Pyrimidine inhibitors synergize with nucleoside analogues to block SARS-CoV-2.

The SARS-CoV-2 virus has infected more than 261 million people and has led to more than 5 million deaths in the past year and a half1 ( https://www.who.org/ ). Individuals with SARS-CoV-2 infection typically develop mild-to-severe flu-like symptoms, whereas infection of a subset of individuals leads to severe-to-fatal clinical outcomes2. Although vaccines have been rapidly developed to combat SARS-CoV-2, there has been a dearth of antiviral therapeutics. There is an urgent need for therapeutics, which has been amplified by the emerging threats of variants that may evade vaccines. Large-scale efforts are underway to identify antiviral drugs. Here we screened approximately 18,000 drugs for antiviral activity using live virus infection in human respiratory cells and validated 122 drugs with antiviral activity and selectivity against SARS-CoV-2. Among these candidates are 16 nucleoside analogues, the largest category of clinically used antivirals. This included the antivirals remdesivir and molnupiravir, which have been approved for use in COVID-19. RNA viruses rely on a high supply of nucleoside triphosphates from the host to efficiently replicate, and we identified a panel of host nucleoside biosynthesis inhibitors as antiviral. Moreover, we found that combining pyrimidine biosynthesis inhibitors with antiviral nucleoside analogues synergistically inhibits SARS-CoV-2 infection in vitro and in vivo against emerging strains of SARS-CoV-2, suggesting a clinical path forward.

Diet-induced obesity and diabetes enhance mortality and reduce vaccine efficacy for SARS-CoV-2.

IMPORTANCE: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused a wide spectrum of diseases in the human population, from asymptomatic infections to death. It is important to study the host differences that may alter the pathogenesis of this virus. One clinical finding in coronavirus disease 2019 (COVID-19) patients is that people with obesity or diabetes are at increased risk of severe illness from SARS-CoV-2 infection. We used a high-fat diet model in mice to study the effects of obesity and type 2 diabetes on SARS-CoV-2 infection as well as how these comorbidities alter the response to vaccination. We find that diabetic/obese mice have increased disease after SARS-CoV-2 infection and they have slower clearance of the virus. We find that the lungs of these mice have increased neutrophils and that removing these neutrophils protects diabetic/obese mice from disease. This demonstrates why these diseases have increased risk of severe disease and suggests specific interventions upon infection.

Target-agnostic drug prediction integrated with medical record analysis uncovers differential associations of statins with increased survival in COVID-19 patients.

Drug repurposing requires distinguishing established drug class targets from novel molecule-specific mechanisms and rapidly derisking their therapeutic potential in a time-critical manner, particularly in a pandemic scenario. In response to the challenge to rapidly identify treatment options for COVID-19, several studies reported that statins, as a drug class, reduce mortality in these patients. However, it is unknown if different statins exhibit consistent function or may have varying therapeutic benefit. A Bayesian network tool was used to predict drugs that shift the host transcriptomic response to SARS-CoV-2 infection towards a healthy state. Drugs were predicted using 14 RNA-sequencing datasets from 72 autopsy tissues and 465 COVID-19 patient samples or from cultured human cells and organoids infected with SARS-CoV-2. Top drug predictions included statins, which were then assessed using electronic medical records containing over 4,000 COVID-19 patients on statins to determine mortality risk in patients prescribed specific statins versus untreated matched controls. The same drugs were tested in Vero E6 cells infected with SARS-CoV-2 and human endothelial cells infected with a related OC43 coronavirus. Simvastatin was among the most highly predicted compounds (14/14 datasets) and five other statins, including atorvastatin, were predicted to be active in > 50% of analyses. Analysis of the clinical database revealed that reduced mortality risk was only observed in COVID-19 patients prescribed a subset of statins, including simvastatin and atorvastatin. In vitro testing of SARS-CoV-2 infected cells revealed simvastatin to be a potent direct inhibitor whereas most other statins were less effective. Simvastatin also inhibited OC43 infection and reduced cytokine production in endothelial cells. Statins may differ in their ability to sustain the lives of COVID-19 patients despite having a shared drug target and lipid-modifying mechanism of action. These findings highlight the value of target-agnostic drug prediction coupled with patient databases to identify and clinically evaluate non-obvious mechanisms and derisk and accelerate drug repurposing opportunities.

p47 licenses activation of the immune deficiency pathway in the tick Ixodes scapularis.

