Two-component spike nanoparticle vaccine protects macaques from SARS-CoV-2 infection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is continuing to disrupt personal lives, global healthcare systems, and economies. Hence, there is an urgent need for a vaccine that prevents viral infection, transmission, and disease. Here, we present a two-component protein-based nanoparticle vaccine that displays multiple copies of the SARS-CoV-2 spike protein. Immunization studies show that this vaccine induces potent neutralizing antibody responses in mice, rabbits, and cynomolgus macaques. The vaccine-induced immunity protects macaques against a high-dose challenge, resulting in strongly reduced viral infection and replication in the upper and lower airways. These nanoparticles are a promising vaccine candidate to curtail the SARS-CoV-2 pandemic.

Cross-Neutralization of a SARS-CoV-2 Antibody to a Functionally Conserved Site Is Mediated by Avidity.

Most antibodies isolated from individuals with coronavirus disease 2019 (COVID-19) are specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, COVA1-16 is a relatively rare antibody that also cross-neutralizes SARS-CoV. Here, we determined a crystal structure of the COVA1-16 antibody fragment (Fab) with the SARS-CoV-2 receptor-binding domain (RBD) and negative-stain electron microscopy reconstructions with the spike glycoprotein trimer to elucidate the structural basis of its cross-reactivity. COVA1-16 binds a highly conserved epitope on the SARS-CoV-2 RBD, mainly through a long complementarity-determining region (CDR) H3, and competes with the angiotensin-converting enzyme 2 (ACE2) receptor because of steric hindrance rather than epitope overlap. COVA1-16 binds to a flexible up conformation of the RBD on the spike and relies on antibody avidity for neutralization. These findings, along with the structural and functional rationale for epitope conservation, provide insights for development of more universal SARS-like coronavirus vaccines and therapies.

Mosaic and mixed HIV-1 glycoprotein nanoparticles elicit antibody responses to broadly neutralizing epitopes.

An effective human immunodeficiency virus 1 (HIV-1) vaccine will most likely have to elicit broadly neutralizing antibodies (bNAbs) to overcome the sequence diversity of the envelope glycoprotein (Env). So far, stabilized versions of Env, such as SOSIP trimers, have been able to induce neutralizing antibody (NAb) responses, but those responses are mainly strain-specific. Here we attempted to broaden NAb responses by using a multivalent vaccine and applying a number of design improvements. First, we used highly stabilized SOSIP.v9 trimers. Second, we removed any holes in the glycan shields and optimized glycan occupancy to avoid strain-specific glycan hole responses. Third, we selected five sequences from the same clade (B), as we observed previously that combining Env trimers from clade A, B and C did not improve cross-reactive responses, as they might have been too diverse. Fourth, to improve antibody (Ab) responses, the Env trimers were displayed on two-component I53-50 nanoparticles (NPs). Fifth, to favor activation of cross-reactive B cells, the five Env trimers were co-displayed on mosaic NPs. Sixth, we immunized rabbits four times with long intervals between vaccinations. These efforts led to the induction of cross-reactive B cells and cross-reactive binding Ab responses, but we only sporadically detected cross-neutralizing responses. We conclude that stabilized HIV-1 Env trimers that are not modified specifically for priming naive B cells are unable to elicit strong bNAb responses, and infer that sequential immunization regimens, most likely starting with specific germline-targeting immunogens, will be necessary to overcome Env's defenses against the induction of NAbs. The antigens described here could be excellent boosting immunogens in a sequential immunization regimen, as responses to bNAb epitopes were induced.

Infection and transmission of SARS-CoV-2 depend on heparan sulfate proteoglycans.

The current pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and outbreaks of new variants highlight the need for preventive treatments. Here, we identified heparan sulfate proteoglycans as attachment receptors for SARS-CoV-2. Notably, neutralizing antibodies against SARS-CoV-2 isolated from COVID-19 patients interfered with SARS-CoV-2 binding to heparan sulfate proteoglycans, which might be an additional mechanism of antibodies to neutralize infection. SARS-CoV-2 binding to and infection of epithelial cells was blocked by low molecular weight heparins (LMWH). Although dendritic cells (DCs) and mucosal Langerhans cells (LCs) were not infected by SARS-CoV-2, both DC subsets efficiently captured SARS-CoV-2 via heparan sulfate proteoglycans and transmitted the virus to ACE2-positive cells. Notably, human primary nasal cells were infected by SARS-CoV-2, and infection was blocked by pre-treatment with LMWH. These data strongly suggest that heparan sulfate proteoglycans are important attachment receptors facilitating infection and transmission, and support the use of LMWH as prophylaxis against SARS-CoV-2 infection.

