Biology subjects

Dam, K.-M. A.

Publications and source records attributed to Dam, K.-M. A..

12 recordsLinked to original sources

bioRxiv2025

mRNA delivery of genetically encoded mosaic-8 pan-sarbecovirus RBD vaccines

Effective pan-sarbecovirus vaccines could prevent future zoonotic spillovers of SARS-like betacoronaviruses. We previously developed protein-based mosaic-8 nanoparticles displaying eight diverse sarbecovirus RBDs, either individually (mosaic-8 RBD-NPs) or as two "quartets" of four tandemly-arranged RBDs (dual-quartet RBD-NPs), which elicited broadly cross-reactive antibodies but require multi-component manufacturing. Here, we address scalability challenges by extending the mosaic-8 concept to mRNA by encoding membrane-bound RBD quartets as dual-quartet RBD-mRNA and dual-quartet RBD-EABR-mRNA, the latter leveraging ESCRT- and ALIX-binding region (EABR) technology for immunogen display on cell surfaces and secreted virus-like particles. Compared with protein-based mosaic-8 immunogens, mRNA-encoded mosaic-8 vaccines induced equivalent or enhanced antibody breadth, neutralization potencies, T-cell responses, and targeting of conserved RBD epitopes. In addition, mRNA-encoded mosaic-8 vaccines elicited more balanced IgG subclass profiles and increased Fc{gamma} receptor-binding IgGs, consistent with potentially superior Fc effector functions. These findings demonstrate successful translation of mosaic-8 RBD-NPs into mRNA/EABR-mRNA vaccines, enabling scalable manufacturing and improving protection against future sarbecovirus outbreaks. Finally, our newly developed technique, Systems Serology-Polyclonal Epitope Mapping (SySPEM), revealed distinct IgG-subclass-specific epitope signatures across mRNA, EABR-mRNA, and protein vaccines, demonstrating that the mode of antigen display can shape epitope recognition. SummaryWe translated a pan-sarbecovirus RBD vaccine from protein nanoparticles to scalable mRNA and EABR-mRNA platforms encoding RBD quartets. Compared with protein-based immunogens, mRNA-based vaccines matched or improved antibody breadth, T-cell responses, Fc functionality, and conserved epitope targeting. A newly-developed Systems Serology-Polyclonal Epitope Mapping (SySPEM) technique revealed that antigen presentation modality shapes IgG subclass-specific epitope recognition.

immunology↗

bioRxiv2025

Bivalent mRNA booster encoding virus-like particles elicits potent polyclass RBD antibodies in pre-vaccinated mice

mRNA vaccines emerged as a leading vaccine technology during the COVID-19 pandemic. However, their sustained protective efficacies were limited by relatively short-lived antibody responses and the emergence of SARS-CoV-2 variants, necessitating frequent and variant-updated boosters. We recently developed the ESCRT- and ALIX-binding region (EABR) mRNA vaccine platform, which encodes engineered immunogens that induce budding of enveloped virus-like particles (eVLPs) from the plasma membrane, thereby resulting in presentation of immunogens on cell surfaces and eVLPs. Prior studies showed that spike (S)-EABR mRNA-LNP immunizations elicited enhanced neutralizing antibody responses against ancestral and variant SARS-CoV-2 compared with conventional S mRNA-LNP in naive mice, but the effectiveness of S-EABR mRNA-LNP boosters in the context of pre-existing immunity has not been investigated. Here, we evaluated monovalent Wuhan-Hu-1 (Wu1) and bivalent (Wu1/BA.5) S-EABR mRNA-LNP boosters in mice pre-vaccinated with conventional Wu1 S mRNA-LNP. Compared to conventional S mRNA-LNP boosters, the EABR approach enhanced monovalent and bivalent mRNA-LNP booster-induced neutralizing responses against Omicron subvariants BA.1, BA.5, BQ.1.1, and XBB.1, with bivalent S-EABR mRNA-LNP consistently eliciting the highest titers. Epitope mapping of polyclonal antisera by deep mutational scanning revealed that bivalent S-EABR mRNA-LNP boosted diverse "polyclass" anti-receptor-binding domain (RBD) responses, suggesting balanced targeting of multiple RBD epitope classes. In contrast, monovalent S, bivalent S, and monovalent S-EABR mRNA-LNP boosters elicited less diverse polyclonal serum responses primarily targeting immunodominant RBD epitopes. Cryo-EM structures demonstrated that bivalent mRNA immunizations promote S heterotrimer formation, potentially enhancing bivalent S-EABR mRNA-LNP booster-induced antibody breadth and polyclass epitope targeting by activating cross-reactive B cells through intra-S crosslinking. These findings support the future design of bivalent or multivalent S-EABR mRNA-LNP boosters as a promising strategy to confer broader, and therefore potentially more durable, protection against emerging SARS-CoV-2 variants and other rapidly evolving viruses.

