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

Abstract: The development of a portfolio of COVID-19 vaccines to vaccinate the global population remains an urgent public health imperative1. Here we demonstrate the capacity of a subunit vaccine, comprising the SARS-CoV-2 spike protein receptor-binding domain displayed on an I53-50 protein nanoparticle scaffold (hereafter designated RBD-NP), to stimulate robust and durable neutralizing-antibody responses and protection against SARS-CoV-2 in rhesus macaques. We evaluated five adjuvants including Essai O/W 1849101, a squalene-in-water emulsion; AS03, an α-tocopherol-containing oil-in-water emulsion; AS37, a Toll-like receptor 7 (TLR7) agonist adsorbed to alum; CpG1018-alum, a TLR9 agonist formulated in alum; and alum. RBD-NP immunization with AS03, CpG1018-alum, AS37 or alum induced substantial neutralizing-antibody and CD4 T cell responses, and conferred protection against SARS-CoV-2 infection in the pharynges, nares and bronchoalveolar lavage. The neutralizing-antibody response to live virus was maintained up to 180 days after vaccination with RBD-NP in AS03 (RBD-NP-AS03), and correlated with protection from infection. RBD-NP immunization cross-neutralized the B.1.1.7 SARS-CoV-2 variant efficiently but showed a reduced response against the B.1.351 variant. RBD-NP-AS03 produced a 4.5-fold reduction in neutralization of B.1.351 whereas the group immunized with RBD-NP-AS37 produced a 16-fold reduction in neutralization of B.1.351, suggesting differences in the breadth of the neutralizing-antibody response induced by these adjuvants. Furthermore, RBD-NP-AS03 was as immunogenic as a prefusion-stabilized spike immunogen (HexaPro) with AS03 adjuvant. These data highlight the efficacy of the adjuvanted RBD-NP vaccine in promoting protective immunity against SARS-CoV-2 and have led to phase I/II clinical trials of this vaccine (NCT04742738 and NCT04750343).

Abstract: A combination of vaccination approaches will likely be necessary to fully control the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Here, we show that modified vaccinia Ankara (MVA) vectors expressing membrane-anchored pre-fusion stabilized spike (MVA/S) but not secreted S1 induced strong neutralizing antibody responses against SARS-CoV-2 in mice. In macaques, the MVA/S vaccination induced strong neutralizing antibodies and CD8+ T cell responses, and conferred protection from SARS-CoV-2 infection and virus replication in the lungs as early as day 2 following intranasal and intratracheal challenge. Single-cell RNA sequencing analysis of lung cells on day 4 after infection revealed that MVA/S vaccination also protected macaques from infection-induced inflammation and B cell abnormalities and lowered induction of interferon-stimulated genes. These results demonstrate that MVA/S vaccination induces neutralizing antibodies and CD8+ T cells in the blood and lungs and is a potential vaccine candidate for SARS-CoV-2.

Abstract: The development of a portfolio of SARS-CoV-2 vaccines to vaccinate the global population remains an urgent public health imperative Here, we demonstrate the capacity of a subunit vaccine under clinical development, comprising the SARS-CoV-2 Spike protein receptor-binding domain displayed on a two-component protein nanoparticle (RBD-NP), to stimulate robust and durable neutralizing antibody (nAb) responses and protection against SARS-CoV-2 in non-human primates We evaluated five different adjuvants combined with RBD-NP including Essai O/W 1849101, a squalene-in-water emulsion; AS03, an alpha-tocopherol-containing squalene-based oil-in-water emulsion used in pandemic influenza vaccines; AS37, a TLR-7 agonist adsorbed to Alum; CpG 1018-Alum (CpG-Alum), a TLR-9 agonist formulated in Alum; or Alum, the most widely used adjuvant All five adjuvants induced substantial nAb and CD4 T cell responses after two consecutive immunizations Durable nAb responses were evaluated for RBD-NP/AS03 immunization and the live-virus nAb response was durably maintained up to 154 days post-vaccination AS03, CpG-Alum, AS37 and Alum groups conferred significant protection against SARS-CoV-2 infection in the pharynges, nares and in the bronchoalveolar lavage The nAb titers were highly correlated with protection against infection Furthermore, RBD-NP when used in conjunction with AS03 was as potent as the prefusion stabilized Spike immunogen, HexaPro Taken together, these data highlight the efficacy of the RBD-NP formulated with clinically relevant adjuvants in promoting robust immunity against SARS-CoV-2 in non-human primates

