Distinct sensitivities to SARS-CoV-2 variants in vaccinated humans and mice.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019 has led to the development of a large number of vaccines, several of which are now approved for use in humans. Understanding vaccine-elicited antibody responses against emerging SARS-CoV-2 variants of concern (VOCs) in real time is key to inform public health policies. Serum neutralizing antibody titers are the current best correlate of protection from SARS-CoV-2 challenge in non-human primates and a key metric to understand immune evasion of VOCs. We report that vaccinated BALB/c mice do not recapitulate faithfully the breadth and potency of neutralizing antibody responses elicited by various vaccine platforms against VOCs, compared with non-human primates or humans, suggesting caution should be exercised when interpreting data obtained with this animal model.

Multivalent designed proteins neutralize SARS-CoV-2 variants of concern and confer protection against infection in mice.

New variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to arise and prolong the coronavirus disease 2019 (COVID-19) pandemic. Here, we used a cell-free expression workflow to rapidly screen and optimize constructs containing multiple computationally designed miniprotein inhibitors of SARS-CoV-2. We found the broadest efficacy was achieved with a homotrimeric version of the 75-residue angiotensin-converting enzyme 2 (ACE2) mimic AHB2 (TRI2-2) designed to geometrically match the trimeric spike architecture. Consistent with the design model, in the cryo-electron microscopy structure TRI2-2 forms a tripod at the apex of the spike protein that engaged all three receptor binding domains simultaneously. TRI2-2 neutralized Omicron (B.1.1.529), Delta (B.1.617.2), and all other variants tested with greater potency than the monoclonal antibodies used clinically for the treatment of COVID-19. TRI2-2 also conferred prophylactic and therapeutic protection against SARS-CoV-2 challenge when administered intranasally in mice. Designed miniprotein receptor mimics geometrically arrayed to match pathogen receptor binding sites could be a widely applicable antiviral therapeutic strategy with advantages over antibodies in greater resistance to viral escape and antigenic drift, and advantages over native receptor traps in lower chances of autoimmune responses.

SARS-CoV-2 breakthrough infections elicit potent, broad, and durable neutralizing antibody responses.

Although infections among vaccinated individuals lead to milder COVID-19 symptoms relative to those in unvaccinated subjects, the specificity and durability of antibody responses elicited by breakthrough cases remain unknown. Here, we demonstrate that breakthrough infections induce serum-binding and -neutralizing antibody responses that are markedly more potent, durable, and resilient to spike mutations observed in variants than those in subjects who received only 2 doses of vaccine. However, we show that breakthrough cases, subjects who were vaccinated after infection, and individuals vaccinated three times have serum-neutralizing activity of comparable magnitude and breadth, indicating that an increased number of exposures to SARS-CoV-2 antigen(s) enhance the quality of antibody responses. Neutralization of SARS-CoV was moderate, however, underscoring the importance of developing vaccines eliciting broad sarbecovirus immunity for pandemic preparedness.

Delta breakthrough infections elicit potent, broad and durable neutralizing antibody responses.

The SARS-CoV-2 Delta variant is currently responsible for most infections worldwide, including among fully vaccinated individuals. Although these latter infections are associated with milder COVID-19 disease relative to unvaccinated subjects, the specificity and durability of antibody responses elicited by Delta breakthrough cases remain unknown. Here, we demonstrate that breakthrough infections induce serum binding and neutralizing antibody responses that are markedly more potent, durable and resilient to spike mutations observed in variants of concern than those observed in subjects who were infected only or received only two doses of COVID-19 vaccine. However, wee show that Delta breakthrough cases, subjects who were vaccinated after SARS-CoV-2 infection and individuals vaccinated three times (without infection) have serum neutralizing activity of comparable magnitude and breadth indicate that multiple types of exposure or increased number of exposures to SARS-CoV-2 antigen(s) enhance spike-specific antibody responses. Neutralization of the genetically divergent SARS-CoV, however, was moderate with all four cohorts examined, except after four exposures to the SARS-CoV-2 spike, underscoring the importance of developing vaccines eliciting broad sarbecovirus immunity for pandemic preparedness.

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.

Stabilization of the SARS-CoV-2 Spike Receptor-Binding Domain Using Deep Mutational Scanning and Structure-Based Design.

