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 ID
= 386) two weeks after prime immunization, significantly higher than those elicited by a molar equivalent amount of soluble S2P (geometric mean ID
= 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
. 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
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.
The emergence of highly transmissible SARS-CoV-2 variants of concern (VOCs) that are resistant to therapeutic antibodies highlights the need for continuing discovery of broadly reactive antibodies. ...We identified four receptor binding domain-targeting antibodies from three early-outbreak convalescent donors with potent neutralizing activity against 23 variants, including the B.1.1.7, B.1.351, P.1, B.1.429, B.1.526, and B.1.617 VOCs. Two antibodies are ultrapotent, with subnanomolar neutralization titers half-maximal inhibitory concentration (IC
) 0.3 to 11.1 nanograms per milliliter; IC
1.5 to 34.5 nanograms per milliliter). We define the structural and functional determinants of binding for all four VOC-targeting antibodies and show that combinations of two antibodies decrease the in vitro generation of escape mutants, suggesting their potential in mitigating resistance development.
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.
Vaccine-associated enhanced respiratory disease (VAERD) was previously observed in some preclinical models of severe acute respiratory syndrome (SARS) and MERS coronavirus vaccines. We used the SARS ...coronavirus 2 (SARS-CoV-2) mouse-adapted, passage 10, lethal challenge virus (MA10) mouse model of acute lung injury to evaluate the immune response and potential for immunopathology in animals vaccinated with research-grade mRNA-1273. Whole-inactivated virus or heat-denatured spike protein subunit vaccines with alum designed to elicit low-potency antibodies and Th2-skewed CD4+ T cells resulted in reduced viral titers and weight loss post challenge but more severe pathological changes in the lung compared to saline-immunized animals. In contrast, a protective dose of mRNA-1273 induced favorable humoral and cellular immune responses that protected from viral replication in the upper and lower respiratory tract upon challenge. A subprotective dose of mRNA-1273 reduced viral replication and limited histopathological manifestations compared to animals given saline. Overall, our findings demonstrate an immunological signature associated with antiviral protection without disease enhancement following vaccination with mRNA-1273.
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•mRNA-1273 elicits neutralizing antibodies and spike-binding germinal center B cells•Humoral immunity is accompanied by spike-specific CD4+ Th1, Tfh, and CD8+ T cells•mRNA-1273 demonstrates protection in the mouse MA10 acute lung injury model•mRNA-1273 vaccination does not result in enhanced disease following infection
As vaccine-enhanced disease to respiratory viruses has been previously observed, a thorough safety evaluation of COVID-19 vaccines in preclinical animal models is essential. Here, DiPiazza and Leist et al. provide evidence for antiviral protection in the absence of lung disease following SARS-CoV-2 challenge in mice immunized with research-grade mRNA-1273.
There is currently no licensed vaccine for respiratory syncytial virus (RSV). Here, we assess the effect of RSV fusion protein (F) conformation on B cell responses in a post hoc comparison of samples ...from the DS-Cav1 prefusion (pre-F) and MEDI7510 postfusion (post-F) vaccine clinical trials. We compared the magnitude and quality of the serological and B cell responses across time points and vaccines. We measured RSV A and B neutralization, F-binding immunoglobulin G titers, and competition assays at week 0 (before vaccination) and week 4 (after vaccination) to evaluate antibody specificity and potency. To compare B cell specificity and activation, we used pre-F and post-F probes in tandem with a 17-color immunophenotyping flow cytometry panel at week 0 (before vaccination) and week 1 (after vaccination). Our data demonstrate that both DS-Cav1 and MEDI7510 vaccination robustly elicit F-specific antibodies and B cells, but DS-Cav1 elicited antibodies that more potently neutralized both RSV A and B. The superior potency was mediated by antibodies that bind antigenic sites on the apex of pre-F that are not present on post-F. In the memory (CD27
) B cell compartment, vaccination with DS-Cav1 or MEDI7510 elicited B cells with different epitope specificities. B cells preferentially binding the pre-F probe were activated in DS-Cav1-vaccinated participants but not in MEDI7510-vaccinated participants. Our findings emphasize the importance of using pre-F as an immunogen in humans because of its deterministic role in eliciting highly potent neutralizing antibodies and memory B cells.
•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.
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