Abstract: The need for SARS-CoV-2 next-generation vaccines has been highlighted by the rise of variants of concern (VoC) and the long-term threat of other coronaviruses. Here, we designed and characterized four categories of engineered nanoparticle immunogens that recapitulate the structural and antigenic properties of prefusion Spike (S), S1 and RBD. These immunogens induced robust S-binding, ACE2-inhibition, and authentic and pseudovirus neutralizing antibodies against SARS-CoV-2 in mice. A Spike-ferritin nanoparticle (SpFN) vaccine elicited neutralizing titers more than 20-fold higher than convalescent donor serum, following a single immunization, while RBD-Ferritin nanoparticle (RFN) immunogens elicited similar responses after two immunizations. Passive transfer of IgG purified from SpFN- or RFN-immunized mice protected K18-hACE2 transgenic mice from a lethal SARS-CoV-2 virus challenge. Furthermore, SpFN- and RFN-immunization elicited ACE2 blocking activity and neutralizing ID50 antibody titers >2,000 against SARS-CoV-1, along with high magnitude neutralizing titers against major VoC. These results provide design strategies for pan-coronavirus vaccine development. HIGHLIGHTSO_LIIterative structure-based design of four Spike-domain Ferritin nanoparticle classes of immunogens C_LIO_LISpFN-ALFQ and RFN-ALFQ immunization elicits potent neutralizing activity against SARS-CoV-2, variants of concern, and SARS-CoV-1 C_LIO_LIPassively transferred IgG from immunized C57BL/6 mice protects K18-hACE2 mice from lethal SARS-CoV-2 challenge C_LI

Abstract: Emerging SARS-CoV-2 variants pose a threat to human immunity induced by natural infection and vaccination. We assessed the recognition of three variants of concern (B.1.1.7, B.1.351 and P.1) in cohorts of COVID-19 patients ranging in disease severity (n = 69) and recipients of the Pfizer/BioNTech vaccine (n = 50). Spike binding and neutralization against all three VOC was substantially reduced in the majority of samples, with the largest 4-7-fold reduction in neutralization being observed against B.1.351. While hospitalized COVID-19 patients and vaccinees maintained sufficient neutralizing titers against all three VOC, 39% of non-hospitalized patients did not neutralize B.1.351. Moreover, monoclonal neutralizing antibodies (NAbs) show sharp reductions in their binding kinetics and neutralizing potential to B.1.351 and P.1, but not to B.1.1.7. These data have implications for the degree to which pre-existing immunity can protect against subsequent infection with VOC and informs policy makers of susceptibility to globally circulating SARS-CoV-2 VOC.

Abstract: The outbreak of a novel coronavirus (2019-nCoV) represents a pandemic threat that has been declared a public health emergency of international concern. The CoV spike (S) glycoprotein is a key target for vaccines, therapeutic antibodies, and diagnostics. To facilitate medical countermeasure development, we determined a 3.5-angstrom-resolution cryo-electron microscopy structure of the 2019-nCoV S trimer in the prefusion conformation. The predominant state of the trimer has one of the three receptor-binding domains (RBDs) rotated up in a receptor-accessible conformation. We also provide biophysical and structural evidence that the 2019-nCoV S protein binds angiotensin-converting enzyme 2 (ACE2) with higher affinity than does severe acute respiratory syndrome (SARS)-CoV S. Additionally, we tested several published SARS-CoV RBD-specific monoclonal antibodies and found that they do not have appreciable binding to 2019-nCoV S, suggesting that antibody cross-reactivity may be limited between the two RBDs. The structure of 2019-nCoV S should enable the rapid development and evaluation of medical countermeasures to address the ongoing public health crisis.

