, the sensitivity of nucleocapsid-protein-specific immunoassays was similar in wild-type and Omicron-infected subjects. In summary, the antigenic changes in the Omicron S lead to reduced immunoreactivity in the current commercial S- and RBD-specific antibody assays, impairing their diagnostic performance. IMPORTANCE This study demonstrates that the antigenic changes of the SARS-CoV-2 Omicron variant affect test results from commercial Spike- and RBD-specific antibody assays, significantly diminishing their sensitivities and diagnostic abilities to assess neutralizing antibodies.

Different Neutralization Profiles After Primary SARS-CoV-2 Omicron BA.1 and BA.2 Infections.

Background and Methods: The SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) Omicron (B.1.1.529) variant is the antigenically most distinct variant to date. As the heavily mutated spike protein enables neutralization escape, we studied serum-neutralizing activities of naïve and vaccinated individuals after Omicron BA.1 or BA.2 sub-lineage infections in live virus neutralization tests with Omicron BA.1, Omicron BA.2, wildtype (WT, B1.1), and Delta (B.1.617.2) strains. Serum samples obtained after WT infections and three-dose mRNA vaccinations with and without prior infection were included as controls. Results: Primary BA.1 infections yielded reduced neutralizing antibody levels against WT, Delta, and Omicron BA.2, while samples from BA.2-infected individuals showed almost no cross-neutralization against the other variants. Serum neutralization of Omicron BA.1 and BA.2 variants was detectable after three-dose mRNA vaccinations, but with reduced titers. Vaccination-breakthrough infections with either Omicron BA.1 or BA.2, however, generated equal cross-neutralizing antibody levels against all SARS-CoV-2 variants tested. Conclusions: Our study demonstrates that although Omicron variants are able to enhance cross-neutralizing antibody levels in pre-immune individuals, primary infections with BA.1 or BA.2 induced mostly variant-specific neutralizing antibodies, emphasizing the differently shaped humoral immunity induced by the two Omicron variants. These data thus contribute substantially to the understanding of antibody responses induced by primary Omicron infections or multiple exposures to different SARS-CoV-2 variants and are of particular importance for developing vaccination strategies in the light of future emerging variants.

Heterogeneous SARS-CoV-2-Neutralizing Activities After Infection and Vaccination.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) with different resistance levels to existing immunity have recently emerged. Antibodies that recognize the SARS-CoV-2 spike (S) protein and exhibit neutralizing activities are considered the best correlate of protection and an understanding of humoral immunity is crucial for controlling the pandemic. We thus analyzed such antibodies in individuals recovered from infection in 2020 as well as vaccinees after two doses of an mRNA vaccine. Methods: Neutralizing antibody responses against three SARS-CoV-2 variants (D614G, VOCs Beta and Delta) were determined in serum samples from 54 infected individuals (24 non-hospitalized, 30 hospitalized) and 34 vaccinees shortly after symptom onset or second vaccination, respectively, as well as six months later. In addition, the effect of the S sequence of the infecting strain on neutralization was studied. Results: Non-hospitalized patients had the lowest neutralization titers against all variants, while those of hospitalized patients equaled or exceeded those of vaccinees. Neutralizing activity was lower against the two VOCs and declined significantly in all cohorts after six months. This decrease was more pronounced in hospitalized and vaccinated individuals than in non-hospitalized patients. Of note, the specific neutralizing activity (NT titer/ELISA value ratio) was higher in the infected cohorts than in vaccinees and did not differ between non-hospitalized and hospitalized patients. Patients infected with viral strains carrying mutations in the N-terminal domain of the spike protein were impaired in Beta VOC neutralization. Conclusions: Specific neutralizing activities were higher in infected than in vaccinated individuals, and no difference in the quality of these antibodies was observed between hospitalized and non-hospitalized patients, despite significantly lower titers in the latter group. Additionally, antibody responses of infected individuals showed greater heterogeneity than those of vaccinees, which was associated with mutations in the spike protein of the infecting strain. Overall, our findings yielded novel insights into SARS-CoV-2-specific neutralizing antibodies, evolving differently after virus infection and COVID-19 vaccination, which is an important issue to consider in ongoing vaccine strategy improvements.