The E3 ubiquitin ligase X-linked inhibitor of apoptosis (XIAP) acts as a molecular rheostat for the immune deficiency (IMD) pathway of the tick Ixodes scapularis How XIAP activates the IMD pathway in response to microbial infection remains ill defined. Here, we identified the XIAP enzymatic substrate p47 as a positive regulator of the I. scapularis IMD network. XIAP polyubiquitylates p47 in a lysine 63-dependent manner and interacts with the p47 ubiquitin-like (UBX) module. p47 also binds to Kenny (IKKγ/NEMO), the regulatory subunit of the inhibitor of nuclear factor (NF)- κB kinase complex. Replacement of the amino acid lysine to arginine within the p47 linker region completely abrogated molecular interactions with Kenny. Furthermore, mitigation of p47 transcription levels through RNA interference in I. scapularis limited Kenny accumulation, reduced phosphorylation of IKKß (IRD5), and impaired cleavage of the NF-κB molecule Relish. Accordingly, disruption of p47 expression increased microbial colonization by the Lyme disease spirochete Borrelia burgdorferi and the rickettsial agent Anaplasma phagocytophilum Collectively, we highlight the importance of ticks for the elucidation of paradigms in arthropod immunology. Manipulating immune signaling cascades within I. scapularis may lead to innovative approaches to reducing the burden of tick-borne diseases.

The Prostaglandin E2-EP3 Receptor Axis Regulates Anaplasma phagocytophilum-Mediated NLRC4 Inflammasome Activation.

Rickettsial agents are sensed by pattern recognition receptors but lack pathogen-associated molecular patterns commonly observed in facultative intracellular bacteria. Due to these molecular features, the order Rickettsiales can be used to uncover broader principles of bacterial immunity. Here, we used the bacterium Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis, to reveal a novel microbial surveillance system. Mechanistically, we discovered that upon A. phagocytophilum infection, cytosolic phospholipase A2 cleaves arachidonic acid from phospholipids, which is converted to the eicosanoid prostaglandin E2 (PGE2) via cyclooxygenase 2 (COX2) and the membrane associated prostaglandin E synthase-1 (mPGES-1). PGE2-EP3 receptor signaling leads to activation of the NLRC4 inflammasome and secretion of interleukin (IL)-1ß and IL-18. Importantly, the receptor-interacting serine/threonine-protein kinase 2 (RIPK2) was identified as a major regulator of the immune response against A. phagocytophilum. Accordingly, mice lacking COX2 were more susceptible to A. phagocytophilum, had a defect in IL-18 secretion and exhibited splenomegaly and damage to the splenic architecture. Remarkably, Salmonella-induced NLRC4 inflammasome activation was not affected by either chemical inhibition or genetic ablation of genes associated with PGE2 biosynthesis and signaling. This divergence in immune circuitry was due to reduced levels of the PGE2-EP3 receptor during Salmonella infection when compared to A. phagocytophilum. Collectively, we reveal the existence of a functionally distinct NLRC4 inflammasome illustrated by the rickettsial agent A. phagocytophilum.

Pyronaridine tetraphosphate is an efficacious antiviral and anti-inflammatory active against multiple highly pathogenic coronaviruses.

IMPORTANCE: Pyronaridine tetraphosphate is on the WHO Essential Medicine List for its importance as a widely available and safe treatment for malaria. We find that pyronaridine is a highly effective antiviral therapeutic across mouse models using multiple variants of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), and the highly pathogenic viruses SARS-CoV-1 and Middle East respiratory syndrome coronavirus responsible for previous coronavirus outbreaks. Additionally, we find that pyronaridine additively combines with current COVID-19 treatments such as nirmatrelvir (protease inhibitor in Paxlovid) and molnupiravir to further inhibit SARS-CoV-2 infections. There are many antiviral compounds that demonstrate efficacy in cellular models, but few that show this level of impact in multiple mouse models and represent a promising therapeutic for the current coronavirus pandemic as well as future outbreaks as well.