SARS-CoV-2 infection activates dendritic cells via cytosolic receptors rather than extracellular TLRs.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), an infectious disease characterized by strong induction of inflammatory cytokines, progressive lung inflammation, and potentially multiorgan dysfunction. It remains unclear how SARS-CoV-2 infection leads to immune activation. The Spike (S) protein of SARS-CoV-2 has been suggested to trigger TLR4 and thereby activate immunity. Here, we have investigated the role of TLR4 in SARS-CoV-2 infection and immunity. Neither exposure of isolated S protein, SARS-CoV-2 pseudovirus nor primary SARS-CoV-2 isolate induced TLR4 activation in a TLR4-expressing cell line. Human monocyte-derived DCs express TLR4 but not angiotensin converting enzyme 2 (ACE2), and DCs were not infected by SARS-CoV-2. Notably, neither S protein nor SARS-CoV-2 induced DC maturation or cytokines, indicating that both S protein and SARS-CoV-2 virus particles do not trigger extracellular TLRs including TLR4. Ectopic expression of ACE2 in DCs led to efficient infection by SARS-CoV-2 and, strikingly, efficient type I IFN and cytokine responses. These data strongly suggest that not extracellular TLRs but intracellular viral sensors are key players in sensing SARS-CoV-2. These data imply that SARS-CoV-2 escapes direct sensing by TLRs, which might underlie the lack of efficient immunity to SARS-CoV-2 early during infection.

Structural and functional evaluation of de novo-designed, two-component nanoparticle carriers for HIV Env trimer immunogens.

Two-component, self-assembling nanoparticles represent a versatile platform for multivalent presentation of viral antigens. Computational design of protein nanoparticles with differing sizes and geometries enables combination with antigens of choice to test novel multimerization concepts in immunization strategies where the goal is to improve the induction and maturation of neutralizing antibody lineages. Here, we describe detailed antigenic, structural, and functional characterization of computationally designed tetrahedral, octahedral, and icosahedral nanoparticle immunogens displaying trimeric HIV envelope glycoprotein (Env) ectodomains. Env trimers, based on subtype A (BG505) or consensus group M (ConM) sequences and engineered with SOSIP stabilizing mutations, were fused to an underlying trimeric building block of each nanoparticle. Initial screening yielded one icosahedral and two tetrahedral nanoparticle candidates, capable of presenting twenty or four copies of the Env trimer. A number of analyses, including detailed structural characterization by cryo-EM, demonstrated that the nanoparticle immunogens possessed the intended structural and antigenic properties. When the immunogenicity of ConM-SOSIP trimers presented on a two-component tetrahedral nanoparticle or as soluble proteins were compared in rabbits, the two immunogens elicited similar serum antibody binding titers against the trimer component. Neutralizing antibody titers were slightly elevated in the animals given the nanoparticle immunogen and were initially more focused to the trimer apex. Altogether, our findings indicate that tetrahedral nanoparticles can be successfully applied for presentation of HIV Env trimer immunogens; however, the optimal implementation to different immunization strategies remains to be determined.

Comparative assessment of multiple COVID-19 serological technologies supports continued evaluation of point-of-care lateral flow assays in hospital and community healthcare settings.