immunology↗

bioRxiv2025

The Interplay of Furin Cleavage and D614G in Modulating SARS-CoV-2 Spike Protein Dynamics

We report a detailed analysis of the full-length SARS-CoV-2 spike dynamics within a native-like membrane environment and variants inaccessible to studies on soluble constructs by conducting hydrogen-deuterium exchange mass spectrometry (HDX-MS) on enveloped virus-like particles (eVLPs) displaying various spike constructs. We find that the previously identified open-interface trimer conformation is sampled in all eVLP-displayed spike variants studied including sequences from engineered vaccine constructs and native viral sequences. The D614G mutation, which arose early in the pandemic, favors the canonical closed-interface prefusion conformation, potentially mitigating premature S1 shedding in the presence of a cleaved furin site and providing an evolutionary advantage to the virus. Remarkably, furin cleavage at the S1/S2 boundary allosterically increases the flexibility of the S2 site, which may facilitate increased TMPRSS2 processing, enhancing viral infectivity. The use of eVLPs in HDX-MS studies provides a powerful platform for studying viral and membrane proteins in near-native environments.

biophysics↗

bioRxiv2024

Mapping essential somatic hypermutations in a CD4-binding site bNAb informs HIV-1 vaccine design

HIV-1 broadly-neutralizing antibodies (bNAbs) targeting the CD4-binding site (CD4bs) contain rare features that pose challenges to elicit these bNAbs through vaccination. The IOMA-class of CD4bs bNAbs includes fewer rare features and somatic hypermutations (SHMs) to achieve broad neutralization, thus presenting a potentially accessible pathway for vaccine-induced bNAb development. Here, we created a library of IOMA variants in which each SHM was individually reverted to the inferred germline (iGL) counterpart to investigate the roles of SHMs in conferring IOMAs neutralization potency and breadth. Impacts on neutralization for each variant were evaluated, and this information was used to design minimally-mutated IOMA-class variants (IOMAmin) that incorporated the fewest SHMs required for achieving IOMAs neutralization breadth. A cryo-EM structure of an IOMAmin variant bound to Env was used to further interpret characteristics of IOMA variants to elucidate how IOMAs structural features correlate with its neutralization mechanism, informing the design of IOMA-targeting immunogens. HighlightsIOMAmin variants with reduced SHMs retain neutralization potency and breadth. Cryo-EM structure reveals IOMAmin preserves key Env interactions despite fewer SHMs. IOMA variants with mutations in CDRH3 and CDRL1 fail to improve neutralization.

immunology↗

bioRxiv2023

Conformational flexibility of HIV-1 envelope glycoproteins modulates transmitted / founder sensitivity to broadly neutralizing antibodies

