Extensive flavivirus E trimer breathing accompanies stem zippering of the post-fusion hairpin.

Flaviviruses enter cells by fusion with endosomal membranes through a rearrangement of the envelope protein E, a class II membrane fusion protein, into fusogenic trimers. The rod-like E subunits bend into "hairpins" to bring the fusion loops next to the C-terminal transmembrane (TM) anchors, with the TM-proximal "stem" element zippering the E trimer to force apposition of the membranes. The structure of the complete class II trimeric hairpin is known for phleboviruses but not for flaviviruses, for which the stem is only partially resolved. Here, we performed comparative analyses of E-protein trimers from the tick-borne encephalitis flavivirus with sequential stem truncations. Our thermostability and antibody-binding data suggest that the stem "zipper" ends at a characteristic flavivirus conserved sequence (CS) that cloaks the fusion loops, with the downstream segment not contributing to trimer stability. We further identified a highly dynamic behavior of E trimers C-terminally truncated upstream the CS, which, unlike fully stem-zippered trimers, undergo rapid deuterium exchange at the trimer interface. These results thus identify important "breathing" intermediates in the E-protein-driven membrane fusion process.

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.

Impact of structural dynamics on biological functions of flaviviruses.

Flaviviruses comprise a number of mosquito- or tick-transmitted human pathogens of global public health importance. Advances in structural biology techniques have contributed substantially to our current understanding of the life cycle of these small enveloped RNA viruses and led to deep insights into details of virus assembly, maturation and cell entry. In addition to large-scale conformational changes and oligomeric rearrangements of envelope proteins during these processes, there is increasing evidence that smaller-scale protein dynamics (referred to as virus "breathing") can confer extra flexibility to these viruses for the fine-tuning of their interactions with the immune system and possibly with cellular factors they encounter in their complex ecological cycles in arthropod and vertebrate hosts. In this review, we discuss how work with tick-borne encephalitis virus has extended our view on flavivirus breathing, leading to the identification of a novel mechanism of antibody-mediated infection enhancement and demonstrating breathing intermediates of the envelope protein in the process of membrane fusion. These data are discussed in the context of other flaviviruses and the perspective of a potential role of virus breathing to cope with the requirements of adaptation and replication in evolutionarily very different hosts.

Bivalent COVID-19 mRNA booster vaccination (BA.1 or BA.4/BA.5) increases neutralization of matched Omicron variants.

We report SARS-CoV-2 neutralizing antibody titers in sera of triple-vaccinated individuals who received a booster dose of an original monovalent or a bivalent BA.1- or BA.4/BA.5-adapted vaccine or had a breakthrough infection with Omicron variants BA.1, BA.2 or BA.4/BA.5. A bivalent BA.4/BA.5 booster or Omicron-breakthrough infection induced increased Omicron-neutralization titers compared with the monovalent booster. The XBB.1.5 variant effectively evaded neutralizing-antibody responses elicited by current vaccines and/or infection with previous variants.

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.

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.

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.

Different Cross-Reactivities of IgM Responses in Dengue, Zika and Tick-Borne Encephalitis Virus Infections.

Flaviviruses circulate worldwide and cause a number of medically relevant human diseases, such as dengue, Zika, yellow fever, and tick-borne encephalitis (TBE). Serology plays an important role in the diagnosis of flavivirus infections, but can be impeded by antigenic cross-reactivities among flaviviruses. Therefore, serological diagnosis of a recent infection can be insufficiently specific, especially in areas where flaviviruses co-circulate and/or vaccination coverage against certain flaviviruses is high. In this study, we developed a new IgM assay format, which is well suited for the specific diagnosis of TBE, Zika and dengue virus infections. In the case of TBE and Zika, the IgM response proved to be highly specific for the infecting virus. In contrast, primary dengue virus infections induced substantial amounts of cross-reactive IgM antibodies, which is most likely explained by structural peculiarities of dengue virus particles. Despite the presence of cross-reactive IgM, the standardized nature and the quantitative read-out of the assay even allowed the serotype-specific diagnosis of recent dengue virus infections in most instances.

Dynamics of CD4 T Cell and Antibody Responses in COVID-19 Patients With Different Disease Severity.

Disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ranges from mild illness to severe respiratory disease and death. In this study, we determined the kinetics of viral loads, antibody responses (IgM, IgG, neutralization) and SARS-CoV-2-specific CD4 T cells by quantifying these parameters in 435 serial respiratory and blood samples collected from a cohort of 29 COVID-19 patients with either moderate or severe disease during the whole period of hospitalization or until death. Remarkably, there was no significant difference in the kinetics and plateau levels of neutralizing antibodies among the groups with different disease severity. In contrast, the dynamics of specific CD4 T cell responses differed considerably, but all patients with moderate or severe disease developed robust SARS-CoV-2-specific responses. Of note, none of the patients had detectable cross-reactive CD4 T cells in the first week after symptom onset, which have been described in 20-50% of unexposed individuals. Our data thus provide novel insights into the kinetics of antibody and CD4 T cell responses as well as viral loads that are key to understanding the role of adaptive immunity in combating the virus during acute infection and provide leads for the timing of immune therapies for COVID-19.

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.

Pre-existing yellow fever immunity impairs and modulates the antibody response to tick-borne encephalitis vaccination.

Flaviviruses have an increasing global impact as arthropod-transmitted human pathogens, exemplified by Zika, dengue, yellow fever (YF), West Nile, Japanese encephalitis, and tick-borne encephalitis (TBE) viruses. Since all flaviviruses are antigenically related, they are prone to phenomena of immunological memory ('original antigenic sin'), which can modulate immune responses in the course of sequential infections and/or vaccinations. In our study, we analyzed the influence of pre-existing YF vaccine-derived immunity on the antibody response to TBE vaccination. By comparing samples from YF pre-vaccinated and flavivirus-naive individuals, we show that YF immunity not only caused a significant impairment of the neutralizing antibody response to TBE vaccination but also a reduction of the specific TBE virus neutralizing activities (NT/ELISA-titer ratios). Our results point to a possible negative effect of pre-existing cross-reactive immunity on the outcome of flavivirus vaccination that may also pertain to other combinations of sequential flavivirus infections and/or vaccinations.