Antibodies targeting a quaternary site on SARS-CoV-2 spike glycoprotein prevent viral receptor engagement by conformational locking.

SARS-CoV-2 continues to evolve, with many variants evading clinically authorized antibodies. To isolate monoclonal antibodies (mAbs) with broadly neutralizing capacities against the virus, we screened serum samples from convalescing COVID-19 patients. We isolated two mAbs, 12-16 and 12-19, which neutralized all SARS-CoV-2 variants tested, including the XBB subvariants, and prevented infection in hamsters challenged with Omicron BA.1 intranasally. Structurally, both antibodies targeted a conserved quaternary epitope located at the interface between the N-terminal domain and subdomain 1, uncovering a site of vulnerability on SARS-CoV-2 spike. These antibodies prevented viral receptor engagement by locking the receptor-binding domain (RBD) of spike in the down conformation, revealing a mechanism of virus neutralization for non-RBD antibodies. Deep mutational scanning showed that SARS-CoV-2 could mutate to escape 12-19, but such mutations are rarely found in circulating viruses. Antibodies 12-16 and 12-19 hold promise as prophylactic agents for immunocompromised persons who do not respond robustly to COVID-19 vaccines.

Characterization of two engineered dimeric Zika virus envelope proteins as immunogens for neutralizing antibody selection and vaccine design.

The envelope protein of Zika virus (ZIKV) exists as a dimer on the mature viral surface and is an attractive antiviral target because it mediates viral entry. However, recombinant soluble wild-type ZIKV envelope (wtZE) might preferentially exist as monomer (monZE). Recently, it has been shown that the A264C substitution could promote formation of dimeric ZIKV envelope protein (ZEA264C), requiring further characterization of purified ZEA264C for its potential applications in vaccine development. We also noted that ZEA264C, connected by disulfide bond, might be different from the noncovalent native envelope dimer on the virion surface. Because the antibody Fc fragment exists as dimer and is widely used for fusion protein construction, here we fused wtZE to human immunoglobulin G1 (IgG1) Fc fragment (ZE-Fc) for noncovalent wtZE dimerization. Using a multistep purification procedure, we separated dimeric ZEA264C and ZE-Fc, revealing that they both exhibit typical ß-sheet-rich secondary structures and stabilities similar to those of monZE. The binding activities of monZE, ZEA264C, and ZE-Fc to neutralizing antibodies targeting different epitopes indicated that ZEA264C and ZE-Fc could better mimic the native dimeric status, especially in terms of the formation of tertiary and quaternary epitopes. Both ZEA264C and ZE-Fc recognize a ZIKV-sensitive cell line as does monZE, indicating that the two constructs are still functional. Furthermore, a murine immunization assay disclose that ZEA264C and ZE-Fc elicit more neutralizing antibody responses than monZE does. These results suggest that the two immunogen candidates ZEA264C and ZE-Fc have potential utility for neutralizing antibody selection and vaccine design against ZIKV.

Conformational Engineering of HIV-1 Env Based on Mutational Tolerance in the CD4 and PG16 Bound States.

HIV-1 infection is initiated by viral Env engaging the host receptor CD4, triggering Env to transition from a "closed" to "open" conformation during the early events of virus-cell membrane fusion. To understand how Env sequence accommodates this conformational change, mutational landscapes decoupled from virus replication were determined for Env from BaL (clade B) and DU422 (clade C) isolates interacting with CD4 or antibody PG16 that preferentially recognizes closed trimers. Sequence features uniquely important to each bound state were identified, including glycosylation and binding sites. Notably, the Env apical domain and trimerization interface are under selective pressure for PG16 binding. Based on this key observation, mutations were found that increase presentation of quaternary epitopes associated with properly conformed trimers when Env is expressed at the plasma membrane. Many mutations reduce electrostatic repulsion at the Env apex and increase PG16 recognition of Env sequences from clades A and B. Other mutations increase hydrophobic packing at the gp120 inner-outer domain interface and were broadly applicable for engineering Env from diverse strains spanning tiers 1, 2, and 3 across clades A, B, C, and BC recombinants. Core mutations predicted to introduce steric strain in the open state show markedly reduced CD4 interactions. Finally, we demonstrate how our methodology can be adapted to interrogate interactions between membrane-associated Env and the matrix domain of Gag. These findings and methods may assist vaccine design.IMPORTANCE HIV-1 Env is dynamic and undergoes large conformational changes that drive fusion of virus and host cell membranes. Three Env proteins in a trimer contact each other at their apical tips to form a closed conformation that presents epitopes recognized by broadly neutralizing antibodies. The apical tips separate, among other changes, to form an open conformation that binds tightly to host receptors. Understanding how Env sequence facilitates these structural changes can inform the biophysical mechanism and aid immunogen design. Using deep mutational scans decoupled from virus replication, we report mutational landscapes for Env from two strains interacting with conformation-dependent binding proteins. Residues in the Env trimer interface and apical domains are preferentially conserved in the closed conformation, and conformational diversity is facilitated by electrostatic repulsion and an underpacked core between domains. Specific mutations are described that enhance presentation of the trimeric closed conformation across diverse HIV-1 strains.

Physiological temperatures reduce dimerization of dengue and Zika virus recombinant envelope proteins.