HIV-1 envelope glycoproteins (Envs) mediate viral entry and are the sole target of neutralizing antibodies. Envs of most primary HIV-1 strains exist in a closed conformation and occasionally sample more open states. Thus, current knowledge guides immunogen design to mimic the closed Env conformation as the preferred target for eliciting broadly neutralizing antibodies (bnAbs) to block HIV-1 entry. Here we show that Env-preferred conformations of 6 out of 13 (46%) transmitted/founder (T/F) strains tested are incompletely closed. As a result, entry of these T/Fs into target cells is sensitive to antibodies that recognize internal epitopes exposed on open Env conformations. A cryo-electron microscopy structure of unliganded, incompletely closed T/F Envs (1059-SOSIP) at 3.6 [A] resolution exhibits an asymmetric configuration of Env protomers with increased sampling of states with incompletely closed trimer apex. Double electron-electron resonance spectroscopy provided further evidence for enriched occupancy of more open Env conformations. Consistent with conformational flexibility, 1059 Envs were associated with resistance to most bnAbs that exhibit reduced potency against functional Env intermediates. To follow the fate of incompletely closed Env in patients, we reconstructed de novo the post-transmission evolutionary pathway of a second T/F Env (CH040), which is sensitive to the V3-targeting antibody 19b and highly resistant to most bnAbs. Evolved viruses exhibited increased resistance to cold, soluble CD4 and 19b, all of which correlate with closing of the adapted Env trimer. Lastly, we show a correlation between efficient neutralization of multiple Env conformations and increased antiviral breadth of CD4-binding site (CD4bs) bnAbs. In particular, N6 bnAb, which uniquely recognizes different Env conformations, efficiently neutralizes 50% of the HIV-1 strains that were resistant to VRC01 and transmitted during the first-in-humans antibody-mediated prevention trial (HVTN 704). VRC01-resistant Envs are incompletely closed based on their sensitivity to cold and on partial sensitivity to antibodies targeting internal, typically occluded, epitopes. Most VRC01-resistant Envs retain the VRC01 epitope according to VRC01 binding to their gp120 subunit at concentrations that have no significant effect on virus entry, and they exhibit cross resistance to other CD4bs bnAbs that poorly recognize functional Env intermediates. Our findings refine current knowledge of Env conformational states and provide guidance for developing new strategies for bnAb immunotherapy and Env-based immunogen design.

microbiology↗

bioRxiv2023

Structural characterization of HIV-1 Env heterotrimers bound to one or two CD4 receptors reveals intermediate Env conformations

HIV-1 envelope (Env) exhibits distinct conformational changes in response to host receptor (CD4) engagement. Env, a trimer of gp120/gp41 heterodimers, has been structurally characterized in a closed, prefusion conformation with closely associated gp120s and coreceptor binding sites on gp120 V3 hidden by V1V2 loops, and in fully-saturated CD4-bound open Env conformations with changes including outwardly rotated gp120s and displaced V1V2 loops. To investigate changes resulting from sub-stoichiometric CD4 binding, we solved 3.4[A] and 3.9[A] single-particle cryo-EM structures of soluble, native-like Envs bound to one or two CD4 molecules. Env trimer bound to one CD4 adopted the closed, prefusion Env state. When bound to two CD4s, the CD4-bound gp120s exhibited an open Env conformation including a four-stranded gp120 bridging sheet and displaced gp120 V1V2 loops that expose the coreceptor sites on V3. The third gp120 adopted an intermediate, occluded-open state that included gp120 outward rotation but maintained the prefusion, three-stranded gp120 bridging sheet and showed only partial V1V2 displacement and V3 exposure. We conclude that engagement of one CD4 molecule was insufficient to stimulate CD4-induced conformational changes, while binding two CD4 molecules led to Env opening in CD4-bound protomers only. Together, these results illuminate HIV-1 Env intermediate conformations and illustrate the structural plasticity of HIV-1 Env.

molecular biology↗

bioRxiv2022

CD4-binding site immunogens elicit heterologous anti-HIV-1 neutralizing antibodies in transgenic and wildtype animals