Abstract: The emergence of SARS-CoV-2 has resulted in >90,000 infections and >3,000 deaths. Coronavirus spike (S) glycoproteins promote entry into cells and are the main target of antibodies. We show that SARS-CoV-2 S uses ACE2 to enter cells and that the receptor-binding domains of SARS-CoV-2 S and SARS-CoV S bind with similar affinities to human ACE2, correlating with the efficient spread of SARS-CoV-2 among humans. We found that the SARS-CoV-2 S glycoprotein harbors a furin cleavage site at the boundary between the S1/S2 subunits, which is processed during biogenesis and sets this virus apart from SARS-CoV and SARS-related CoVs. We determined cryo-EM structures of the SARS-CoV-2 S ectodomain trimer, providing a blueprint for the design of vaccines and inhibitors of viral entry. Finally, we demonstrate that SARS-CoV S murine polyclonal antibodies potently inhibited SARS-CoV-2 S mediated entry into cells, indicating that cross-neutralizing antibodies targeting conserved S epitopes can be elicited upon vaccination.

Abstract: Background The ongoing outbreak of the recently emerged novel coronavirus (2019-nCoV) poses a challenge for public health laboratories as virus isolates are unavailable while there is growing evidence that the outbreak is more widespread than initially thought, and international spread through travellers does already occur. Aim We aimed to develop and deploy robust diagnostic methodology for use in public health laboratory settings without having virus material available. Methods Here we present a validated diagnostic workflow for 2019-nCoV, its design relying on close genetic relatedness of 2019-nCoV with SARS coronavirus, making use of synthetic nucleic acid technology. Results The workflow reliably detects 2019-nCoV, and further discriminates 2019-nCoV from SARS-CoV. Through coordination between academic and public laboratories, we confirmed assay exclusivity based on 297 original clinical specimens containing a full spectrum of human respiratory viruses. Control material is made available through European Virus Archive – Global (EVAg), a European Union infrastructure project. Conclusion The present study demonstrates the enormous response capacity achieved through coordination of academic and public laboratories in national and European research networks.

Abstract: Coronavirus disease 2019 (COVID-19) is an acute infection of the respiratory tract that emerged in late 20191,2. Initial outbreaks in China involved 13.8% of cases with severe courses, and 6.1% of cases with critical courses3. This severe presentation may result from the virus using a virus receptor that is expressed predominantly in the lung2,4; the same receptor tropism is thought to have determined the pathogenicity—but also aided in the control—of severe acute respiratory syndrome (SARS) in 20035. However, there are reports of cases of COVID-19 in which the patient shows mild upper respiratory tract symptoms, which suggests the potential for pre- or oligosymptomatic transmission6–8. There is an urgent need for information on virus replication, immunity and infectivity in specific sites of the body. Here we report a detailed virological analysis of nine cases of COVID-19 that provides proof of active virus replication in tissues of the upper respiratory tract. Pharyngeal virus shedding was very high during the first week of symptoms, with a peak at 7.11 × 108 RNA copies per throat swab on day 4. Infectious virus was readily isolated from samples derived from the throat or lung, but not from stool samples—in spite of high concentrations of virus RNA. Blood and urine samples never yielded virus. Active replication in the throat was confirmed by the presence of viral replicative RNA intermediates in the throat samples. We consistently detected sequence-distinct virus populations in throat and lung samples from one patient, proving independent replication. The shedding of viral RNA from sputum outlasted the end of symptoms. Seroconversion occurred after 7 days in 50% of patients (and by day 14 in all patients), but was not followed by a rapid decline in viral load. COVID-19 can present as a mild illness of the upper respiratory tract. The confirmation of active virus replication in the upper respiratory tract has implications for the containment of COVID-19. Detailed virological analysis of nine cases of coronavirus disease 2019 (COVID-19) provides proof of active replication of the SARS-CoV-2 virus in tissues of the upper respiratory tract.

