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The COVID-19 pandemic highlights an urgent need for vaccines that confer protection from SARS-CoV-2 infection. One approach to an effective COVID-19 vaccine may be through the display of SARS-CoV-2 spikes on the surface of virus-like particles, in a manner structurally mimicking spikes on a native virus. Here we report the development of Newcastle disease virus-like particles (NDVLPs) displaying the prefusion-stabilized SARS-CoV-2 spike ectodomain (S2P). Immunoassays with SARS-CoV-2-neutralizing antibodies revealed the antigenicity of S2P-NDVLP to be generally similar to that of soluble S2P, and negative-stain electron microscopy showed S2P on the NDVLP surface to be displayed with a morphology corresponding to its prefusion conformation. Mice immunized with S2P-NDVLP showed substantial neutralization titers (geometric mean ID50 = 386) two weeks after prime immunization, significantly higher than those elicited by a molar equivalent amount of soluble S2P (geometric mean ID50 = 17). Neutralizing titers at Week 5, two weeks after a boost immunization with S2P-NDVLP doses ranging from 2.0 to 250 μg, extended from 2125 to 4552, and these generally showed a higher ratio of neutralization versus ELISA than observed with soluble S2P. Overall, S2P-NDVLP appears to be a promising COVID-19 vaccine candidate capable of eliciting substantial neutralizing activity.

Respiratory syncytial virus (RSV) causes substantial morbidity and mortality in children and older adults. An effective vaccine must elicit neutralizing antibodies targeting the RSV fusion (F) protein, which exists in two major conformations, pre-fusion (pre-F) and post-fusion (post-F). Although 50% of the surface is shared, pre-F contains highly neutralization-sensitive antigenic sites not present on post-F. Recent advancement of several subunit F-based vaccine trials has spurred interest in quantifying and understanding the protective potential of antibodies directed to individual antigenic sites. Monoclonal antibody competition ELISAs are being used to measure these endpoints, but the impact of F conformation and competition from antibodies binding to adjacent antigenic sites has not been thoroughly investigated. Since this information is critical for interpreting clinical trial outcomes and defining serological correlates of protection, we optimized assays to evaluate D25-competing antibodies (DCA) to antigenic site Ø on pre-F, and compared readouts of palivizumab-competing antibodies (PCA) to site II on both pre-F and post-F. We show that antibodies to adjacent antigenic sites can contribute to DCA and PCA readouts, and that cross-competition from non-targeted sites is especially confounding when PCA is measured using a post-F substrate. While measuring DCA and PCA levels may be useful to delineate the role of antibodies targeting the apex and side of the F protein, respectively, the assay limitations and caveats should be considered when conducting immune monitoring during vaccine trials and defining correlates of protection.

A vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is needed to control the coronavirus disease 2019 (COVID-19) global pandemic. Structural studies have led to the development of mutations that stabilize Betacoronavirus spike proteins in the prefusion state, improving their expression and increasing immunogenicity 1 . This principle has been applied to design mRNA-1273, an mRNA vaccine that encodes a SARS-CoV-2 spike protein that is stabilized in the prefusion conformation. Here we show that mRNA-1273 induces potent neutralizing antibody responses to both wild-type (D614) and D614G mutant 2 SARS-CoV-2 as well as CD8 + T cell responses, and protects against SARS-CoV-2 infection in the lungs and noses of mice without evidence of immunopathology. mRNA-1273 is currently in a phase III trial to evaluate its efficacy. mRNA-1273, an mRNA vaccine that encodes a stabilized prefusion-state severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, elicits robust immune responses and protects mice against replication of SARS-CoV-2 in the upper and lower airways.