Diet Induced Obesity and Diabetes Enhance Mortality and Reduces Vaccine Efficacy for SARS-CoV-2.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), the causative agent of Coronavirus disease 2019 (COVID-19), emerged in Wuhan, China, in December 2019. As of October 2022, there have been over 625 million confirmed cases of COVID-19, including over 6.5 million deaths. Epidemiological studies have indicated that comorbidities of obesity and diabetes mellitus are associated with increased morbidity and mortality following SARS-CoV-2 infection. We determined how the comorbidities of obesity and diabetes affect morbidity and mortality following SARS-CoV-2 infection in unvaccinated and adjuvanted spike nanoparticle (NVX-CoV2373) vaccinated mice. We find that obese/diabetic mice infected with SARS-CoV-2 have increased morbidity and mortality compared to age matched normal mice. Mice fed a high-fat diet (HFD) then vaccinated with NVX-CoV2373 produce equivalent neutralizing antibody titers to those fed a normal diet (ND). However, the HFD mice have reduced viral clearance early in infection. Analysis of the inflammatory immune response in HFD mice demonstrates a recruitment of neutrophils that was correlated with increased mortality and reduced clearance of the virus. Depletion of neutrophils in diabetic/obese vaccinated mice reduced disease severity and protected mice from lethality. This model recapitulates the increased disease severity associated with obesity and diabetes in humans with COVID-19 and is an important comorbidity to study with increasing obesity and diabetes across the world.

Immunogenicity and protection of a variant nanoparticle vaccine that confers broad neutralization against SARS-CoV-2 variants.

SARS-CoV-2 variants have emerged with elevated transmission and a higher risk of infection for vaccinated individuals. We demonstrate that a recombinant prefusion-stabilized spike (rS) protein vaccine based on Beta/B.1.351 (rS-Beta) produces a robust anamnestic response in baboons against SARS-CoV-2 variants when given as a booster one year after immunization with NVX-CoV2373. Additionally, rS-Beta is highly immunogenic in mice and produces neutralizing antibodies against WA1/2020, Beta/B.1.351, and Omicron/BA.1. Mice vaccinated with two doses of Novavax prototype NVX-CoV2373 (rS-WU1) or rS-Beta alone, in combination, or heterologous prime-boost, are protected from challenge. Virus titer is undetectable in lungs in all vaccinated mice, and Th1-skewed cellular responses are observed. We tested sera from a panel of variant spike protein vaccines and find broad neutralization and inhibition of spike:ACE2 binding from the rS-Beta and rS-Delta vaccines against a variety of variants including Omicron. This study demonstrates that rS-Beta vaccine alone or in combination with rS-WU1 induces antibody-and cell-mediated responses that are protective against challenge with SARS-CoV-2 variants and offers broader neutralizing capacity than a rS-WU1 prime/boost regimen alone. Together, these nonhuman primate and murine data suggest a Beta variant booster dose could elicit a broad immune response to fight new and future SARS-CoV-2 variants.

Structural characterization and inhibition of the Plasmodium Atg8-Atg3 interaction.

The autophagy-related proteins are thought to serve multiple functions in Plasmodium and are considered essential to parasite survival and development. We have studied two key interacting proteins, Atg8 and Atg3, of the autophagy pathway in Plasmodium falciparum. These proteins are vital for the formation and elongation of the autophagosome and essential to the process of macroautophagy. Autophagy may be required for conversion of the sporozoite into erythrocytic-infective merozoites and may be crucial for other functions during asexual blood stages. Here we describe the identification of an Atg8 family interacting motif (AIM) in Plasmodium Atg3, which binds Plasmodium Atg8. We determined the co-crystal structure of PfAtg8 with a short Atg3¹°³â»¹¹° peptide, corresponding to this motif, to 2.2 Å resolution. Our in vitro interaction studies are in agreement with our X-ray crystal structure. Furthermore they suggest an important role for a unique Apicomplexan loop absent from human Atg8 homologues. Prevention of the protein-protein interaction of full length PfAtg8 with PfAtg3 was achieved at low micromolar concentrations with a small molecule, 1,2,3-trihydroxybenzene. Together our structural and interaction studies represent a starting point for future antimalarial drug discovery and design for this novel protein-protein interaction.

Phage-like particle vaccines are highly immunogenic and protect against pathogenic coronavirus infection and disease.