Evolution and activation mechanism of the flavivirus class II membrane-fusion machinery.

The flavivirus envelope glycoproteins prM and E drive the assembly of icosahedral, spiky immature particles that bud across the membrane of the endoplasmic reticulum. Maturation into infectious virions in the trans-Golgi network involves an acid-pH-driven rearrangement into smooth particles made of (prM/E)2 dimers exposing a furin site for prM cleavage into "pr" and "M". Here we show that the prM "pr" moiety derives from an HSP40 cellular chaperonin. Furthermore, the X-ray structure of the tick-borne encephalitis virus (pr/E)2 dimer at acidic pH reveals the E 150-loop as a hinged-lid that opens at low pH to expose a positively-charged pr-binding pocket at the E dimer interface, inducing (prM/E)2 dimer formation to generate smooth particles in the Golgi. Furin cleavage is followed by lid-closure upon deprotonation in the neutral-pH extracellular environment, expelling pr while the 150-loop takes the relay in fusion loop protection, thus revealing the elusive flavivirus mechanism of fusion activation.

Increased in vitro neutralizing activity of SARS-CoV-2 IgA1 dimers compared to monomers and IgG.

Here, we expressed two neutralizing monoclonal antibodies (Abs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; H4 and B38) in three formats: IgG1, IgA1 monomers (m), and IgA1 dimers (d) in glycoengineered Nicotiana benthamiana plants. All six Ab variants assembled properly and exhibited a largely homogeneous glycosylation profile. Despite modest variation in antigen binding between Ab formats, SARS-CoV-2 neutralization (NT) potency significantly increased in the following manner: IgG1 < IgA1-m < IgA1-d, with an up to 240-fold NT increase of dimers compared to corresponding monomers. Our results underscore that both IgA's structural features and multivalency positively impact NT potency. In addition, they emphasize the versatile use of plants for the rapid expression of complex human proteins.

Highly active engineered IgG3 antibodies against SARS-CoV-2.

Monoclonal antibodies (mAbs) that efficiently neutralize SARS-CoV-2 have been developed at an unprecedented speed. Notwithstanding, there is a vague understanding of the various Ab functions induced beyond antigen binding by the heavy-chain constant domain. To explore the diverse roles of Abs in SARS-CoV-2 immunity, we expressed a SARS-CoV-2 spike protein (SP) binding mAb (H4) in the four IgG subclasses present in human serum (IgG1-4) using glyco-engineered Nicotiana benthamiana plants. All four subclasses, carrying the identical antigen-binding site, were fully assembled in planta and exhibited a largely homogeneous xylose- and fucose-free glycosylation profile. The Ab variants ligated to the SP with an up to fivefold increased binding activity of IgG3. Furthermore, all H4 subtypes were able to neutralize SARS-CoV-2. However, H4-IgG3 exhibited an up to 50-fold superior neutralization potency compared with the other subclasses. Our data point to a strong protective effect of IgG3 Abs in SARS-CoV-2 infection and suggest that superior neutralization might be a consequence of cross-linking the SP on the viral surface. This should be considered in therapy and vaccine development. In addition, we underscore the versatile use of plants for the rapid expression of complex proteins in emergency cases.

An Absolutely Conserved Tryptophan in the Stem of the Envelope Protein E of Flaviviruses Is Essential for the Formation of Stable Particles.

