Mosaic sarbecovirus nanoparticles elicit cross-reactive responses in pre-vaccinated animals.

Immunization with mosaic-8b (nanoparticles presenting 8 SARS-like betacoronavirus [sarbecovirus] receptor-binding domains [RBDs]) elicits more broadly cross-reactive antibodies than homotypic SARS-CoV-2 RBD-only nanoparticles and protects against sarbecoviruses. To investigate original antigenic sin (OAS) effects on mosaic-8b efficacy, we evaluated the effects of prior COVID-19 vaccinations in non-human primates and mice on anti-sarbecovirus responses elicited by mosaic-8b, admix-8b (8 homotypics), or homotypic SARS-CoV-2 immunizations, finding the greatest cross-reactivity for mosaic-8b. As demonstrated by molecular fate mapping, in which antibodies from specific cohorts of B cells are differentially detected, B cells primed by WA1 spike mRNA-LNP dominated antibody responses after RBD-nanoparticle boosting. While mosaic-8b- and homotypic-nanoparticles boosted cross-reactive antibodies, de novo antibodies were predominantly induced by mosaic-8b, and these were specific for variant RBDs with increased identity to RBDs on mosaic-8b. These results inform OAS mechanisms and support using mosaic-8b to protect COVID-19-vaccinated/infected humans against as-yet-unknown SARS-CoV-2 variants and animal sarbecoviruses with human spillover potential.

Elicitation of broadly protective sarbecovirus immunity by receptor-binding domain nanoparticle vaccines.

Understanding vaccine-elicited protection against SARS-CoV-2 variants and other sarbecoviruses is key for guiding public health policies. We show that a clinical stage multivalent SARS-CoV-2 spike receptor-binding domain nanoparticle (RBD-NP) vaccine protects mice from SARS-CoV-2 challenge after a single immunization, indicating a potential dose-sparing strategy. We benchmarked serum neutralizing activity elicited by RBD-NPs in non-human primates against a lead prefusion-stabilized SARS-CoV-2 spike (HexaPro) using a panel of circulating mutants. Polyclonal antibodies elicited by both vaccines are similarly resilient to many RBD residue substitutions tested, although mutations at and surrounding position 484 have negative consequences for neutralization. Mosaic and cocktail nanoparticle immunogens displaying multiple sarbecovirus RBDs elicit broad neutralizing activity in mice and protect mice against SARS-CoV challenge even in the absence of SARS-CoV RBD in the vaccine. This study provides proof of principle that multivalent sarbecovirus RBD-NPs induce heterotypic protection and motivates advancing such broadly protective sarbecovirus vaccines to the clinic.

Identification of a therapeutic interfering particle-A single-dose SARS-CoV-2 antiviral intervention with a high barrier to resistance.

Viral-deletion mutants that conditionally replicate and inhibit the wild-type virus (i.e., defective interfering particles, DIPs) have long been proposed as single-administration interventions with high genetic barriers to resistance. However, theories predict that robust, therapeutic DIPs (i.e., therapeutic interfering particles, TIPs) must conditionally spread between cells with R0 >1. Here, we report engineering of TIPs that conditionally replicate with SARS-CoV-2, exhibit R0 >1, and inhibit viral replication 10- to 100-fold. Inhibition occurs via competition for viral replication machinery, and a single administration of TIP RNA inhibits SARS-CoV-2 sustainably in continuous cultures. Strikingly, TIPs maintain efficacy against neutralization-resistant variants (e.g., B.1.351). In hamsters, both prophylactic and therapeutic intranasal administration of lipid-nanoparticle TIPs durably suppressed SARS-CoV-2 by 100-fold in the lungs, reduced pro-inflammatory cytokine expression, and prevented severe pulmonary edema. These data provide proof of concept for a class of single-administration antivirals that may circumvent current requirements to continually update medical countermeasures against new variants.

Elicitation of Potent Neutralizing Antibody Responses by Designed Protein Nanoparticle Vaccines for SARS-CoV-2.