The response by vaccine developers to the COVID-19 pandemic has been extraordinary with effective vaccines authorized for emergency use in the United States within 1 year of the appearance of the first COVID-19 cases. However, the emergence of SARS-CoV-2 variants and obstacles with the global rollout of new vaccines highlight the need for platforms that are amenable to rapid tuning and stable formulation to facilitate the logistics of vaccine delivery worldwide. We developed a "designer nanoparticle" platform using phage-like particles (PLPs) derived from bacteriophage lambda for a multivalent display of antigens in rigorously defined ratios. Here, we engineered PLPs that display the receptor-binding domain (RBD) protein from SARS-CoV-2 and MERS-CoV, alone (RBDSARS-PLPs and RBDMERS-PLPs) and in combination (hCoV-RBD PLPs). Functionalized particles possess physiochemical properties compatible with pharmaceutical standards and retain antigenicity. Following primary immunization, BALB/c mice immunized with RBDSARS- or RBDMERS-PLPs display serum RBD-specific IgG endpoint and live virus neutralization titers that, in the case of SARS-CoV-2, were comparable to those detected in convalescent plasma from infected patients. Further, these antibody levels remain elevated up to 6 months post-prime. In dose-response studies, immunization with as little as one microgram of RBDSARS-PLPs elicited robust neutralizing antibody responses. Finally, animals immunized with RBDSARS-PLPs, RBDMERS-PLPs, and hCoV-RBD PLPs were protected against SARS-CoV-2 and/or MERS-CoV lung infection and disease. Collectively, these data suggest that the designer PLP system provides a platform for facile and rapid generation of single and multi-target vaccines.

Preclinical characterization of amubarvimab and romlusevimab, a pair of non-competing neutralizing monoclonal antibody cocktail, against SARS-CoV-2.

Monoclonal antibodies (mAbs) targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein have demonstrated clinical efficacy in preventing or treating coronavirus disease 2019 (COVID-19), resulting in the emergency use authorization (EUA) for several SARS-CoV-2 targeting mAb by regulatory authority. However, the continuous virus evolution requires diverse mAb options to combat variants. Here we describe two fully human mAbs, amubarvimab (BRII-196) and romlusevimab (BRII-198) that bind to non-competing epitopes on the receptor binding domain (RBD) of spike protein and effectively neutralize SARS-CoV-2 variants. A YTE modification was introduced to the fragment crystallizable (Fc) region of both mAbs to prolong serum half-life and reduce effector function. The amubarvimab and romlusevimab combination retained activity against most mutations associated with reduced susceptibility to previously authorized mAbs and against variants containing amino acid substitutions in their epitope regions. Consistently, the combination of amubarvimab and romlusevimab effectively neutralized a wide range of viruses including most variants of concern and interest in vitro. In a Syrian golden hamster model of SARS-CoV-2 infection, animals receiving combination of amubarvimab and romlusevimab either pre- or post-infection demonstrated less weight loss, significantly decreased viral load in the lungs, and reduced lung pathology compared to controls. These preclinical findings support their development as an antibody cocktail therapeutic option against COVID-19 in the clinic.

Target-agnostic drug prediction integrated with medical record analysis uncovers differential associations of statins with increased survival in COVID-19 patients.