The major envelope protein E of flaviviruses contains an ectodomain that is connected to the transmembrane domain by the so-called "stem" region. In mature flavivirus particles, the stem is composed of two or three mostly amphipathic α-helices and a conserved sequence element (CS) with an undefined role in the viral life cycle. A tryptophan is the only residue within this region which is not only conserved in all vector-borne flaviviruses, but also in the group with no known vector. We investigated the importance of this residue in different stages of the viral life cycle by a mutagenesis-based approach using tick-borne encephalitis virus (TBEV). Replacing W421 by alanine or histidine strongly reduced the release of infectious virions and their thermostability, whereas fusion-related entry functions and virus maturation were still intact. Serial passaging of the mutants led to the emergence of a same-site compensatory mutation to leucine that largely restored these properties of the wildtype. The conserved tryptophan in CS (or another big hydrophobic amino acid at the same position) is thus essential for the assembly and infectivity of flaviviruses by being part of a network required for conferring stability to infectious particles.

Tick-Borne Encephalitis Specific Lymphocyte Response after Allogeneic Hematopoietic Stem Cell Transplantation Predicts Humoral Immunity after Vaccination.

The aim of this prospective study was to assess lymphocyte proliferative and cytokine response prior to and following tick-borne encephalitis (TBE) immunization among patients after allogeneic hematopoietic stem cell transplantation (HSCT). Seventeen adult patients 11-13 months after HSCT and eight unvaccinated healthy adults received up to three TBE vaccinations. Following in vitro stimulation with TBE-antigen, lymphocyte proliferation and cytokine secretion (IL-2, IL-10, IL-13, TNF-alpha, IFN-gamma, GM-CSF) were analyzed by thymidine incorporation assay and the Luminex system. Ten patients (59%) showed significant baseline TBE-specific lymphocyte proliferation (stimulation index (SI) > 3) prior to vaccination, but none of the unvaccinated controls (p = 0.002). All patients with a TBE-specific antibody response after two vaccinations (at least 2-fold increase of neutralization test titers) exhibited a strong TBE-specific lymphocyte proliferative response at baseline (SI > 10). Patients with sibling donors had a significantly stronger baseline TBE-specific lymphocyte proliferative and IL-13 cytokine response than patients with unrelated donors (p < 0.05). In conclusion, a relevant proportion of patients showed TBE-specific lymphocyte proliferative and cytokine responses prior to vaccination after HSCT, which predicted the humoral response to the vaccine. Patients with vaccinated sibling donors were more likely to elicit a cellular immune response than patients with unrelated donors of unknown vaccination status.

Distinguishing features of current COVID-19 vaccines: knowns and unknowns of antigen presentation and modes of action.

COVID-19 vaccines were developed with an unprecedented pace since the beginning of the pandemic. Several of them have reached market authorization and mass production, leading to their global application on a large scale. This enormous progress was achieved with fundamentally different vaccine technologies used in parallel. mRNA, adenoviral vector as well as inactivated whole-virus vaccines are now in widespread use, and a subunit vaccine is in a final stage of authorization. They all rely on the native viral spike protein (S) of SARS-CoV-2 for inducing potently neutralizing antibodies, but the presentation of this key antigen to the immune system differs substantially between the different categories of vaccines. In this article, we review the relevance of structural modifications of S in different vaccines and the different modes of antigen expression after vaccination with genetic adenovirus-vector and mRNA vaccines. Distinguishing characteristics and unknown features are highlighted in the context of protective antibody responses and reactogenicity of vaccines.

High-throughput sequencing of two European strains of tick-borne encephalitis virus (TBEV), Hochosterwitz and 1993/783.

Tick-borne encephalitis virus (TBEV) is a medically important arbovirus, widespread in Europe and Asia. The virus is primarily transmitted to humans and animals by bites from ticks and, in rare cases, by consumption of unpasteurized dairy products. The aim of this study was to sequence and characterize two TBEV strains with amplicon sequencing by designing overlapping primers. The amplicon sequencing, via Illumina MiSeq, covering nearly the entire TBEV genome, was successful: We retrieved and characterized the complete polyprotein sequence of two TBEV strains, Hochosterwitz and 1993/783 from Austria and Sweden, respectively. In this study the previous phylogenetic analysis of both strains was confirmed to be of the European subtypes of TBEV (TBEV-Eu) by whole genome sequencing. The Hochosterwitz strain clustered with the two strains KrM 93 and KrM 213 from South Korea, and the 1993/783 strain clustered together with the NL/UH strain from the Netherlands. Our study confirms the suitability and rapidness of the high-throughput sequencing method used to produce complete TBEV genomes from TBEV samples of high viral load giving high-molecular-weight cDNA with large overlapping amplicons.