The unprecedented global demand for SARS-CoV-2 vaccines has demonstrated the need for highly effective vaccine candidates that are thermostable and amenable to large-scale manufacturing. Nanoparticle immunogens presenting the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein (S) in repetitive arrays are being advanced as second-generation vaccine candidates, as they feature robust manufacturing characteristics and have shown promising immunogenicity in preclinical models. Here, we used previously reported deep mutational scanning (DMS) data to guide the design of stabilized variants of the RBD. The selected mutations fill a cavity in the RBD that has been identified as a linoleic acid binding pocket. Screening of several designs led to the selection of two lead candidates that expressed at higher yields than the wild-type RBD. These stabilized RBDs possess enhanced thermal stability and resistance to aggregation, particularly when incorporated into an icosahedral nanoparticle immunogen that maintained its integrity and antigenicity for 28 days at 35-40°C, while corresponding immunogens displaying the wild-type RBD experienced aggregation and loss of antigenicity. The stabilized immunogens preserved the potent immunogenicity of the original nanoparticle immunogen, which is currently being evaluated in a Phase I/II clinical trial. Our findings may improve the scalability and stability of RBD-based coronavirus vaccines in any format and more generally highlight the utility of comprehensive DMS data in guiding vaccine design.

SARS-CoV-2 immune evasion by the B.1.427/B.1.429 variant of concern.

A novel variant of concern (VOC) named CAL.20C (B.1.427/B.1.429), which was originally detected in California, carries spike glycoprotein mutations S13I in the signal peptide, W152C in the N-terminal domain (NTD), and L452R in the receptor-binding domain (RBD). Plasma from individuals vaccinated with a Wuhan-1 isolate-based messenger RNA vaccine or from convalescent individuals exhibited neutralizing titers that were reduced 2- to 3.5-fold against the B.1.427/B.1.429 variant relative to wild-type pseudoviruses. The L452R mutation reduced neutralizing activity in 14 of 34 RBD-specific monoclonal antibodies (mAbs). The S13I and W152C mutations resulted in total loss of neutralization for 10 of 10 NTD-specific mAbs because the NTD antigenic supersite was remodeled by a shift of the signal peptide cleavage site and the formation of a new disulfide bond, as revealed by mass spectrometry and structural studies.

SARS-CoV-2 immune evasion by variant B.1.427/B.1.429.

SARS-CoV-2 entry is mediated by the spike (S) glycoprotein which contains the receptor-binding domain (RBD) and the N-terminal domain (NTD) as the two main targets of neutralizing antibodies (Abs). A novel variant of concern (VOC) named CAL.20C (B.1.427/B.1.429) was originally detected in California and is currently spreading throughout the US and 29 additional countries. It is unclear whether antibody responses to SARS-CoV-2 infection or to the prototypic Wuhan-1 isolate-based vaccines will be impacted by the three B.1.427/B.1.429 S mutations: S13I, W152C and L452R. Here, we assessed neutralizing Ab responses following natural infection or mRNA vaccination using pseudoviruses expressing the wildtype or the B.1.427/B.1.429 S protein. Plasma from vaccinated or convalescent individuals exhibited neutralizing titers, which were reduced 3-6 fold against the B.1.427/B.1.429 variant relative to wildtype pseudoviruses. The RBD L452R mutation reduced or abolished neutralizing activity of 14 out of 35 RBD-specific monoclonal antibodies (mAbs), including three clinical-stage mAbs. Furthermore, we observed a complete loss of B.1.427/B.1.429 neutralization for a panel of mAbs targeting the N-terminal domain due to a large structural rearrangement of the NTD antigenic supersite involving an S13I-mediated shift of the signal peptide cleavage site. These data warrant closer monitoring of signal peptide variants and their involvement in immune evasion and show that Abs directed to the NTD impose a selection pressure driving SARS-CoV-2 viral evolution through conventional and unconventional escape mechanisms.

Adjuvanting a subunit COVID-19 vaccine to induce protective immunity.

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).

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

Understanding the ability of SARS-CoV-2 vaccine-elicited antibodies to neutralize and protect against emerging variants of concern and other sarbecoviruses is key for guiding vaccine development decisions and public health policies. We show that a clinical stage multivalent SARS-CoV-2 receptor-binding domain nanoparticle vaccine (SARS-CoV-2 RBD-NP) protects mice from SARS-CoV-2-induced disease after a single shot, indicating that the vaccine could allow dose-sparing. SARS-CoV-2 RBD-NP elicits high antibody titers in two non-human primate (NHP) models against multiple distinct RBD antigenic sites known to be recognized by neutralizing antibodies. We benchmarked NHP serum neutralizing activity elicited by RBD-NP against a lead prefusion-stabilized SARS-CoV-2 spike immunogen using a panel of single-residue spike mutants detected in clinical isolates as well as the B.1.1.7 and B.1.351 variants of concern. Polyclonal antibodies elicited by both vaccines are resilient to most RBD mutations tested, but the E484K substitution has similar negative consequences for neutralization, and exhibit modest but comparable neutralization breadth against distantly related sarbecoviruses. We demonstrate that mosaic and cocktail sarbecovirus RBD-NPs elicit broad sarbecovirus neutralizing activity, including against the SARS-CoV-2 B.1.351 variant, and protect mice against severe SARS-CoV challenge even in the absence of the SARS-CoV RBD in the vaccine. This study provides proof of principle that sarbecovirus RBD-NPs induce heterotypic protection and enables advancement of broadly protective sarbecovirus vaccines to the clinic.