Importance: Drug repurposing requires distinguishing established drug class targets from novel molecule-specific mechanisms and rapidly derisking their therapeutic potential in a time-critical manner, particularly in a pandemic scenario. In response to the challenge to rapidly identify treatment options for COVID-19, several studies reported that statins, as a drug class, reduce mortality in these patients. However, it is unknown if different statins exhibit consistent function or may have varying therapeutic benefit. Objectives: To test if different statins differ in their ability to exert protective effects based on molecular computational predictions and electronic medical record analysis. Main Outcomes and Measures: A Bayesian network tool was used to predict drugs that shift the host transcriptomic response to SARS-CoV-2 infection towards a healthy state. Drugs were predicted using 14 RNA-sequencing datasets from 72 autopsy tissues and 465 COVID-19 patient samples or from cultured human cells and organoids infected with SARS-CoV-2, with a total of 2,436 drugs investigated. Top drug predictions included statins, which were then assessed using electronic medical records containing over 4,000 COVID-19 patients on statins to determine mortality risk in patients prescribed specific statins versus untreated matched controls. The same drugs were tested in Vero E6 cells infected with SARS-CoV-2 and human endothelial cells infected with a related OC43 coronavirus. Results: Simvastatin was among the most highly predicted compounds (14/14 datasets) and five other statins, including atorvastatin, were predicted to be active in > 50% of analyses. Analysis of the clinical database revealed that reduced mortality risk was only observed in COVID-19 patients prescribed a subset of statins, including simvastatin and atorvastatin. In vitro testing of SARS-CoV-2 infected cells revealed simvastatin to be a potent direct inhibitor whereas most other statins were less effective. Simvastatin also inhibited OC43 infection and reduced cytokine production in endothelial cells. Conclusions and Relevance: Different statins may differ in their ability to sustain the lives of COVID-19 patients despite having a shared drug target and lipid-modifying mechanism of action. These findings highlight the value of target-agnostic drug prediction coupled with patient databases to identify and clinically evaluate non-obvious mechanisms and derisk and accelerate drug repurposing opportunities.

Safety and immunogenicity following a homologous booster dose of a SARS-CoV-2 recombinant spike protein vaccine (NVX-CoV2373): a secondary analysis of a randomised, placebo-controlled, phase 2 trial.

BACKGROUND: Emerging SARS-CoV-2 variants and evidence of waning vaccine efficacy present substantial obstacles towards controlling the COVID-19 pandemic. Booster doses of SARS-CoV-2 vaccines might address these concerns by amplifying and broadening the immune responses seen with initial vaccination regimens. We aimed to assess the immunogenicity and safety of a homologous booster dose of a SARS-CoV-2 recombinant spike protein vaccine (NVX-CoV2373). METHODS: This secondary analysis of a phase 2, randomised study assessed a single booster dose of a SARS-CoV-2 recombinant spike protein vaccine with Matrix-M adjuvant (NVX-CoV2373) in healthy adults aged 18-84 years, recruited from 17 clinical centres in the USA and Australia. Eligible participants had a BMI of 17-35 kg/m2 and, for women, were heterosexually inactive or using contraception. Participants who had a history of SARS-CoV or SARS-CoV-2, confirmed diagnosis of COVID-19, serious chronic medical conditions, or were pregnant or breastfeeding were excluded. Approximately 6 months following their primary two-dose vaccination series (administered day 0 and day 21), participants who received placebo for their primary vaccination series received a placebo booster (group A) and participants who received NVX-CoV2373 for their primary vaccination series (group B) were randomly assigned (1:1) again, via centralised interactive response technology system, to receive either placebo (group B1) or a single booster dose of NVX-CoV2373 (5 µg SARS-CoV-2 rS with 50 µg Matrix-M adjuvant; group B2) via intramuscular injection; randomisation was stratified by age and study site. Vaccinations were administered by designated site personnel who were masked to treatment assignment, and participants and other site staff were also masked. Administration personnel also assessed the outcome. The primary endpoints are safety (unsolicited adverse events) and reactogenicity (solicited local and systemic) events and immunogenicity (serum IgG antibody concentrations for the SARS-CoV-2 rS protein antigen) assessed 14 days after the primary vaccination series (day 35) and 28 days following booster (day 217). Safety was analysed in all participants in groups A, B1, and B2, according to the treatment received; immunogenicity was analysed in the per-protocol population (ie, participants in groups A, B1, and B2) who received all assigned doses and who did not test SARS-CoV-2-positive or received an authorised vaccine, analysed according to treatment assignment). This trial is registered with ClinicalTrials.gov, NCT04368988. FINDINGS: 1610 participants were screened from Aug 24, 2020, to Sept 25, 2020. 1282 participants were enrolled, of whom 173 were assigned again to placebo (group A), 106 were re-randomised to NVX-CoV2373-placebo (group B1), and 104 were re-randomised to NVX-CoV2373-NVX-CoV2373 (group B2); after accounting for exclusions and incorrect administration, 172 participants in group A, 102 in group B1, and 105 in group B2 were analysed for safety. Following the active booster, the proportion of participants with available data reporting local (80 [82%] of 97 participants had any adverse event; 13 [13%] had a grade ≥3 event) and systemic (75 [77%] of 98 participants had any adverse event; 15 [15%] had a grade ≥3 event) reactions was higher than after primary vaccination (175 [70%] of 250 participants had any local adverse event, 13 [5%] had a grade ≥3 event; 132 [53%] of 250 had any systemic adverse event, 14 [6%] had a grade ≥3 event). Local and systemic events were transient in nature (median duration 1·0-2·5 days). In the per-protocol immunogenicity population at day 217 (167 participants in group A, 101 participants in group B1, 101 participants in group B2), IgG geometric mean titres (GMT) had increased by 4·7-fold and MN50 GMT by 4·1-fold for the ancestral SARS-CoV-2 strain compared with the day 35 titres. INTERPRETATION: Administration of a booster dose of NVX-CoV2373 resulted in an incremental increase in reactogenicity. For both the prototype strain and all variants evaluated, immune responses following the booster were similar to or higher than those associated with high levels of efficacy in phase 3 studies of the vaccine. These data support the use of NVX-CoV2373 in booster programmes. FUNDING: Novavax and the Coalition for Epidemic Preparedness Innovations.

