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

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

Brouwer et al. present preclinical evidence in support of a COVID-19 vaccine candidate, designed as a self-assembling two-component protein nanoparticle displaying multiple copies of the SARS-CoV-2 spike protein, which induces strong neutralizing antibody responses and protects from high-dose SARS-CoV-2 challenge.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 i

Nanoparticle vaccines for inducing HIV-1 neutralizing antibodies

The enormous sequence diversity between human immunodeficiency virus type 1 (HIV-1) strains poses a major roadblock for generating a broadly protective vaccine. Many experimental HIV-1 vaccine efforts are therefore aimed at eliciting broadly neutralizing antibodies (bNAbs) that are capable of neutralizing the majority of circulating HIV-1 strains. The envelope glycoprotein (Env) trimer on the viral membrane is the sole target of bNAbs and the key component of vaccination approaches aimed at eliciting bNAbs. Multimeric presentation of Env on nanoparticles often plays a critical role in these strategies. Here, we will discuss the different aspects of nanoparticles in Env vaccination, including recent insights in immunological processes underlying their perceived advantages, the different nanoparticle platforms and the various immunogenicity studies that employed nanoparticles to improve (neutralizing) antibody responses against Env

Curcuminoids as EBV Lytic Activators for Adjuvant Treatment in EBV-Positive Carcinomas

Epstein-Barr virus (EBV) persists in nasopharyngeal (NPC) and gastric carcinomas (EBVaGC) in a tightly latent form. Cytolytic virus activation (CLVA) therapy employs gemcitabine and valproic acid (GCb+VPA) to reactivate latent EBV into the lytic phase and antiviral valganciclovir to enhance cell death and prevent virus production. CLVA treatment has proven safe in phase-I/II trials with promising clinical responses in patients with recurrent NPC. However, a major challenge is to maximize EBV lytic reactivation by CLVA. Curcumin, a dietary spice used in Asian countries, is known for its antitumor property and therapeutic potential. Novel curcuminoids that were developed to increase efficacy and bioavailability may serve as oral CLVA adjuvants. We investigated the potential of curcumin and its analogs (curcuminoids) to trigger the EBV lytic cycle in EBVaGC and NPC cells. EBV-reactivating effects were measured by immunoblot and immunofluorescence using monoclonal antibodies specific for EBV lytic proteins. Two of the hit compounds (41, EF24) with high lytic inducing activity were further studied for their synergistic or antagonistic effects when combined with GCb+VPA and analyzed by cytotoxicity and mRNA profiling assays to measure the EBV reactivation. Curcuminoid as a single agent significantly induced EBV reactivation in recombinant GC and NPC lines. The drug effects were dose- and time-dependent. Micromolar concentration of curcuminoid EF24 enhanced the CLVA effect in all cell systems except SNU719, a naturally infected EBVaGC cell that carries a more tightly latent viral genome. These findings indicated that EF24 has potential as EBV lytic activator and may serve as an adjuvant in CLVA treatment

Probing Affinity, Avidity, Anticooperativity, and Competition in Antibody and Receptor Binding to the SARS-CoV-2 Spike by Single Particle Mass Analyses

Determining how antibodies interact with the spike (S) protein of the SARS-CoV-2 virus is critical for combating COVID-19. Structural studies typically employ simplified, truncated constructs that may not fully recapitulate the behavior of the original complexes. Here, we combine two single particle mass analysis techniques (mass photometry and charge-detection mass spectrometry) to enable the measurement of full IgG binding to the trimeric SARS-CoV-2 S ectodomain. Our experiments reveal that antibodies targeting the S-trimer typically prefer stoichiometries lower than the symmetry-predicted 3:1 binding. We determine that this behavior arises from the interplay of steric clashes and avidity effects that are not reflected in common antibody constructs (i.e., Fabs). Surprisingly, these substoichiometric complexes are fully effective at blocking ACE2 binding despite containing free receptor binding sites. Our results highlight the importance of studying antibody/antigen interactions using complete, multimeric constructs and showcase the utility of single particle mass analyses in unraveling these complex interactions

Probing Affinity, Avidity, Anticooperativity, and Competition in Antibody and Receptor Binding to the SARS-CoV-2 Spike by Single Particle Mass Analyses

Determining how antibodies interact with the spike (S) protein of the SARS-CoV-2 virus is critical for combating COVID-19. Structural studies typically employ simplified, truncated constructs that may not fully recapitulate the behavior of the original complexes. Here, we combine two single particle mass analysis techniques (mass photometry and charge-detection mass spectrometry) to enable the measurement of full IgG binding to the trimeric SARS-CoV-2 S ectodomain. Our experiments reveal that antibodies targeting the S-trimer typically prefer stoichiometries lower than the symmetry-predicted 3:1 binding. We determine that this behavior arises from the interplay of steric clashes and avidity effects that are not reflected in common antibody constructs (i.e., Fabs). Surprisingly, these substoichiometric complexes are fully effective at blocking ACE2 binding despite containing free receptor binding sites. Our results highlight the importance of studying antibody/antigen interactions using complete, multimeric constructs and showcase the utility of single particle mass analyses in unraveling these complex interactions

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

A third SARS-CoV-2 spike vaccination improves neutralization of variants-of-concern

The emergence of SARS-CoV-2 variants that are more resistant to antibody-mediated neutralization pose a new hurdle in combating the COVID-19 pandemic. Although vaccines based on the original Wuhan sequence have been shown to be effective at preventing COVID-19, their efficacy is likely to be decreased against more neutralization-resistant variants-of-concern (VOC), in particular, the Beta variant originating in South Africa. We assessed, in mice, rabbits, and non-human primates, whether a third vaccination with experimental Wuhan-based Spike vaccines could alleviate this problem. Our data show that a third immunization improves neutralizing antibody titers against the variants-of-concern, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2). After three vaccinations, the level of neutralization against Beta was similar to the level of neutralization against the original strain after two vaccinations, suggesting that simply providing a third immunization could nullify the reduced activity of current vaccines against VOC