Technologies that define the atomic-level structure of neutralization-sensitive epitopes on viral surface proteins are transforming vaccinology and guiding new vaccine development approaches. Previously, iterative rounds of protein engineering were performed to preserve the prefusion conformation of the respiratory syncytial virus (RSV) fusion (F) glycoprotein, resulting in a stabilized subunit vaccine candidate (DS-Cav1), which showed promising results in mice and macaques. Here, phase I human immunogenicity data reveal a more than 10-fold boost in neutralizing activity in serum from antibodies targeting prefusion-specific surfaces of RSV F. These findings represent a clinical proof of concept for structure-based vaccine design, suggest that development of a successful RSV vaccine will be feasible, and portend an era of precision vaccinology.

Antigens displayed on self-assembling nanoparticles can stimulate strong immune responses and have been playing an increasingly prominent role in structure-based vaccines. However, the development of such immunogens is often complicated by inefficiencies in their production. To alleviate this issue, we developed a plug-and-play platform using the spontaneous isopeptide-bond formation of the SpyTag:SpyCatcher system to display trimeric antigens on self-assembling nanoparticles, including the 60-subunit Aquifex aeolicus lumazine synthase (LuS) and the 24-subunit Helicobacter pylori ferritin. LuS and ferritin coupled to SpyTag expressed well in a mammalian expression system when an N- linked glycan was added to the nanoparticle surface. The respiratory syncytial virus fusion (F) glycoprotein trimer—stabilized in the prefusion conformation and fused with SpyCatcher—could be efficiently conjugated to LuS-SpyTag or ferritin-SpyTag, enabling multivalent display of F trimers with prefusion antigenicity. Similarly, F-glycoprotein trimers from human parainfluenza virus-type 3 and spike-glycoprotein trimers from SARS-CoV-2 could be displayed on LuS nanoparticles with decent yield and antigenicity. Notably, murine vaccination with 0.08 µg of SARS-CoV-2 spike-LuS nanoparticle elicited similar neutralizing responses as 2.0 µg of spike, which was ~ 25-fold higher on a weight-per-weight basis. The versatile platform described here thus allows for multivalent plug-and-play presentation on self-assembling nanoparticles of trimeric viral antigens, with SARS-CoV-2 spike-LuS nanoparticles inducing particularly potent neutralizing responses.

Respiratory syncytial virus (RSV) causes substantial morbidity in children and the elderly, with no vaccine available. A vaccine eliciting a substantial and sustainable boost in neutralizing antibodies is likely required to provide protection in at-risk populations. The fusion (F) glycoprotein is a major vaccine target because it is highly conserved between RSV subtypes A and B, it mediates viral entry, and it displays distinct antigenic sites that elicit highly potent neutralizing antibodies. RSV F exists in two major conformational states: the active pre-fusion (pre-F) and the inactive post-fusion (post-F) conformation. Multiple vaccine trials using post-F as a subunit vaccine resulted in a modest boost in neutralization and the induction of non-neutralizing antibodies and conferred little to no protection from disease. In 2013, the stabilization of pre-F conformation through cavity-filling mutations and disulfide bonds (DS-Cav1) revealed surfaces unique to the pre-F conformation that are targets for highly neutralizing antibodies. To compare and contrast the serological and B cell profiles of pre-F versus post-F subunit vaccine-elicited responses, we assessed the profile of binding and neutralizing antibodies before and after vaccination from subjects enrolled in a phase I pre-F subunit vaccine trial (DS-Cav1, VRC 317) or a phase IIb post-F subunit vaccine trial (MEDI7510, MedImmune D4420C00005). Unlike vaccination with MEDI7510, DS-Cav1 vaccination elicited antibodies that bound to sites exclusive to pre-F in addition to sites shared by both immunogens. As a result of the higher potency of antibodies binding these pre-F exclusive sites, DS-Cav1 elicited a more robust increase in neutralizing activity. We confirmed this profile of B cell and antibody specificity by quantifying RSV F-specific B cells in peripheral blood mononuclear cells (PBMC) using fluorescently labeled, tetramerized pre-F and post-F probes and a 17-color flow cytometry panel. Together, our results implicate that the induction of pre-F exclusive antibodies is the primary mechanism for supranormal induction of neutralizing activity. We further investigated the longevity of the response to DS-Cav1 and found that RSV-specific antibody was sustained above the week 0 baseline for up to ten months. Similarly, DS-Cav1 vaccination activated pre-F-specific IgG+ and IgA+ memory B cells, with pre-F probe-binding frequencies elevated above the week 0 baseline ten months post-vaccination. Durability was maintained regardless of dose, number of vaccinations, or adjuvant, indicating that a single dose of DS-Cav1 in RSV-experienced individuals may confer protection that extends throughout an entire RSV season. Our results provide a proof-of-concept for the use of structure-based vaccine design to optimize the induction of potent, durable anti-viral immunity.