Adjuvanting a subunit SARS-CoV-2 nanoparticle vaccine to induce protective immunity in non-human primates.

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.

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.

Elicitation of potent neutralizing antibody responses by designed protein nanoparticle vaccines for SARS-CoV-2.

A safe, effective, and scalable vaccine is urgently needed to halt the ongoing SARS-CoV-2 pandemic. Here, 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 copies of the SARS-CoV-2 spike (S) glycoprotein receptor-binding domain (RBD) in a highly immunogenic array and induce neutralizing antibody titers roughly ten-fold higher than the prefusion-stabilized S ectodomain trimer despite a more than five-fold lower dose. Antibodies elicited by the nanoparticle immunogens target multiple distinct epitopes on the RBD, suggesting that they may not be easily susceptible to escape mutations, and exhibit a significantly 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 protein components and assembled nanoparticles, especially compared to the SARS-CoV-2 prefusion-stabilized S trimer, suggest that manufacture of the nanoparticle vaccines will be highly scalable. These results highlight the utility of robust antigen display platforms for inducing potent neutralizing antibody responses and have launched cGMP manufacturing efforts to advance the lead RBD nanoparticle vaccine into the clinic.

Deep Mutational Scanning of SARS-CoV-2 Receptor Binding Domain Reveals Constraints on Folding and ACE2 Binding.

The receptor binding domain (RBD) of the SARS-CoV-2 spike glycoprotein mediates viral attachment to ACE2 receptor and is a major determinant of host range and a dominant target of neutralizing antibodies. Here, we experimentally measure how all amino acid mutations to the RBD affect expression of folded protein and its affinity for ACE2. Most mutations are deleterious for RBD expression and ACE2 binding, and we identify constrained regions on the RBD's surface that may be desirable targets for vaccines and antibody-based therapeutics. But a substantial number of mutations are well tolerated or even enhance ACE2 binding, including at ACE2 interface residues that vary across SARS-related coronaviruses. However, we find no evidence that these ACE2-affinity-enhancing mutations have been selected in current SARS-CoV-2 pandemic isolates. We present an interactive visualization and open analysis pipeline to facilitate use of our dataset for vaccine design and functional annotation of mutations observed during viral surveillance.

Deep mutational scanning of SARS-CoV-2 receptor binding domain reveals constraints on folding and ACE2 binding.

The receptor binding domain (RBD) of the SARS-CoV-2 spike glycoprotein mediates viral attachment to ACE2 receptor, and is a major determinant of host range and a dominant target of neutralizing antibodies. Here we experimentally measure how all amino-acid mutations to the RBD affect expression of folded protein and its affinity for ACE2. Most mutations are deleterious for RBD expression and ACE2 binding, and we identify constrained regions on the RBD's surface that may be desirable targets for vaccines and antibody-based therapeutics. But a substantial number of mutations are well tolerated or even enhance ACE2 binding, including at ACE2 interface residues that vary across SARS-related coronaviruses. However, we find no evidence that these ACE2-affinity enhancing mutations have been selected in current SARS-CoV-2 pandemic isolates. We present an interactive visualization and open analysis pipeline to facilitate use of our dataset for vaccine design and functional annotation of mutations observed during viral surveillance.

PfCap380 as a marker for Plasmodium falciparum oocyst development in vivo and in vitro.