Passive transfer of broadly neutralizing anti-HIV-1 antibodies (bNAbs) protects against infection, and therefore eliciting bNAbs by vaccination is a major goal of HIV-1 vaccine efforts. bNAbs that target the CD4-binding site (CD4bs) on HIV-1 Env are among the most broadly active, but to date, responses elicited against this epitope in vaccinated animals have lacked potency and breadth. We hypothesized that CD4bs bNAbs resembling the antibody IOMA might be easier to elicit than other CD4bs antibodies that exhibit higher somatic mutation rates, a difficult-to-achieve mechanism to accommodate Envs N276gp120 N-glycan, and rare 5-residue light chain complementarity determining region 3s (CDRL3s). As an initial test of this idea, we developed IOMA germline-targeting Env immunogens and evaluated a sequential immunization regimen in transgenic mice expressing germline-reverted IOMA. These mice developed CD4bs epitope-specific responses with heterologous neutralization, and cloned antibodies overcame neutralization roadblocks including accommodating the N276gp120 glycan, with some neutralizing selected HIV-1 strains more potently than IOMA. The immunization regimen also elicited CD4bs-specific responses in animals containing polyclonal antibody repertoires. Thus, germline-targeting of IOMA-class antibody precursors represents a potential vaccine strategy to induce CD4bs bNAbs.

immunology↗

bioRxiv2022

Timeline of changes in spike conformational dynamics in emergent SARS-CoV-2 variants reveal progressive stabilization of trimer stalk and enhanced NTD dynamics

SARS-CoV-2 emergent variants are characterized by increased transmissibility and each show multiple mutations predominantly localized to the spike (S) protein. Here, amide hydrogen/deuterium exchange mass spectrometry has been applied to track correlative changes in S dynamics from multiple SARS-CoV-2 variants. Our results highlight large differences across variants at two loci with impacts on S dynamics and stability. A significant enhancement in stabilization first occurred with the emergence of D614G S followed by smaller, progressive stabilization in Omicron BA.1 S traced through Alpha S and Delta S variants. Stabilization preceded progressive enhancement in dynamics in the N-terminal domain, wherein Omicron BA.1 S showed the largest magnitude increases relative to other preceding variants. Changes in stabilization and dynamics resulting from specific S mutations detail the evolutionary trajectory of S protein in emerging variants. These carry major implications for SARS-CoV-2 viral fitness and offer new insights into variant-specific therapeutic development.

biophysics↗

bioRxiv2022

Antibody recognition of CD4-induced open HIV-1 Env trimers

HIV-1 envelope (Env), a heterotrimer of gp120-gp41 subunits, mediates fusion of the viral and host cell membranes after interactions with the host receptor CD4 and a coreceptor. CD4 binding induces rearrangements in Env trimer, resulting in a CD4-induced (CD4i) open Env conformation. Structural studies of antibodies isolated from infected donors have defined antibody-Env interactions, with one class of antibodies specifically recognizing the CD4i open Env conformation. Here, we characterize a group of monoclonal antibodies isolated from HIV-1 infected donors (V2i mAbs) that display characteristics of CD4i antibodies. Binding experiments demonstrate that the V2i mAbs preferentially recognize CD4-bound open Env trimers. Structural characterizations of V2i mAb-Env-CD4 trimer complexes using single-particle cryo-electron microscopy show recognition by V2i mAbs using different angles of approach to the gp120 V1V2 domain and the {beta}2/{beta}3 strands on a CD4i open conformation Env with no direct interactions of the mAbs with CD4. We also characterize CG10, a CD4i antibody that was raised in mice immunized with a gp120-CD4 complex, complexed with Env trimer and CD4. CG10 exhibits similar characteristics to the V2i antibodies: i.e., recognition of the open Env conformation, but shows direct contacts to both CD4 and gp120. Structural comparisons of these and previously characterized CD4i antibody interactions with Env provide a suggested mechanism for how these antibodies are elicited during HIV-1 infection. ImportanceThe RV144 HIV-1 clinical vaccination trial showed mild protection against viral infection. Antibody responses to the V1V2 region of HIV-1 Env gp120 were correlated inversely with the risk of infection. In addition, antibodies targeting V1V2 have been correlated with protections from SIV and SHIV infections in non-human primates. We structurally characterized V2i antibodies directed against V1V2 isolated from HIV-1 infected humans in complex with open Env trimers bound to the host receptor CD4. We also characterized a CD4i antibody that interacts with CD4 as well as the gp120 subunit of an open Env trimer. Our study suggests how V2i and CD4i antibodies were elicited during HIV-1 infection.