A safe, effective, and scalable vaccine is needed to halt the ongoing SARS-CoV-2 pandemic. We describe the structure-based design of self-assembling protein nanoparticle immunogens that elicit potent and protective antibody responses against SARS-CoV-2 in mice. The nanoparticle vaccines display 60 SARS-CoV-2 spike receptor-binding domains (RBDs) in a highly immunogenic array and induce neutralizing antibody titers 10-fold higher than the prefusion-stabilized spike despite a 5-fold lower dose. Antibodies elicited by the RBD nanoparticles target multiple distinct epitopes, suggesting they may not be easily susceptible to escape mutations, and exhibit a lower binding:neutralizing ratio than convalescent human sera, which may minimize the risk of vaccine-associated enhanced respiratory disease. The high yield and stability of the assembled nanoparticles suggest that manufacture of the nanoparticle vaccines will be highly scalable. These results highlight the utility of robust antigen display platforms and have launched cGMP manufacturing efforts to advance the SARS-CoV-2-RBD nanoparticle vaccine into the clinic.

A Thermostable mRNA Vaccine against COVID-19.

There is an urgent need for vaccines against coronavirus disease 2019 (COVID-19) because of the ongoing SARS-CoV-2 pandemic. Among all approaches, a messenger RNA (mRNA)-based vaccine has emerged as a rapid and versatile platform to quickly respond to this challenge. Here, we developed a lipid nanoparticle-encapsulated mRNA (mRNA-LNP) encoding the receptor binding domain (RBD) of SARS-CoV-2 as a vaccine candidate (called ARCoV). Intramuscular immunization of ARCoV mRNA-LNP elicited robust neutralizing antibodies against SARS-CoV-2 as well as a Th1-biased cellular response in mice and non-human primates. Two doses of ARCoV immunization in mice conferred complete protection against the challenge of a SARS-CoV-2 mouse-adapted strain. Additionally, ARCoV is manufactured as a liquid formulation and can be stored at room temperature for at least 1 week. ARCoV is currently being evaluated in phase 1 clinical trials.

Mammalian Near-Infrared Image Vision through Injectable and Self-Powered Retinal Nanoantennae.

Mammals cannot see light over 700 nm in wavelength. This limitation is due to the physical thermodynamic properties of the photon-detecting opsins. However, the detection of naturally invisible near-infrared (NIR) light is a desirable ability. To break this limitation, we developed ocular injectable photoreceptor-binding upconversion nanoparticles (pbUCNPs). These nanoparticles anchored on retinal photoreceptors as miniature NIR light transducers to create NIR light image vision with negligible side effects. Based on single-photoreceptor recordings, electroretinograms, cortical recordings, and visual behavioral tests, we demonstrated that mice with these nanoantennae could not only perceive NIR light, but also see NIR light patterns. Excitingly, the injected mice were also able to differentiate sophisticated NIR shape patterns. Moreover, the NIR light pattern vision was ambient-daylight compatible and existed in parallel with native daylight vision. This new method will provide unmatched opportunities for a wide variety of emerging bio-integrated nanodevice designs and applications. VIDEO ABSTRACT.

Multivalent antigen display on nanoparticle immunogens increases B cell clonotype diversity and neutralization breadth to pneumoviruses.

Nanoparticles for multivalent display and delivery of vaccine antigens have emerged as a promising avenue for enhancing B cell responses to protein subunit vaccines. Here, we evaluated B cell responses in rhesus macaques immunized with prefusion-stabilized respiratory syncytial virus (RSV) F glycoprotein trimer compared with nanoparticles displaying 10 or 20 copies of the same antigen. We show that multivalent display skews antibody specificities and drives epitope-focusing of responding B cells. Antibody cloning and repertoire sequencing revealed that focusing was driven by the expansion of clonally distinct B cells through recruitment of diverse precursors. We identified two antibody lineages that developed either ultrapotent neutralization or pneumovirus cross-neutralization from precursor B cells with low initial affinity for the RSV-F immunogen. This suggests that increased avidity by multivalent display facilitates the activation and recruitment of these cells. Diversification of the B cell response by multivalent nanoparticle immunogens has broad implications for vaccine design.

Heavy-chain CDR3-engineered B cells facilitate in vivo evaluation of HIV-1 vaccine candidates.