There is a clear requirement for an accurate SARS-CoV-2 antibody test, both as a complement to existing diagnostic capabilities and for determining community seroprevalence. We therefore evaluated the performance of a variety of antibody testing technologies and their potential use as diagnostic tools. Highly specific in-house ELISAs were developed for the detection of anti-spike (S), -receptor binding domain (RBD) and -nucleocapsid (N) antibodies and used for the cross-comparison of ten commercial serological assays-a chemiluminescence-based platform, two ELISAs and seven colloidal gold lateral flow immunoassays (LFIAs)-on an identical panel of 110 SARS-CoV-2-positive samples and 50 pre-pandemic negatives. There was a wide variation in the performance of the different platforms, with specificity ranging from 82% to 100%, and overall sensitivity from 60.9% to 87.3%. However, the head-to-head comparison of multiple sero-diagnostic assays on identical sample sets revealed that performance is highly dependent on the time of sampling, with sensitivities of over 95% seen in several tests when assessing samples from more than 20 days post onset of symptoms. Furthermore, these analyses identified clear outlying samples that were negative in all tests, but were later shown to be from individuals with mildest disease presentation. Rigorous comparison of antibody testing platforms will inform the deployment of point-of-care technologies in healthcare settings and their use in the monitoring of SARS-CoV-2 infections.

Feasibility and performance of smartphone-based daily micro-surveys among patients recovering from cancer surgery.

AIMS: Daily micro-surveys, or the high-frequency administration of patient-reported outcome measures (PROMs), may provide real-time, unbiased assessments of health-related quality of life (HRQoL). We evaluated the feasibility and accuracy of daily micro-surveys using a smartphone platform among patients recovering from cancer surgery. METHODS: In a prospective study (2017-2019), patients undergoing cancer surgery downloaded a smartphone application that administered daily micro-surveys comprising five randomly selected items from the Short Form-36 (SF-36). Micro-surveys were administered without replacement until the entire SF-36 was administered weekly. The full-length SF-36 was also administered preoperatively and 4, 12, and 24 weeks postoperatively. We assessed response and completion rates between the micro-surveys and full-length SF-36, as well as agreement of responses using Bland-Altman (B&A) analyses. RESULTS: Ninety-five patients downloaded the application and were followed for a mean of 131 days [SD ± 85]. Response rates for the full-length SF-36 and micro-surveys was 76% [95%CI 69, 83], and 34% [95%CI 26, 39]. Despite lower response rates, more SF-36 surveys were collected using the daily micro-surveys compared to the intermittent full-length SF-36 (9.9 [95%CI 8.4, 12.6] vs. 3.0 [95%CI 2.8, 3.3], respectively). B&A analyses demonstrated lack of agreement between micro-surveys and SF-36. However, agreement improved with higher micro-survey completion rate. Eighty-five percent of participants reported that daily micro-surveys were not burdensome. CONCLUSION: This study suggests that collection of daily micro-surveys among patients recovering from cancer surgery is feasible using smartphones in the early postoperative period. Future implementation of daily micro-surveys may more granularly describe momentary HRQoL changes through a greater volume of self-reported survey data.

Site-Specific Steric Control of SARS-CoV-2 Spike Glycosylation.

A central tenet in the design of vaccines is the display of native-like antigens in the elicitation of protective immunity. The abundance of N-linked glycans across the SARS-CoV-2 spike protein is a potential source of heterogeneity among the many different vaccine candidates under investigation. Here, we investigate the glycosylation of recombinant SARS-CoV-2 spike proteins from five different laboratories and compare them against S protein from infectious virus, cultured in Vero cells. We find patterns that are conserved across all samples, and this can be associated with site-specific stalling of glycan maturation that acts as a highly sensitive reporter of protein structure. Molecular dynamics simulations of a fully glycosylated spike support a model of steric restrictions that shape enzymatic processing of the glycans. These results suggest that recombinant spike-based SARS-CoV-2 immunogen glycosylation reproducibly recapitulates signatures of viral glycosylation.

Anti-Immune Complex Antibodies are Elicited During Repeated Immunization with HIV Env Immunogens.

Vaccination strategies against HIV-1 aim to elicit broadly neutralizing antibodies (bnAbs) using prime-boost regimens with HIV envelope (Env) immunogens. Early antibody responses to easily accessible epitopes on these antigens are directed to non-neutralizing epitopes instead of bnAb epitopes. Autologous neutralizing antibody responses appear upon boosting once immunodominant epitopes are saturated. Here we report another type of antibody response that arises after repeated immunizations with HIV Env immunogens and present the structures of six anti-immune complexes discovered using polyclonal epitope mapping. The anti-immune complex antibodies target idiotopes composed of framework regions of antibodies bound to Env. This work sheds light on current vaccine development efforts for HIV, as well as for other pathogens, in which repeated exposure to antigen is required.