immunology↗

bioRxiv2022

HIV-1 CD4-binding site germline antibody-Env structures inform vaccine design

BG24, a VRC01-class broadly neutralizing antibody (bNAb) against HIV-1 Env with relatively few somatic hypermutations (SHMs), represents a promising target for vaccine strategies to elicit CD4-binding site (CD4bs) bNAbs. To understand how SHMs correlate with BG24 neutralization of HIV-1, we solved 4.1 [A] and 3.4 [A] single-particle cryo-EM structures of two inferred germline (iGL) BG24 precursors complexed with engineered Env-based immunogens lacking CD4bs N-glycans. Structures revealed critical Env contacts by BG24iGL and identified antibody light chain structural features that impede Env recognition. In addition, biochemical data and cryo-EM structures of BG24iGL variants bound to Envs with CD4bs glycans present provided insights into N-glycan accommodation, including structural modes of light chain adaptations in the presence of the N276gp120 glycan. Together, these findings revealed Env regions critical for germline antibody recognition and potential sites to alter in immunogen design.

biochemistry↗

bioRxiv2021

Neutralizing antibodies induced in immunized macaques recognize the CD4-binding site on an occluded-open HIV-1 envelope trimer

Broadly-neutralizing antibodies (bNAbs) against HIV-1 Env can protect from infection. We characterize Ab1303 and Ab1573, heterologously-neutralizing CD4-binding site (CD4bs) antibodies, isolated from sequentially-immunized macaques. Ab1303/Ab1573 binding is observed only when Env trimers are not constrained in the closed, prefusion conformation. Fab-Env cryo-EM structures show that both antibodies recognize the CD4bs on Env trimer with an occluded-open conformation between closed, as targeted by bNAbs, and fully-open, as recognized by CD4. The occluded-open Env trimer conformation includes outwardly-rotated gp120 subunits, but unlike CD4-bound Envs, does not exhibit V1V2 displacement, 4-stranded gp120 bridging sheet, or co-receptor binding site exposure. Inter-protomer distances within trimers measured by double electron-electron resonance spectroscopy suggest an equilibrium between occluded-open and closed Env conformations, consistent with Ab1303/Ab1573 binding stabilizing an existing conformation. Studies of Ab1303/Ab1573 demonstrate that CD4bs neutralizing antibodies that bind open Env trimers can be raised by immunization, thereby informing immunogen design and antibody therapeutic efforts.

immunology↗

bioRxiv2020

Structural classification of neutralizing antibodies against the SARS-CoV-2 spike receptor-binding domain suggests vaccine and therapeutic strategies

The COVID-19 pandemic presents an urgent health crisis. Human neutralizing antibodies (hNAbs) that target the host ACE2 receptor-binding domain (RBD) of the SARS-CoV-2 spike1-5 show therapeutic promise and are being evaluated clincally6-8. To determine structural correlates of SARS-CoV-2 neutralization, we solved 8 new structures of distinct COVID-19 hNAbs5 in complex with SARS-CoV-2 spike trimer or RBD. Structural comparisons allowed classification into categories: (1) VH3-53 hNAbs with short CDRH3s that block ACE2 and bind only to "up" RBDs, (2) ACE2-blocking hNAbs that bind both "up" and "down" RBDs and can contact adjacent RBDs, (3) hNAbs that bind outside the ACE2 site and recognize "up" and "down" RBDs, and (4) Previously-described antibodies that do not block ACE2 and bind only "up" RBDs9. Class 2 comprised four hNAbs whose epitopes bridged RBDs, including a VH3-53 hNAb that used a long CDRH3 with a hydrophobic tip to bridge between adjacent "down" RBDs, thereby locking spike into a closed conformation. Epitope/paratope mapping revealed few interactions with host-derived N-glycans and minor contributions of antibody somatic hypermutations to epitope contacts. Affinity measurements and mapping of naturally-occurring and in vitro-selected spike mutants in 3D provided insight into the potential for SARS-CoV-2 escape from antibodies elicited during infection or delivered therapeutically. These classifications and structural analyses provide rules for assigning current and future human RBD-targeting antibodies into classes, evaluating avidity effects, suggesting combinations for clinical use, and providing insight into immune responses against SARS-CoV-2.

molecular biology↗