The spread of dengue (DENV) and Zika virus (ZIKV) is a major public health concern. The primary target of antibodies that neutralize DENV and ZIKV is the envelope (E) glycoprotein, and there is interest in using soluble recombinant E (sRecE) proteins as subunit vaccines. However, the most potent neutralizing antibodies against DENV and ZIKV recognize epitopes on the virion surface that span two or more E proteins. Therefore, to create effective DENV and ZIKV vaccines, presentation of these quaternary epitopes may be necessary. The sRecE proteins from DENV and ZIKV crystallize as native-like dimers, but studies in solution suggest that these dimers are marginally stable. To better understand the challenges associated with creating stable sRecE dimers, we characterized the thermostability of sRecE proteins from ZIKV and three DENV serotypes, DENV2-4. All four proteins irreversibly unfolded at moderate temperatures (46-53 °C). At 23 °C and low micromolar concentrations, DENV2 and ZIKV were primarily dimeric, and DENV3-4 were primarily monomeric, whereas at 37 °C, all four proteins were predominantly monomeric. We further show that the dissociation constant for DENV2 dimerization is very temperature-sensitive, ranging from <1 µm at 25 °C to 50 µm at 41 °C, due to a large exothermic enthalpy of binding of -79 kcal/mol. We also found that quaternary epitope antibody binding to DENV2-4 and ZIKV sRecE is reduced at 37 °C. Our observation of reduced sRecE dimerization at physiological temperature highlights the need for stabilizing the dimer as part of its development as a subunit vaccine.

Rational Engineering and Characterization of an mAb that Neutralizes Zika Virus by Targeting a Mutationally Constrained Quaternary Epitope.

Following the recent emergence of Zika virus (ZIKV), many murine and human neutralizing anti-ZIKV antibodies have been reported. Given the risk of virus escape mutants, engineering antibodies that target mutationally constrained epitopes with therapeutically relevant potencies can be valuable for combating future outbreaks. Here, we applied computational methods to engineer an antibody, ZAb_FLEP, that targets a highly networked and therefore mutationally constrained surface formed by the envelope protein dimer. ZAb_FLEP neutralized a breadth of ZIKV strains and protected mice in distinct in vivo models, including resolving vertical transmission and fetal mortality in infected pregnant mice. Serial passaging of ZIKV in the presence of ZAb_FLEP failed to generate viral escape mutants, suggesting that its epitope is indeed mutationally constrained. A single-particle cryo-EM reconstruction of the Fab-ZIKV complex validated the structural model and revealed insights into ZAb_FLEP's neutralization mechanism. ZAb_FLEP has potential as a therapeutic in future outbreaks.

Analysis of Individuals from a Dengue-Endemic Region Helps Define the Footprint and Repertoire of Antibodies Targeting Dengue Virus 3 Type-Specific Epitopes.

The four dengue virus serotypes (DENV1 to 4) cause dengue, a major public health problem worldwide. Individuals exposed to primary DENV infections develop serotype-specific neutralizing antibodies, including strongly neutralizing antibodies targeting quaternary epitopes. To date, no studies have measured the levels and kinetics of serum antibodies directed to such epitopes among populations in regions where dengue is endemic. Here, we use a recombinant DENV4 (rDENV4/3-M14) displaying a major DENV3 type-specific quaternary epitope recognized by human monoclonal antibody 5J7 to measure the proportion, magnitude, and kinetics of DENV3 type-specific neutralizing antibody responses targeting this epitope. Primary DENV3 sera from 30 individuals in a dengue hospital-based study in Nicaragua were studied 3, 6, 12, and 18 months post-infection, alongside samples collected annually 1 to 4 years post-primary DENV3 infection from 10 individuals in a cohort study in Nicaragua. We found substantial individual variation in the proportion of DENV3 type-specific neutralizing antibody titers attributed to the 5J7 epitope (range, 0 to 100%), with the mean significantly increasing from 22.6% to 41.4% from 3 to 18 months. We extended the transplanted DENV3 5J7 epitope on the virion (rDENV4/3-M16), resulting in increased recognition in several individuals, helping define the footprint of the epitope. However, 37% and 13% of the subjects still showed little to no recognition of the 5J7 epitope at 3 and 18 months, respectively, indicating that one or more additional DENV3 type-specific epitopes exist. Overall, this study demonstrates how DENV-immune plasma from populations from areas of endemicity, when coupled with structurally guided recombinant viruses, can help characterize the epitope-specific neutralizing antibody response in natural DENV infections, with direct implications for design and evaluation of dengue vaccines.IMPORTANCE The four serotypes of dengue virus cause dengue, a major public health burden worldwide, yet it has been challenging to develop a vaccine that is safe and equally effective against all four serotypes. More in-depth characterization of natural human neutralizing antibody responses is needed to identify determinants of protective antibody responses to all DENV serotypes. Here, we use hospital and cohort studies in a region where dengue is endemic to assess the proportion and kinetics of the DENV3 neutralizing antibody response directed to a quaternary epitope on DENV3 recognized by strongly neutralizing human monoclonal antibody 5J7, which was transplanted into a DENV4 backbone. We show that many individuals recognized the 5J7 epitope, but to various degrees over time, suggesting that additional DENV3-specific epitopes likely exist. Thus, characterization of epitope-specific neutralizing antibody responses in natural DENV infections can help define the footprint and repertoire of antibodies directed to DENV3 type-specific epitopes, with implications for dengue vaccine development.