An aluminum hydroxide:CpG adjuvant enhances protection elicited by a SARS-CoV-2 receptor binding domain vaccine in aged mice.

Global deployment of vaccines that can provide protection across several age groups is still urgently needed to end the COVID-19 pandemic, especially in low- and middle-income countries. Although vaccines against SARS-CoV-2 based on mRNA and adenoviral vector technologies have been rapidly developed, additional practical and scalable SARS-CoV-2 vaccines are required to meet global demand. Protein subunit vaccines formulated with appropriate adjuvants represent an approach to address this urgent need. The receptor binding domain (RBD) is a key target of SARS-CoV-2 neutralizing antibodies but is poorly immunogenic. We therefore compared pattern recognition receptor (PRR) agonists alone or formulated with aluminum hydroxide (AH) and benchmarked them against AS01B and AS03-like emulsion-based adjuvants for their potential to enhance RBD immunogenicity in young and aged mice. We found that an AH and CpG adjuvant formulation (AH:CpG) produced an 80-fold increase in anti-RBD neutralizing antibody titers in both age groups relative to AH alone and protected aged mice from the SARS-CoV-2 challenge. The AH:CpG-adjuvanted RBD vaccine elicited neutralizing antibodies against both wild-type SARS-CoV-2 and the B.1.351 (beta) variant at serum concentrations comparable to those induced by the licensed Pfizer-BioNTech BNT162b2 mRNA vaccine. AH:CpG induced similar cytokine and chemokine gene enrichment patterns in the draining lymph nodes of both young adult and aged mice and enhanced cytokine and chemokine production in human mononuclear cells of younger and older adults. These data support further development of AH:CpG-adjuvanted RBD as an affordable vaccine that may be effective across multiple age groups.

Durable protection against the SARS-CoV-2 Omicron variant is induced by an adjuvanted subunit vaccine.

Despite the remarkable efficacy of COVID-19 vaccines, waning immunity and the emergence of SARS-CoV-2 variants such as Omicron represents a global health challenge. Here, we present data from a study in nonhuman primates demonstrating durable protection against the Omicron BA.1 variant induced by a subunit SARS-CoV-2 vaccine comprising the receptor binding domain of the ancestral strain (RBD-Wu) on the I53-50 nanoparticle adjuvanted with AS03, which was recently authorized for use in individuals 18 years or older. Vaccination induced neutralizing antibody (nAb) titers that were maintained at high concentrations for at least 1 year after two doses, with a pseudovirus nAb geometric mean titer (GMT) of 1978 and a live virus nAb GMT of 1331 against the ancestral strain but not against the Omicron BA.1 variant. However, a booster dose at 6 to 12 months with RBD-Wu or RBD-ß (RBD from the Beta variant) displayed on I53-50 elicited high neutralizing titers against the ancestral and Omicron variants. In addition, we observed persistent neutralization titers against a panel of sarbecoviruses, including SARS-CoV. Furthermore, there were substantial and persistent memory T and B cell responses reactive to Beta and Omicron variants. Vaccination resulted in protection against Omicron infection in the lung and suppression of viral burden in the nares at 6 weeks after the final booster immunization. Even at 6 months after vaccination, we observed protection in the lung and rapid control of virus in the nares. These results highlight the durable and cross-protective immunity elicited by the AS03-adjuvanted RBD-I53-50 nanoparticle vaccine.