Development and characterization of specific anti-Usutu virus chicken-derived single chain variable fragment antibodies.

Usutu virus belongs to the Japanese encephalitis serogroup within the Flaviviridae family. Mammals may become incidental hosts after the bite of an infected mosquito while birds act as the main reservoir. Human cases have become more common recently and elicit various outcomes ranging from asymptomatic to severe illness including encephalitis. Problematically, antisera against Usutu virus cross-react with other flaviviruses such as the co-circulating West Nile virus. As an approach to generate Usutu virus-specific antibodies, we immunized chickens with purified Usutu virus envelope protein domain III, isolated the spleen mRNA and generated an scFv phage display library. The most potent binders for Usutu virus domain III were selected via biopanning and their affinity to domain III was examined using SPR. Four scFvs bound the domain III of Usutu virus in the nanomolar region; two bound the protein over 40 times more strongly than West Nile virus domain III. We further characterized these scFv antibodies for suitability in standard laboratory tests such as western blots, ELISA, and neutralization tests. Four specific and one cross-reactive antibody performed well in western blots with domain III and the full-length envelope protein of Usutu virus and West Nile virus. All antibodies bound in virus ELISA assays to Usutu virus strain Vienna-2001. However, none of the antibodies neutralized either Usutu virus or West Nile virus. These antibody candidates could be crucial in future diagnostic tests to distinguish Usutu virus from other flaviviruses and might even offer virus neutralization after a conversion to Fab or IgG.

Humoral immune response to tick-borne encephalitis vaccination in allogeneic blood and marrow graft recipients.

The aim of this prospective study was to characterize the humoral immune response to TBE vaccination after hematopoietic stem cell transplantation (HSCT). Nineteen adult patients 11-13 months after HSCT and 15 age-matched immunocompetent adults received up to three TBE vaccinations. Antibodies against TBE virus were measured by neutralization test (NT). As primary endpoint, the antibody response (NT titer of ≥10 and at least a twofold increase from baseline 4 weeks after second vaccination) was compared between patients and controls using Fisher exact test. Prior vaccination, 15 (79%) HSCT patients still had detectable neutralizing antibodies. At primary endpoint, the antibody response was significantly lower in patients than in controls (35% versus 93%; p < 0.001). The CD4+ cell count was a predictor for an antibody response in patients (p = 0.019). Interestingly, the majority of HSCT patients still had detectable antibodies prior vaccination. Following vaccination, antibody response in HSCT patients was associated with the CD4+ cell count.

Impact of flavivirus vaccine-induced immunity on primary Zika virus antibody response in humans.