An important consequence of infection with a SARS-CoV-2 variant is protective humoral immunity against other variants. However, the basis for such cross-protection at the molecular level is incompletely understood. Here, we characterized the repertoire and epitope specificity of antibodies elicited by infection with the Beta, Gamma and WA1 ancestral variants and assessed their cross-reactivity to these and the more recent Delta and Omicron variants. We developed a method to obtain immunoglobulin sequences with concurrent rapid production and functional assessment of monoclonal antibodies from hundreds of single B cells sorted by flow cytometry. Infection with any variant elicited similar cross-binding antibody responses exhibiting a conserved hierarchy of epitope immunodominance. Furthermore, convergent V gene usage and similar public B cell clones were elicited regardless of infecting variant. These convergent responses despite antigenic variation may account for the continued efficacy of vaccines based on a single ancestral variant. Vaccines against the WA1 SARS-CoV2 strain confer protection against other variants. However, the mechanisms underlying cross-protection are not fully understood. Here, the authors develop a method for rapid analysis of single B cells from patient samples and show that infection with a variant elicits convergent, public B cell responses to other variants.

•RSV F-specific antibodies did not correlate with protection against life-threatening RSV disease in infants.•Pre-existing acute RSV neutralizing antibodies did not correlate with protection against life-threatening RSV disease.•Acute RSV F-specific antibodies negatively correlated with patient age.•Convalescent RSV F-specific antibodies positively correlated with patient age for children over 2 months of age.•Acute RSV antibody titers showed no association with nasal RSV load. Humoral immune response against the pre-fusion (pre-F) conformation of respiratory syncytial virus (RSV) F protein has been proposed to play a protective role against infection. An RSV pre-F maternal vaccine has been recently approved in several countries to protect young infants against RSV. We aimed to assess serum IgG titers against the pre-F and post-F conformations of RSV F protein and their association with life-threatening RSV disease (LTD) in previously healthy infants. A prospective cohort study including hospitalized infants <12 months with a first RSV infection was conducted during 2017–2019. Patients with LTD required intensive care and mechanical respiratory assistance. RSV pre-F exclusive and post-F antibody responses were determined by post-F competition and non-competition immunoassays, respectively, and neutralizing activity was measured by plaque reduction neutralization test. Fifty-eight patients were included; the median age was 3.5 months and 41 % were females. Fifteen patients developed LTD. RSV F-specific antibody titers positively correlated with neutralizing antibody titers in acute and convalescent phases but, importantly, they did not associate with LTD. Acute RSV pre-F exclusive and post-F IgG titers negatively correlated with patient age (P = 0.0007 and P < 0.0001), while a positive correlation was observed between the fold changes in RSV F-specific antibody titers between convalescent and acute phase and patient age (P = 0.0014 and P < 0.0001). Infants ≤2 months exhibited significantly lower fold-changes in RSV F-specific and neutralizing antibody titers between convalescence and acute phase than older infants. Additionally, acute RSV antibody titers showed no correlation with nasal RSV load and, furthermore, nasal viral load was not associated with the development of LTD. This study highlights that protection against life-threatening RSV disease is not necessarily antibody-dependent. Further characterization of the immune response against RSV and its role in protection against severe disease is important for the development of the safest possible preventive strategies.