BACKGROUND: Despite the importance of the Plasmodium berghei oocyst capsule protein (PbCap380) in parasite survival, very little is known about the orthologous Plasmodium falciparum capsule protein (PfCap380). The goal of this work was to study the growth of P. falciparum oocysts using PfCap380 as a developmental marker. METHODS: To study P. falciparum oocyst development using both in vivo (mosquito-derived) and in vitro (culture-derived) growth conditions, antibodies (polyclonal antisera) were raised against PfCap380. For studies on in vivo oocysts, mature P. falciparum gametocytes were fed to Anopheles stephensi mosquitoes. For studies on in vitro parasites, P. falciparum gametocytes were induced and matured for subsequent ookinete production. Ookinetes were purified and then tested for binding affinity to basal lamina components and transformation into early oocysts, which were grown on reconstituted basal lamia coated wells with novel oocyst media. To monitor in vivo oocyst development, immunofluorescence assays (IFA) were performed using anti-PfCap380 antisera on Pf-infected mosquito midguts. IFA were also performed on culture-derived oocysts to follow in vitro oocyst development. RESULTS: The anti-PfCap380 antisera allowed detection of early midgut oocysts starting at 2 days after gametocyte infection, while circumsporozoite protein was definitively observed on day 6. For in vitro culture, significant transformation of gametocytes to ookinetes (24%) and of ookinetes to early oocysts (85%) was observed. After screening several basal lamina components, collagen IV provided greatest binding of ookinetes and transformation into early oocysts. Finally, PfCap380 expression was observed on the surface of culture-derived oocysts but not on gametocytes or ookinetes. CONCLUSIONS: This study presents developmental monitoring of P. falciparum oocysts produced in vivo and in vitro. The anti-PfCap380 antisera serves as an important reagent for developmental studies of oocysts from the mosquito midgut and also from oocyst culture using in vitro methodology. The present data demonstrate that PfCap380 is a useful marker to follow the development and maturation of in vivo and in vitro produced oocysts as early as 2 days after zygote formation. Further in vitro studies focused on oocyst and sporozoite maturation will support the manufacturing of whole sporozoites for malaria vaccines.

Assessing drug efficacy against Plasmodium falciparum liver stages in vivo.

Malaria eradication necessitates new tools to fight the evolving and complex Plasmodium pathogens. These tools include prophylactic drugs that eliminate Plasmodium liver stages and consequently prevent clinical disease, decrease transmission, and reduce the propensity for resistance development. Currently, the identification of these drugs relies on in vitro P. falciparum liver stage assays or in vivo causal prophylaxis assays using rodent malaria parasites; there is no method to directly test in vivo liver stage activity of candidate antimalarials against the human malaria-causing parasite P. falciparum. Here, we use a liver-chimeric humanized mouse (FRG huHep) to demonstrate in vivo P. falciparum liver stage development and describe the efficacy of clinically used and candidate antimalarials with prophylactic activity. We show that daily administration of atovaquone-proguanil (ATQ-PG; ATQ, 30 mg/kg, and PG, 10 mg/kg) protects 5 of 5 mice from liver stage infection, consistent with the use in humans as a causal prophylactic drug. Single-dose primaquine (60 mg/kg) has similar activity to that observed in humans, demonstrating the activity of this drug (and its active metabolites) in FRG huHep mice. We also show that DSM265, a selective Plasmodial dihydroorotate dehydrogenase inhibitor with causal prophylactic activity in humans, reduces liver stage burden in FRG huHep mice. Finally, we measured liver stage-to-blood stage transition of the parasite, the ultimate readout of prophylactic activity and measurement of infective capacity of parasites in the liver, to show that ATQ-PG reduces blood stage patency to below the limit of quantitation by quantitative PCR (qPCR). The FRG huHep model, thus, provides a platform for preclinical evaluation of drug candidates for liver stage causal prophylactic activity, pharmacokinetic/pharmacodynamics studies, and biological studies to investigate the mechanism of action of liver stage active antimalarials.

An Opsonic Phagocytosis Assay for Plasmodium falciparum Sporozoites.

Plasmodium falciparum malaria remains the deadliest parasitic disease worldwide. Vaccines targeting the preerythrocytic sporozoite and liver stages have the potential to entirely prevent blood-stage infection and disease, as well as onward transmission. Sporozoite surface and secreted proteins are leading candidates for inclusion in a preerythrocytic stage-specific, antibody-based vaccine. Preclinical functional assays to identify humoral correlates of protection in vitro and to validate novel sporozoite protein targets for inclusion in multisubunit vaccines currently do not consider the interaction of sporozoite-targeting antibodies with other components of the immune system. Here, we describe the development of a simple flow cytometric assay to quantitatively assess the ability of antibodies directed against P. falciparum sporozoites to facilitate their phagocytosis. We demonstrate that this sporozoite opsonic phagocytosis assay (SOPA) is compatible with both monoclonal antibodies and human immune serum and can be performed using cryopreserved P. falciparum sporozoites. This simple, accessible assay will aid with the assessment of antibody responses to vaccination with Plasmodium antigens and their interaction with phagocytic cells of the immune system.