V2-glycan/apex broadly neutralizing antibodies (bnAbs) recognize a closed quaternary epitope of the HIV-1 envelope glycoprotein (Env). This closed structure is necessary to elicit apex antibodies and useful to guide the maturation of other bnAb classes. To compare antigens designed to maintain this conformation, we evaluated apex-specific responses in mice engrafted with a diverse repertoire of B cells expressing the HCDR3 of the apex bnAb VRC26.25. Engineered B cells affinity matured, guiding the improvement of VRC26.25 itself. We found that soluble Env (SOSIP) variants differed significantly in their ability to raise anti-apex responses. A transmembrane SOSIP (SOSIP-TM) delivered as an mRNA-lipid nanoparticle elicited more potent neutralizing responses than multimerized SOSIP proteins. Importantly, SOSIP-TM elicited neutralizing sera from B cells engineered with the predicted VRC26.25-HCDR3 progenitor, which also affinity matured. Our data show that HCDR3-edited B cells facilitate efficient in vivo comparisons of Env antigens and highlight the potential of an HCDR3-focused vaccine approach.

Co-immunization with hemagglutinin stem immunogens elicits cross-group neutralizing antibodies and broad protection against influenza A viruses.

Current influenza vaccines predominantly induce immunity to the hypervariable hemagglutinin (HA) head, requiring frequent vaccine reformulation. Conversely, the immunosubdominant yet conserved HA stem harbors a supersite that is targeted by broadly neutralizing antibodies (bnAbs), representing a prime target for universal vaccines. Here, we showed that the co-immunization of two HA stem immunogens derived from group 1 and 2 influenza A viruses elicits cross-group protective immunity and neutralizing antibody responses in mice, ferrets, and nonhuman primates (NHPs). Immunized mice were protected from multiple group 1 and 2 viruses, and all animal models showed broad serum-neutralizing activity. A bnAb isolated from an immunized NHP broadly neutralized and protected against diverse viruses, including H5N1 and H7N9. Genetic and structural analyses revealed strong homology between macaque and human bnAbs, illustrating common biophysical constraints for acquiring cross-group specificity. Vaccine elicitation of stem-directed cross-group-protective immunity represents a step toward the development of broadly protective influenza vaccines.

Neutralizing monoclonal antibodies elicited by mosaic RBD nanoparticles bind conserved sarbecovirus epitopes.

Increased immune evasion by SARS-CoV-2 variants of concern highlights the need for new therapeutic neutralizing antibodies. Immunization with nanoparticles co-displaying spike receptor-binding domains (RBDs) from eight sarbecoviruses (mosaic-8 RBD-nanoparticles) efficiently elicits cross-reactive polyclonal antibodies against conserved sarbecovirus RBD epitopes. Here, we identified monoclonal antibodies (mAbs) capable of cross-reactive binding and neutralization of animal sarbecoviruses and SARS-CoV-2 variants by screening single mouse B cells secreting IgGs that bind two or more sarbecovirus RBDs. Single-particle cryo-EM structures of antibody-spike complexes, including a Fab-Omicron complex, mapped neutralizing mAbs to conserved class 1/4 RBD epitopes. Structural analyses revealed neutralization mechanisms, potentials for intra-spike trimer cross-linking by IgGs, and induced changes in trimer upon Fab binding. In addition, we identified a mAb-resembling Bebtelovimab, an EUA-approved human class 3 anti-RBD mAb. These results support using mosaic RBD-nanoparticle vaccination to generate and identify therapeutic pan-sarbecovirus and pan-variant mAbs.

Innate immune mechanisms of mRNA vaccines.

The lipid nanoparticle (LNP)-encapsulated, nucleoside-modified mRNA platform has been used to generate safe and effective vaccines in record time against COVID-19. Here, we review the current understanding of the manner whereby mRNA vaccines induce innate immune activation and how this contributes to protective immunity. We discuss innate immune sensing of mRNA vaccines at the cellular and intracellular levels and consider the contribution of both the mRNA and the LNP components to their immunogenicity. A key message that is emerging from recent observations is that the LNP carrier acts as a powerful adjuvant for this novel vaccine platform. In this context, we highlight important gaps in understanding and discuss how new insight into the mechanisms underlying the effectiveness of mRNA-LNP vaccines may enable tailoring mRNA and carrier molecules to develop vaccines with greater effectiveness and milder adverse events in the future.

Lipid nanoparticles enhance the efficacy of mRNA and protein subunit vaccines by inducing robust T follicular helper cell and humoral responses.