A PCSK9-binding antibody that structurally mimics the EGF(A) domain of LDL-receptor reduces LDL cholesterol in vivo.

Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) regulates LDL cholesterol levels by inhibiting LDL receptor (LDLr)-mediated cellular LDL uptake. We have identified a fragment antigen-binding (Fab) 1D05 which binds PCSK9 with nanomolar affinity. The fully human antibody 1D05-IgG2 completely blocks the inhibitory effects of wild-type PCSK9 and two gain-of-function human PCSK9 mutants, S127R and D374Y. The crystal structure of 1D05-Fab bound to PCSK9 reveals that 1D05-Fab binds to an epitope on the PCSK9 catalytic domain which includes the entire LDLr EGF(A) binding site. Notably, the 1D05-Fab CDR-H3 and CDR-H2 loops structurally mimic the EGF(A) domain of LDLr. In a transgenic mouse model (CETP/LDLr-hemi), in which plasma lipid and PCSK9 profiles are comparable to those of humans, 1D05-IgG2 reduces plasma LDL cholesterol to 40% and raises hepatic LDLr protein levels approximately fivefold. Similarly, in healthy rhesus monkeys, 1D05-IgG2 effectively reduced LDL cholesterol 20%-50% for over 2 weeks, despite its relatively short terminal half-life (t(1/2) = 3.2 days). Importantly, the decrease in circulating LDL cholesterol corresponds closely to the reduction in free PCSK9 levels. Together these results clearly demonstrate that the LDL-lowering effect of the neutralizing anti-PCSK9 1D05-IgG2 antibody is mediated by reducing the amount of PCSK9 that can bind to the LDLr.

A proprotein convertase subtilisin-like/kexin type 9 (PCSK9) C-terminal domain antibody antigen-binding fragment inhibits PCSK9 internalization and restores low density lipoprotein uptake.

PCSK9 binds to the low density lipoprotein receptor (LDLR) and leads to LDLR degradation and inhibition of plasma LDL cholesterol clearance. Consequently, the role of PCSK9 in modulating circulating LDL makes it a promising therapeutic target for treating hypercholesterolemia and coronary heart disease. Although the C-terminal domain of PCSK9 is not involved in LDLR binding, the location of several naturally occurring mutations within this region suggests that it has an important role for PCSK9 function. Using a phage display library, we identified an anti-PCSK9 Fab (fragment antigen binding), 1G08, with subnanomolar affinity for PCSK9. In an assay measuring LDL uptake in HEK293 and HepG2 cells, 1G08 Fab reduced 50% the PCSK9-dependent inhibitory effects on LDL uptake. Importantly, we found that 1G08 did not affect the PCSK9-LDLR interaction but inhibited the internalization of PCSK9 in these cells. Furthermore, proteolysis and site-directed mutagenesis studies demonstrated that 1G08 Fab binds a region of beta-strands encompassing Arg-549, Arg-580, Arg-582, Glu-607, Lys-609, and Glu-612 in the PCSK9 C-terminal domain. Consistent with these results, 1G08 fails to bind PCSK9DeltaC, a truncated form of PCSK9 lacking the C-terminal domain. Additional studies revealed that lack of the C-terminal domain compromised the ability of PCSK9 to internalize into cells, and to inhibit LDL uptake. Together, the present study demonstrate that the PCSK9 C-terminal domain contribute to its inhibition of LDLR function mainly through its role in the cellular uptake of PCSK9 and LDLR complex. 1G08 Fab represents a useful new tool for delineating the mechanism of PCSK9 uptake and LDLR degradation.

Pyrimidine biosynthesis inhibitors synergize with nucleoside analogs to block SARS-CoV-2 infection.