Defining bottlenecks and opportunities for Lassa virus neutralization by structural profiling of vaccine-induced polyclonal antibody responses.

Lassa fever continues to be a major public health burden in West Africa, yet effective therapies or vaccines are lacking. The isolation of protective neutralizing antibodies against the Lassa virus glycoprotein complex (GPC) justifies the development of vaccines that can elicit strong neutralizing antibody responses. However, Lassa vaccine candidates have generally been unsuccessful at doing so, and the associated antibody responses to these vaccines remain poorly characterized. Here, we establish an electron microscopy-based epitope mapping workflow that enables high-resolution structural characterization of polyclonal antibodies to the GPC. By applying this method to rabbits vaccinated with a recombinant GPC vaccine and a GPC-derived virus-like particle, we reveal determinants of neutralization that involve epitopes of the GPC-A competition cluster. Furthermore, by identifying undescribed immunogenic off-target epitopes, we expose the challenges that recombinant GPC vaccines face. By enabling detailed polyclonal antibody characterization, our work ushers in a next generation of more rational Lassa vaccine design.

Defining bottlenecks and opportunities for Lassa virus neutralization by structural profiling of vaccine-induced polyclonal antibody responses.

Lassa fever continues to be a major public health burden in endemic countries in West Africa, yet effective therapies or vaccines are lacking. The isolation of potent and protective neutralizing antibodies against the Lassa virus glycoprotein complex (GPC) justifies the development of vaccines that can elicit strong neutralizing antibody responses. However, Lassa vaccines candidates have generally been unsuccessful in doing so and the associated antibody responses to these vaccines remain poorly characterized. Here, we establish an electron-microscopy based epitope mapping pipeline that enables high-resolution structural characterization of polyclonal antibodies to GPC. By applying this method to rabbits vaccinated with a recombinant GPC vaccine and a GPC-derived virus-like particle, we reveal determinants of neutralization which involve epitopes of the GPC-C, GPC-A, and GP1-A competition clusters. Furthermore, by identifying previously undescribed immunogenic off-target epitopes, we expose challenges that recombinant GPC vaccines face. By enabling detailed polyclonal antibody characterization, our work ushers in a next generation of more rational Lassa vaccine design.

New Monoclonal Antibodies Specific for Different Epitopes of the Spike Protein of SARS-CoV-2 and Its Major Variants: Additional Tools for a More Specific COVID-19 Diagnosis.

The emergence of the new pathogen SARS-CoV-2 determined a rapid need for monoclonal antibodies (mAbs) to detect the virus in biological fluids as a rapid tool to identify infected individuals to be treated or quarantined. The majority of commercially available antigenic tests for SARS-CoV-2 rely on the detection of N antigen in biologic fluid using anti-N antibodies, and their capacity to specifically identify subjects infected by SARS-CoV-2 is questionable due to several structural analogies among the N proteins of different coronaviruses. In order to produce new specific antibodies, BALB/c mice were immunized three times at 20-day intervals with a recombinant spike (S) protein. The procedure used was highly efficient, and 40 different specific mAbs were isolated, purified and characterized, with 13 ultimately being selected for their specificity and lack of cross reactivity with other human coronaviruses. The specific epitopes recognized by the selected mAbs were identified through a peptide library and/or by recombinant fragments of the S protein. In particular, the selected mAbs recognized different linear epitopes along the S1, excluding the receptor binding domain, and along the S2 subunits of the S protein of SARS-CoV-2 and its major variants of concern. We identified combinations of anti-S mAbs suitable for use in ELISA or rapid diagnostic tests, with the highest sensitivity and specificity coming from proof-of-concept tests using recombinant antigens, SARS-CoV-2 or biological fluids from infected individuals, that represent important additional tools for the diagnosis of COVID-19.

A Lassa virus mRNA vaccine confers protection but does not require neutralizing antibody in a guinea pig model of infection.