Adjuvants are critical for improving the quality and magnitude of adaptive immune responses to vaccination. Lipid nanoparticle (LNP)-encapsulated nucleoside-modified mRNA vaccines have shown great efficacy against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but the mechanism of action of this vaccine platform is not well-characterized. Using influenza virus and SARS-CoV-2 mRNA and protein subunit vaccines, we demonstrated that our LNP formulation has intrinsic adjuvant activity that promotes induction of strong T follicular helper cell, germinal center B cell, long-lived plasma cell, and memory B cell responses that are associated with durable and protective antibodies in mice. Comparative experiments demonstrated that this LNP formulation outperformed a widely used MF59-like adjuvant, AddaVax. The adjuvant activity of the LNP relies on the ionizable lipid component and on IL-6 cytokine induction but not on MyD88- or MAVS-dependent sensing of LNPs. Our study identified LNPs as a versatile adjuvant that enhances the efficacy of traditional and next-generation vaccine platforms.

Nanoparticle Vaccines Based on the Receptor Binding Domain (RBD) and Heptad Repeat (HR) of SARS-CoV-2 Elicit Robust Protective Immune Responses.

Various vaccine strategies have been proposed in response to the global COVID-19 pandemic, each with unique strategies for eliciting immune responses. Here, we developed nanoparticle vaccines by covalently conjugating the self-assembled 24-mer ferritin to the receptor binding domain (RBD) and/or heptad repeat (HR) subunits of the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) spike (S) protein. Compared to monomer vaccines, nanoparticle vaccines elicited more robust neutralizing antibodies and cellular immune responses. RBD and RBD-HR nanoparticle vaccinated hACE2 transgenic mice vaccinated with RBD and/or RBD-HR nanoparticles exhibited reduced viral load in the lungs after SARS-CoV-2 challenge. RBD-HR nanoparticle vaccines also promoted neutralizing antibodies and cellular immune responses against other coronaviruses. The nanoparticle vaccination of rhesus macaques induced neutralizing antibodies, and T and B cell responses prior to boost immunization; these responses persisted for more than three months. RBD- and HR-based nanoparticles thus present a promising vaccination approach against SARS-CoV-2 and other coronaviruses.

Multifaceted Effects of Antigen Valency on B Cell Response Composition and Differentiation In Vivo.

How antigen valency affects B cells in vivo during immune responses is not well understood. Here, using HIV immunogens with defined valencies ranging from 1 to 60, we investigated the role of antigen valency during different phases of B cell responses in vivo. Highly multimerized immunogens preferentially rapidly activated cognate B cells, with little affinity discrimination. This led to strong early induction of the transcription factors IRF4 (interferon regulatory factor 4) and Bcl6, driving both early extrafollicular plasma cell and germinal center responses, in a CD4+ T-cell-dependent manner, involving B cells with a broad range of affinities. Low-valency antigens induced smaller effector B cell responses, with preferential recruitment of high-affinity B cells. Thus, antigen valency has multifaceted effects on B cell responses and can dictate affinity thresholds and competitive landscapes for B cells in vivo, with implications for vaccine design.

Pre-twisting for improved genome modification and miRNA targeting.

Two reports by Dhuri et al. and Oyaghire et al., respectively, show that, through installing chiral centers at the backbone of the artificial nucleic acid, peptide nucleic acid (PNA), enhanced miRNA targeting and genome modification can be achieved, with important implications in fighting cancers and ß-thalassemia.

Immunization with Components of the Viral Fusion Apparatus Elicits Antibodies That Neutralize Epstein-Barr Virus in B Cells and Epithelial Cells.

Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with epithelial-cell cancers and B cell lymphomas. An effective EBV vaccine is not available. We found that antibodies to the EBV glycoprotein gH/gL complex were the principal components in human plasma that neutralized infection of epithelial cells and that antibodies to gH/gL and gp42 contributed to B cell neutralization. Immunization of mice and nonhuman primates with nanoparticle vaccines that displayed components of the viral-fusion machinery EBV gH/gL or gH/gL/gp42 elicited antibodies that potently neutralized both epithelial-cell and B cell infection. Immune serum from nonhuman primates inhibited EBV-glycoprotein-mediated fusion of epithelial cells and B cells and targeted an epitope critical for virus-cell fusion. Therefore, unlike the leading EBV gp350 vaccine candidate, which only protects B cells from infection, these EBV nanoparticle vaccines elicit antibodies that inhibit the virus-fusion apparatus and provide cell-type-independent protection from virus infection.