The ongoing COVID-19 pandemic has highlighted the dearth of approved drugs to treat viral infections, with only ∼90 FDA approved drugs against human viral pathogens. To identify drugs that can block SARS-CoV-2 replication, extensive drug screening to repurpose approved drugs is underway. Here, we screened ∼18,000 drugs for antiviral activity using live virus infection in human respiratory cells. Dose-response studies validate 122 drugs with antiviral activity and selectivity against SARS-CoV-2. Amongst these drug candidates are 16 nucleoside analogs, the largest category of clinically used antivirals. This included the antiviral Remdesivir approved for use in COVID-19, and the nucleoside Molnupirivir, which is undergoing clinical trials. RNA viruses rely on a high supply of nucleoside triphosphates from the host to efficiently replicate, and we identified a panel of host nucleoside biosynthesis inhibitors as antiviral, and we found that combining pyrimidine biosynthesis inhibitors with antiviral nucleoside analogs synergistically inhibits SARS-CoV-2 infection in vitro and in vivo suggesting a clinical path forward.

SARS-CoV-2 spike glycoprotein vaccine candidate NVX-CoV2373 immunogenicity in baboons and protection in mice.

The COVID-19 pandemic continues to spread throughout the world with an urgent need for a safe and protective vaccine to effectuate herd protection and control the spread of SARS-CoV-2. Here, we report the development of a SARS-CoV-2 subunit vaccine (NVX-CoV2373) from the full-length spike (S) protein that is stable in the prefusion conformation. NVX-CoV2373 S form 27.2-nm nanoparticles that are thermostable and bind with high affinity to the human angiotensin-converting enzyme 2 (hACE2) receptor. In mice, low-dose NVX-CoV2373 with saponin-based Matrix-M adjuvant elicit high titer anti-S IgG that blocks hACE2 receptor binding, neutralize virus, and protects against SARS-CoV-2 challenge with no evidence of vaccine-associated enhanced respiratory disease. NVX-CoV2373 also elicits multifunctional CD4+ and CD8+ T cells, CD4+ follicular helper T cells (Tfh), and antigen-specific germinal center (GC) B cells in the spleen. In baboons, low-dose levels of NVX-CoV2373 with Matrix-M was also highly immunogenic and elicited high titer anti-S antibodies and functional antibodies that block S-protein binding to hACE2 and neutralize virus infection and antigen-specific T cells. These results support the ongoing phase 1/2 clinical evaluation of the safety and immunogenicity of NVX-CoV2373 with Matrix-M (NCT04368988).

Alum:CpG adjuvant enables SARS-CoV-2 RBD-induced protection in aged mice and synergistic activation of human elder type 1 immunity.

Global deployment of vaccines that can provide protection across several age groups is still urgently needed to end the COVID-19 pandemic especially for low- and middle-income countries. While vaccines against SARS-CoV-2 based on mRNA and adenoviral-vector technologies have been rapidly developed, additional practical and scalable SARS-CoV-2 vaccines are needed to meet global demand. In this context, protein subunit vaccines formulated with appropriate adjuvants represent a promising approach to address this urgent need. Receptor-binding domain (RBD) is a key target of neutralizing antibodies (Abs) but is poorly immunogenic. We therefore compared pattern recognition receptor (PRR) agonists, including those activating STING, TLR3, TLR4 and TLR9, alone or formulated with aluminum hydroxide (AH), and benchmarked them to AS01B and AS03-like emulsion-based adjuvants for their potential to enhance RBD immunogenicity in young and aged mice. We found that the AH and CpG adjuvant formulation (AH:CpG) demonstrated the highest enhancement of anti-RBD neutralizing Ab titers in both age groups (∼80-fold over AH), and protected aged mice from the SARS-CoV-2 challenge. Notably, AH:CpG-adjuvanted RBD vaccine elicited neutralizing Abs against both wild-type SARS-CoV-2 and B.1.351 variant at serum concentrations comparable to those induced by the authorized mRNA BNT162b2 vaccine. AH:CpG induced similar cytokine and chemokine gene enrichment patterns in the draining lymph nodes of both young adult and aged mice and synergistically enhanced cytokine and chemokine production in human young adult and elderly mononuclear cells. These data support further development of AH:CpG-adjuvanted RBD as an affordable vaccine that may be effective across multiple age groups. ONE SENTENCE SUMMARY: Alum and CpG enhance SARS-CoV-2 RBD protective immunity, variant neutralization in aged mice and Th1-polarizing cytokine production by human elder leukocytes.