Lassa virus is a member of the Arenaviridae family, which causes human infections ranging from asymptomatic to severe hemorrhagic disease with a high case fatality rate. We have designed and generated lipid nanoparticle encapsulated, modified mRNA vaccines that encode for the wild-type Lassa virus strain Josiah glycoprotein complex or the prefusion stabilized conformation of the Lassa virus glycoprotein complex. Hartley guinea pigs were vaccinated with two 10 µg doses, 28 days apart, of either construct. Vaccination induced strong binding antibody responses, specific to the prefusion conformation of glycoprotein complex, which were significantly higher in the prefusion stabilized glycoprotein complex construct group and displayed strong Fc-mediated effects. However, Lassa virus-neutralizing antibody activity was detected in some but not all animals. Following the challenge with a lethal dose of the Lassa virus, all vaccinated animals were protected from death and severe disease. Although the definitive mechanism of protection is still unknown, and assessment of the cell-mediated immune response was not investigated in this study, these data demonstrate the promise of mRNA as a vaccine platform against the Lassa virus and that protection against Lassa virus can be achieved in the absence of virus-neutralizing antibodies.

Structural conservation of Lassa virus glycoproteins and recognition by neutralizing antibodies.

Lassa fever is an acute hemorrhagic fever caused by the zoonotic Lassa virus (LASV). The LASV glycoprotein complex (GPC) mediates viral entry and is the sole target for neutralizing antibodies. Immunogen design is complicated by the metastable nature of recombinant GPCs and the antigenic differences among phylogenetically distinct LASV lineages. Despite the sequence diversity of the GPC, structures of most lineages are lacking. We present the development and characterization of prefusion-stabilized, trimeric GPCs of LASV lineages II, V, and VII, revealing structural conservation despite sequence diversity. High-resolution structures and biophysical characterization of the GPC in complex with GP1-A-specific antibodies suggest their neutralization mechanisms. Finally, we present the isolation and characterization of a trimer-preferring neutralizing antibody belonging to the GPC-B competition group with an epitope that spans adjacent protomers and includes the fusion peptide. Our work provides molecular detail information on LASV antigenic diversity and will guide efforts to design pan-LASV vaccines.

Nasal skin reconstruction: Time to rethink the reconstructive ladder?

BACKGROUND: Nasal scarring can compromise aesthetics and function given its complex three-dimensional structure and central location. This study aimed to measure patients' satisfaction after reconstruction for nasal defects following Mohs micrographic surgery. METHODS: Patients presenting with nasal nonmelanoma skin cancer at Memorial Sloan Kettering Cancer Center New York, USA and Catharina Hospital Eindhoven, Netherlands from April 2017 to November 2019 were asked to participate. Reconstruction type, complications, and patients satisfaction were assessed. Patients completed the FACE-Q Skin Cancer - Satisfaction with Facial Appearance scale (preoperative and 1-year postoperative) and the Appraisal of Scars scale (1-year postoperative). RESULTS: A total of 128 patients completed the preand postoperative scales. There were 35 (27%) surgical defects repaired with primary closures, 71 (55.5%) with flaps, and 22 (17.2%) with full-thickness skin grafts (FTSG). Patients that underwent a flap or FTSG reconstruction had higher scar satisfaction scores than primary closures (p = 0.03). A trend was seen with patients following flap reconstructions scoring 7.8 points higher than primary closures and patients with upper nose defects scoring 6.4 points higher than lower nose defects. Males were significantly more satisfied than females. No significant difference was observed in the preoperative and postoperative facial appearance scores between the three groups (p = 0.39). CONCLUSION: Patients are more satisfied in the long term with their scars after flap reconstructions compared to primary closures. Therefore, nasal skin reconstruction may not follow the traditional reconstructive ladder and more complex approaches may lead to higher long-term scar satisfaction.

Diagnostic performance of two serological assays for the detection of SARS-CoV-2 specific antibodies: surveillance after vaccination.