Enhancing plant biotechnology by nanoparticle delivery of nucleic acids.

Plant biotechnology plays a crucial role in developing modern agriculture and plant science research. However, the delivery of exogenous genetic material into plants has been a long-standing obstacle. Nanoparticle-based delivery systems are being established to address this limitation and are proving to be a feasible, versatile, and efficient approach to facilitate the internalization of functional RNA and DNA by plants. The nanoparticle-based delivery systems can also be designed for subcellular delivery and controlled release of the biomolecular cargo. In this review, we provide a concise overview of the recent advances in nanocarriers for the delivery of biomolecules into plants, with a specific focus on applications to enhance RNA interference, foreign gene transfer, and genome editing in plants.

B Cells Are the Dominant Antigen-Presenting Cells that Activate Naive CD4+ T Cells upon Immunization with a Virus-Derived Nanoparticle Antigen.

B cells can present antigens to CD4+ T cells, but it is thought that dendritic cells (DCs) are the primary initiators of naive CD4+ T cell responses. Nanoparticles, including virus-like particles (VLPs), are attractive candidates as carriers for vaccines and drug delivery. Using RNA phage Qß-derived VLP (Qß-VLP) as a model antigen, we found that antigen-specific B cells were the dominant antigen-presenting cells that initiated naive CD4+ T cell activation. B cells were sufficient to induce T follicular helper cell development in the absence of DCs. Qß-specific B cells promoted CD4+ T cell proliferation and differentiation via cognate interactions and through Toll-like receptor signaling-mediated cytokine production. Antigen-specific B cells were also involved in initiating CD4+ T cell responses during immunization with inactivated influenza virus. These findings have implications for the rational design of nanoparticles as vaccine candidates, particularly for therapeutic vaccines that aim to break immune tolerance.

Cationic cholesterol-dependent LNP delivery to lung stem cells, the liver, and heart.

Adding a cationic helper lipid to a lipid nanoparticle (LNP) can increase lung delivery and decrease liver delivery. However, it remains unclear whether charge-dependent tropism is universal or, alternatively, whether it depends on the component that is charged. Here, we report evidence that cationic cholesterol-dependent tropism can differ from cationic helper lipid-dependent tropism. By testing how 196 LNPs delivered mRNA to 22 cell types, we found that charged cholesterols led to a different lung:liver delivery ratio than charged helper lipids. We also found that combining cationic cholesterol with a cationic helper lipid led to mRNA delivery in the heart as well as several lung cell types, including stem cell-like populations. These data highlight the utility of exploring charge-dependent LNP tropism.

Thiophene-based lipids for mRNA delivery to pulmonary and retinal tissues.

Lipid nanoparticles (LNPs) largely rely on ionizable lipids to yield successful nucleic acid delivery via electrostatic disruption of the endosomal membrane. Here, we report the identification and evaluation of ionizable lipids containing a thiophene moiety (Thio-lipids). The Thio-lipids can be readily synthesized via the Gewald reaction, allowing for modular lipid design with functional constituents at various positions of the thiophene ring. Through the rational design of ionizable lipid structure, we prepared 47 Thio-lipids and identified some structural criteria required in Thio-lipids for efficient mRNA (messenger RNA) encapsulation and delivery in vitro and in vivo. Notably, none of the tested lipids have a pH-response profile like traditional ionizable lipids, potentially due to the electron delocalization in the thiophene core. Placement of the tails and localization of the ionizable headgroup in the thiophene core can endow the nanoparticles with the capability to reach various tissues. Using high-throughput formulation and barcoding techniques, we optimized the formulations to select two top lipids-20b and 29d-and investigated their biodistribution in mice. Lipid 20b enabled LNPs to transfect the liver and spleen, and 29d LNP transfected the lung and spleen. Unexpectedly, LNP with lipid 20b was especially potent in mRNA delivery to the retina with no acute toxicity, leading to the successful delivery to the photoreceptors and retinal pigment epithelium in non-human primates.