Abstract: Exome sequencing of tumor samples and matched normal controls has the potential to rapidly identify protein-altering mutations across hundreds of patients, potentially enabling the discovery of recurrent events driving tumor development and growth (International Cancer Genome Consortium 2010; Stratton 2011). Yet the analysis of such data presents significant challenges. Sequencing coverage is nonuniform across targeted regions and from one sample to the next (Ng et al. 2009; Bainbridge et al. 2010; Teer et al. 2010). Many regions achieve high read depth (more than 100×), which can confound variant callers and depth-based filters if not properly addressed (Ku et al. 2011). Repetitive and paralogous sequences can give rise to numerous false positives. The detection of somatic mutations in tumor genomes is even more challenging. The genomes of primary tumors are genetically heterogeneous (Ding et al. 2010), with frequent rearrangements (Campbell et al. 2008) and copy number alterations (CNAs) (Beroukhim et al. 2010). Further, somatic mutations are relatively rare compared with germline variation, often representing <0.1% of variants in a tumor genome (Ley et al. 2008; Mardis et al. 2009). Simply subtracting variants in the matched normal from variants in the tumor (Wei et al. 2011) is poorly suited for the analysis of exome sequence data, because it fails to account for regions that were undersampled in the normal. Accurate mutation detection requires a direct, simultaneous comparison of tumor–normal pairs at every position in the exome, but few algorithms to do so have been described. Numerous algorithms have been developed to assess genome-wide copy number using whole-genome sequencing (WGS) data. Most of these approaches (Campbell et al. 2008; Alkan et al. 2009; Chiang et al. 2009; Yoon et al. 2009; Abyzov et al. 2011) would be confounded by exome data sets, because of the biases introduced by hybridization and the sparse and uneven coverages throughout the genome. However, when both DNA samples in a tumor–normal pair were captured and sequenced under identical hybridization conditions, we reasoned that it might be possible to detect somatic CNAs (SCNAs) as deviations from the log-ratio of sequence coverage depth within a tumor–normal pair, and then quantify the deviations statistically. Such an approach would provide a gene-centric view of copy number in a tumor sample, though it would be limited to the ∼1% of the genome captured by current exome platforms. Previously, we published VarScan (Koboldt et al. 2009), an algorithm for variant detection in next-generation sequencing data. We have since released a new tool, VarScan 2 (http://varscan.sourceforge.net), with several improvements, including the ability to identify somatic mutation, loss of heterozygosity (LOH), and CNA events in tumor–normal pairs. VarScan 2 analyzes sequence data from a tumor sample and its corresponding normal sample simultaneously, applying heuristic methods and a statistical test to detect variants—single nucleotide variants (SNVs) and insertions/deletions (indels)—and classify them by somatic status. By direct comparison of normalized sequence depth, our method also detects SCNAs in the tumor genome. Here, we utilize VarScan 2 for the analysis of exome sequence data from 151 patients with high-grade serous ovarian adenocarcinoma (HGS-OVCa) that were initially characterized within the Cancer Genome Atlas (TCGA) project (Cancer Genome Atlas Research Network 2011). We present a robust pipeline for the detection of both germline (inherited) and somatic (acquired) mutations by exome sequencing and describe filtering approaches for detecting variants with high sensitivity and specificity. To evaluate the performance of our SCNA detection algorithm, we compare our results to copy number data from high-density SNP array and WGS approaches. Our results demonstrate the accuracy of VarScan 2 for somatic mutation and CNA detection and enable a new survey of the genetic landscape in ovarian carcinoma.