BACKGROUND: Zika virus has recently spread to South- and Central America, causing congenital birth defects and neurological complications. Many people at risk are flavivirus pre-immune due to prior infections with other flaviviruses (e.g. dengue virus) or flavivirus vaccinations. Since pre-existing cross-reactive immunity can potentially modulate antibody responses to Zika virus infection and may affect the outcome of disease, we analyzed fine-specificity as well as virus-neutralizing and infection-enhancing activities of antibodies induced by a primary Zika virus infection in flavivirus-naïve as well as yellow fever- and/or tick-borne encephalitis-vaccinated individuals. METHODOLOGY: Antibodies in sera from convalescent Zika patients with and without vaccine-induced immunity were assessed by ELISA with respect to Zika virus-specificity and flavivirus cross-reactivity. Functional analyses included virus neutralization and infection-enhancement. The contribution of IgM and cross-reactive antibodies to these properties was determined by depletion experiments. PRINCIPAL FINDINGS: Pre-existing flavivirus immunity had a strong influence on the antibody response in primary Zika virus infections, resulting in higher titers of broadly flavivirus cross-reactive antibodies and slightly lower levels of Zika virus-specific IgM. Antibody-dependent enhancement (ADE) of Zika virus was mediated by sub-neutralizing concentrations of specific IgG but not by cross-reactive antibodies. This effect was potently counteracted by the presence of neutralizing IgM. Broadly cross-reactive antibodies were able to both neutralize and enhance infection of dengue virus but not Zika virus, indicating a different exposure of conserved sequence elements in the two viruses. CONCLUSIONS: Our data point to an important role of flavivirus-specific IgM during the transient early stages of infection, by contributing substantially to neutralization and by counteracting ADE. In addition, our results highlight structural differences between strains of Zika and dengue viruses that are used for analyzing infection-enhancement by cross-reactive antibodies. These findings underscore the possible impact of specific antibody patterns on flavivirus disease and vaccination efficacy.

CD4 T Cell Determinants in West Nile Virus Disease and Asymptomatic Infection.

West Nile (WN) virus infection of humans is frequently asymptomatic, but can also lead to WN fever or neuroinvasive disease. CD4 T cells and B cells are critical in the defense against WN virus, and neutralizing antibodies, which are directed against the viral glycoprotein E, are an accepted correlate of protection. For the efficient production of these antibodies, B cells interact directly with CD4 helper T cells that recognize peptides from E or the two other structural proteins (capsid-C and membrane-prM/M) of the virus. However, the specific protein sites yielding such helper epitopes remain unknown. Here, we explored the CD4 T cell response in humans after WN virus infection using a comprehensive library of overlapping peptides covering all three structural proteins. By measuring T cell responses in 29 individuals with either WN virus disease or asymptomatic infection, we showed that CD4 T cells focus on peptides in specific structural elements of C and at the exposed surface of the pre- and postfusion forms of the E protein. Our data indicate that these immunodominant epitopes are recognized in the context of multiple different HLA molecules. Furthermore, we observed that immunodominant antigen regions are structurally conserved and similarly targeted in other mosquito-borne flaviviruses, including dengue, yellow fever, and Zika viruses. Together, these findings indicate a strong impact of virion protein structure on epitope selection and antigenicity, which is an important issue to consider in future vaccine design.

Structural Influence on the Dominance of Virus-Specific CD4 T Cell Epitopes in Zika Virus Infection.

Zika virus (ZIKV) has recently caused explosive outbreaks in Pacific islands, South- and Central America. Like with other flaviviruses, protective immunity is strongly dependent on potently neutralizing antibodies (Abs) directed against the viral envelope protein E. Such Ab formation is promoted by CD4 T cells through direct interaction with B cells that present epitopes derived from E or other structural proteins of the virus. Here, we examined the extent and epitope dominance of CD4 T cell responses to capsid (C) and envelope proteins in Zika patients. All patients developed ZIKV-specific CD4 T cell responses, with substantial contributions of C and E. In both proteins, immunodominant epitopes clustered at sites that are structurally conserved among flaviviruses but have highly variable sequences, suggesting a strong impact of protein structural features on immunodominant CD4 T cell responses. Our data are particularly relevant for designing flavivirus vaccines and their evaluation in T cell assays and provide insights into the importance of viral protein structure for epitope selection and antigenicity.

A novel mechanism of antibody-mediated enhancement of flavivirus infection.