Massive vaccination programs are being carried out to limit the SARS-CoV-2 pandemic that started in December 2019. Serological tests are of major importance as an indicator of circulation of the virus and to assess how vaccine-induced immunity progresses. An Enzyme-Linked Immunosorbent Assay (ELISA) and a Lateral Flow Assay (LFA) have been developed based on the SARS-CoV-2 recombinant Receptor Binding Domain (RBD) and the combination of Spike and Nucleoprotein, respectively. The validation with 1272 serum samples by comparison with INgezim COVID 19 DR showed good diagnostic performance (sensitivity: 93.2%-97.2%; specificity: 98.3%-99.3%) for detection of previous contact with SARS-CoV-2. Moreover, according to our results, these assays can help in the serosurveillance during and after vaccination, by detecting the humoral immune response as soon as 15 days postvaccination and identifying low-respondents. Hence, these tests could play a key role in the progression to a COVID-19 free world, helping to adjust future vaccination protocols.

Co-display of diverse spike proteins on nanoparticles broadens sarbecovirus neutralizing antibody responses.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants poses continuous challenges in combating the virus. Here, we describe vaccination strategies to broaden SARS-CoV-2 and sarbecovirus immunity by combining spike proteins based on different viruses or viral strains displayed on two-component protein nanoparticles. First, we combined spike proteins based on ancestral and Beta SARS-CoV-2 strains to broaden SARS-CoV-2 immune responses. Inclusion of Beta spike improved neutralizing antibody responses against SARS-CoV-2 Beta, Gamma, and Omicron BA.1 and BA.4/5. A third vaccination with ancestral SARS-CoV-2 spike also improved cross-neutralizing antibody responses against SARS-CoV-2 variants, in particular against the Omicron sublineages. Second, we combined SARS-CoV and SARS-CoV-2 spike proteins to broaden sarbecovirus immune responses. Adding SARS-CoV spike to a SARS-CoV-2 spike vaccine improved neutralizing responses against SARS-CoV and SARS-like bat sarbecoviruses SHC014 and WIV1. These results should inform the development of broadly active SARS-CoV-2 and pan-sarbecovirus vaccines and highlight the versatility of two-component nanoparticles for displaying diverse antigens.

Induction of cross-neutralizing antibodies by a permuted hepatitis C virus glycoprotein nanoparticle vaccine candidate.

Hepatitis C virus (HCV) infection affects approximately 58 million people and causes ~300,000 deaths yearly. The only target for HCV neutralizing antibodies is the highly sequence diverse E1E2 glycoprotein. Eliciting broadly neutralizing antibodies that recognize conserved cross-neutralizing epitopes is important for an effective HCV vaccine. However, most recombinant HCV glycoprotein vaccines, which usually include only E2, induce only weak neutralizing antibody responses. Here, we describe recombinant soluble E1E2 immunogens that were generated by permutation of the E1 and E2 subunits. We displayed the E2E1 immunogens on two-component nanoparticles and these nanoparticles induce significantly more potent neutralizing antibody responses than E2. Next, we generated mosaic nanoparticles co-displaying six different E2E1 immunogens. These mosaic E2E1 nanoparticles elicit significantly improved neutralization compared to monovalent E2E1 nanoparticles. These results provide a roadmap for the generation of an HCV vaccine that induces potent and broad neutralization.

Lassa virus glycoprotein nanoparticles elicit neutralizing antibody responses and protection.

The Lassa virus is endemic in parts of West Africa, and it causes hemorrhagic fever with high mortality. The development of a recombinant protein vaccine has been hampered by the instability of soluble Lassa virus glycoprotein complex (GPC) trimers, which disassemble into monomeric subunits after expression. Here, we use two-component protein nanoparticles consisting of trimeric and pentameric subunits to stabilize GPC in a trimeric conformation. These GPC nanoparticles present twenty prefusion GPC trimers on the surface of an icosahedral particle. Cryo-EM studies of GPC nanoparticles demonstrated a well-ordered structure and yielded a high-resolution structure of an unliganded GPC. These nanoparticles induced potent humoral immune responses in rabbits and protective immunity against the lethal Lassa virus challenge in guinea pigs. Additionally, we isolated a neutralizing antibody that mapped to the putative receptor-binding site, revealing a previously undefined site of vulnerability. Collectively, these findings offer potential approaches to vaccine and therapeutic design for the Lassa virus.