Older adults face several challenges when transitioning from acute hospitals to community-based care. The PHARMacist Discharge Care (PHARM-DC) intervention is a pharmacist-led Transitions of Care (TOC) program intended to reduce 30-day hospital readmissions and emergency department visits at two large hospitals. This study used the Consolidated Framework for Implementation Research (CFIR) framework to evaluate pharmacist perceptions of the PHARM-DC intervention. In total, ten semi-structured interviews and one focus group were completed across both hospitals. At Site A, six interviews were conducted with intervention pharmacists and pharmacists in administrative roles. Also at Site A, one focus group comprised of five intervention pharmacists was conducted. At Site B, interviews were conducted with four intervention pharmacists and pharmacists in administrative roles. Three overarching themes were identified: PHARM-DC and Institutional Context, Importance of PHARM-DC Adaptability, and Recommendations for PHARM-DC Improvement and Sustainability. Increasing pharmacist support for technical tasks and navigating pharmacist-patient language barriers were important to intervention implementation and delivery. Identifying cost-savings and quantifying outcomes as a result of the intervention were particularly important when considering how to sustain and expand the PHARM-DC intervention. The PHARM-DC intervention can successfully be implemented at two institutions with considerable variations in TOC initiatives, resources, and staffing. Future implementation of PHARM-DC interventions should consider the themes identified, including an examination of institution-specific contextual factors such as the roles that pharmacy technicians may play in TOC interventions, the importance of intervention adaptability to account for patient needs and institutional resources, and pharmacist recommendations for intervention improvement and sustainability.

Introduction Older adults face several challenges when transitioning from acute hospitals to community-based care. The PHARMacist Discharge Care (PHARM-DC) intervention is a pharmacist-led Transitions of Care (TOC) program intended to reduce 30-day hospital readmissions and emergency department visits at two large hospitals. This study used the Consolidated Framework for Implementation Research (CFIR) framework to evaluate pharmacist perceptions of the PHARM-DC intervention. Methods Intervention pharmacists and pharmacy administrators were purposively recruited by study team members located within each participating institution. Study team members located within each institution coordinated with two study authors unaffiliated with the institutions implementing the intervention to conduct interviews and focus groups remotely via telecommunication software. Interviews were recorded and transcribed, with transcriptions imported into NVivo for qualitative analysis. Qualitative analysis was performed using an iterative process to identify “a priori” constructs based on CFIR domains (intervention characteristics, outer setting, inner setting, characteristics of the individuals involved, and the process of implementation) and to create overarching themes as identified during coding. Results In total, ten semi-structured interviews and one focus group were completed across both hospitals. At Site A, six interviews were conducted with intervention pharmacists and pharmacists in administrative roles. Also at Site A, one focus group comprised of five intervention pharmacists was conducted. At Site B, interviews were conducted with four intervention pharmacists and pharmacists in administrative roles. Three overarching themes were identified: PHARM-DC and Institutional Context, Importance of PHARM-DC Adaptability, and Recommendations for PHARM-DC Improvement and Sustainability. Increasing pharmacist support for technical tasks and navigating pharmacist-patient language barriers were important to intervention implementation and delivery. Identifying cost-savings and quantifying outcomes as a result of the intervention were particularly important when considering how to sustain and expand the PHARM-DC intervention. Conclusion The PHARM-DC intervention can successfully be implemented at two institutions with considerable variations in TOC initiatives, resources, and staffing. Future implementation of PHARM-DC interventions should consider the themes identified, including an examination of institution-specific contextual factors such as the roles that pharmacy technicians may play in TOC interventions, the importance of intervention adaptability to account for patient needs and institutional resources, and pharmacist recommendations for intervention improvement and sustainability. Trial registration NCT04071951 .