Antibody-dependent enhancement of viral infection is a well-described phenomenon that is based on the cellular uptake of infectious virus-antibody complexes following their interaction with Fcγ receptors expressed on myeloid cells. Here we describe a novel mechanism of antibody-mediated enhancement of infection by a flavivirus (tick-borne encephalitis virus) in transformed and primary human cells, which is independent of the presence of Fcγ receptors. Using chemical cross-linking and immunoassays, we demonstrate that the monoclonal antibody (mab) A5, recognizing an epitope at the interface of the dimeric envelope protein E, causes dimer dissociation and leads to the exposure of the fusion loop (FL). Under normal conditions of infection, this process is triggered only after virus uptake by the acidic pH in endosomes, resulting in the initiation of membrane fusion through the interaction of the FL with the endosomal membrane. Analysis of virus binding and cellular infection, together with inhibition by the FL-specific mab 4G2, indicated that the FL, exposed after mab A5- induced dimer-dissociation, mediated attachment of the virus to the plasma membrane also at neutral pH, thereby increasing viral infectivity. Since antibody-induced enhancement of binding was not only observed with cells but also with liposomes, it is likely that increased infection was due to FL-lipid interactions and not to interactions with cellular plasma membrane proteins. The novel mechanism of antibody-induced infection enhancement adds a new facet to the complexity of antibody interactions with flaviviruses and may have implications for yet unresolved effects of polyclonal antibody responses on biological properties of these viruses.

Protein structure shapes immunodominance in the CD4 T cell response to yellow fever vaccination.

The live attenuated yellow fever (YF) vaccine is a highly effective human vaccine and induces long-term protective neutralizing antibodies directed against the viral envelope protein E. The generation of such antibodies requires the help of CD4 T cells which recognize peptides derived from proteins in virus particles internalized and processed by E-specific B cells. The CD4 T helper cell response is restricted to few immunodominant epitopes, but the mechanisms of their selection are largely unknown. Here, we report that CD4 T cell responses elicited by the YF-17D vaccine are focused to hotspots of two helices of the viral capsid protein and to exposed strands and loops of E. We found that the locations of immunodominant epitopes within three-dimensional protein structures exhibit a high degree of overlap between YF virus and the structurally homologous flavivirus tick-borne encephalitis virus, although amino acid sequence identity of the epitope regions is only 15-45%. The restriction of epitopes to exposed E protein surfaces and their strikingly similar positioning within proteins of distantly related flaviviruses are consistent with a strong influence of protein structure that shapes CD4 T cell responses and provide leads for a rational design of immunogens for vaccination.

Flavivirus structural heterogeneity: implications for cell entry.

The explosive spread of Zika virus is the most recent example of the threat imposed to human health by flaviviruses. High-resolution structures are available for several of these arthropod-borne viruses, revealing alternative icosahedral organizations of immature and mature virions. Incomplete proteolytic maturation, however, results in a cloud of highly heterogeneous mosaic particles. This heterogeneity is further expanded by a dynamic behavior of the viral envelope glycoproteins. The ensemble of heterogeneous and dynamic infectious particles circulating in infected hosts offers a range of alternative possible receptor interaction sites at their surfaces, potentially contributing to the broad flavivirus host-range and variation in tissue tropism. The potential synergy between heterogeneous particles in the circulating cloud thus provides an additional dimension to understand the unanticipated properties of Zika virus in its recent outbreaks.

Structural basis of potent Zika-dengue virus antibody cross-neutralization.

Zika virus is a member of the Flavivirus genus that had not been associated with severe disease in humans until the recent outbreaks, when it was linked to microcephaly in newborns in Brazil and to Guillain-Barré syndrome in adults in French Polynesia. Zika virus is related to dengue virus, and here we report that a subset of antibodies targeting a conformational epitope isolated from patients with dengue virus also potently neutralize Zika virus. The crystal structure of two of these antibodies in complex with the envelope protein of Zika virus reveals the details of a conserved epitope, which is also the site of interaction of the envelope protein dimer with the precursor membrane (prM) protein during virus maturation. Comparison of the Zika and dengue virus immunocomplexes provides a lead for rational, epitope-focused design of a universal vaccine capable of eliciting potent cross-neutralizing antibodies to protect simultaneously against both Zika and dengue virus infections.