Effect of mRNA-LNP components of two globally-marketed COVID-19 vaccines on efficacy and stability.

During the COVID-19 pandemic, Pfizer-BioNTech and Moderna successfully developed nucleoside-modified mRNA lipid nanoparticle (LNP) vaccines. SARS-CoV-2 spike protein expressed by those vaccines are identical in amino acid sequence, but several key components are distinct. Here, we compared the effect of ionizable lipids, untranslated regions (UTRs), and nucleotide composition of the two vaccines, focusing on mRNA delivery, antibody generation, and long-term stability. We found that the ionizable lipid, SM-102, in Moderna's vaccine performs better than ALC-0315 in Pfizer-BioNTech's vaccine for intramuscular delivery of mRNA and antibody production in mice and long-term stability at 4 °C. Moreover, Pfizer-BioNTech's 5' UTR and Moderna's 3' UTR outperform their counterparts in their contribution to transgene expression in mice. We further found that varying N1-methylpseudouridine content at the wobble position of mRNA has little effect on vaccine efficacy. These findings may contribute to the further improvement of nucleoside-modified mRNA-LNP vaccines and therapeutics.

Utility of Continuous Paravertebral Block After Retroperitoneal Abdominal Aortic Aneurysm Repair.

BACKGROUND: Open abdominal aortic aneurysm (AAA) repairs can be associated with significant pain and morbidity. Previous studies have demonstrated utility of adjunctive epidural analgesia (EA) in addition to general anesthesia (GA) to reduce pain and blunt the maladaptive surgical stress response. However, EA may be complicated by epidural hematomas and severe hypotension. Recently, we started using continuous paravertebral block (PVB) for perioperative analgesia after retroperitoneal AAA repair. PVB has some distinct advantages over EA such as unilateral localization, reduced risk of hypotension, and minimal risk of epidural hematoma in the setting of systemic heparinization. This study aimed to examine the utility of PVB by comparing total opioid consumption in the postoperative period among patients who received GA + PVB and those who received GA alone. METHODS: This retrospective matched cohort study included 62 patients who underwent elective retroperitoneal AAA repair between January 2019 and August 2022. Thirty-one subjects managed with GA + PVB were compared with 31 control subjects treated with GA alone, matched on following criteria: age, sex, and cross-clamp location. Outcome measures included total opioid analgesics administered during their inhospital postoperative course, time to extubation, time to return to baseline activity, time to normal bowel function, and length of stay. Opioid doses were converted to morphine milligram equivalents (MMEs). RESULTS: The GA + PVB group required significantly less opioid analgesics (81 ± 53 MME) than the GA group (171 ± 121 MME) (P < 0.001). Compared to GA alone, GA + PVB was superior in every clinical metric examined: time to extubation (3 vs. 1 hr, P < 0.001), recovery of bowel function (3 vs. 2 days, P = 0.002), recovery of baseline physical activity (4 vs. 2 days, P = 0.019), and length of stay (5 vs. 3 days, P < 0.001). CONCLUSIONS: Continuous paravertebral block provides better pain management with significantly decreased opioid requirements in the postoperative period compared to GA-alone for patients undergoing elective retroperitoneal AAA repair.

Mechanisms of SARS-CoV-2 entry into cells.

The unprecedented public health and economic impact of the COVID-19 pandemic caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been met with an equally unprecedented scientific response. Much of this response has focused, appropriately, on the mechanisms of SARS-CoV-2 entry into host cells, and in particular the binding of the spike (S) protein to its receptor, angiotensin-converting enzyme 2 (ACE2), and subsequent membrane fusion. This Review provides the structural and cellular foundations for understanding the multistep SARS-CoV-2 entry process, including S protein synthesis, S protein structure, conformational transitions necessary for association of the S protein with ACE2, engagement of the receptor-binding domain of the S protein with ACE2, proteolytic activation of the S protein, endocytosis and membrane fusion. We define the roles of furin-like proteases, transmembrane protease, serine 2 (TMPRSS2) and cathepsin L in these processes, and delineate the features of ACE2 orthologues in reservoir animal species and S protein adaptations that facilitate efficient human transmission. We also examine the utility of vaccines, antibodies and other potential therapeutics targeting SARS-CoV-2 entry mechanisms. Finally, we present key outstanding questions associated with this critical process.

Clinical Antiviral Drug Arbidol Inhibits Infection by SARS-CoV-2 and Variants through Direct Binding to the Spike Protein.

Arbidol (ARB) is a broad-spectrum antiviral drug approved in Russia and China for the treatment of influenza. ARB was tested in patients as a drug candidate for the treatment at the early onset of COVID-19 caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Despite promising clinical results and multiple ongoing trials, preclinical data are lacking and the molecular mechanism of action of ARB against SARS-CoV-2 remains unknown. Here, we demonstrate that ARB binds to the spike viral fusion glycoprotein of the SARS-CoV-2 Wuhan strain as well as its more virulent variants from the United Kingdom (strain B.1.1.7) and South Africa (strain B.1.351). We pinpoint the ARB binding site on the S protein to the S2 membrane fusion domain and use an infection assay with Moloney murine leukemia virus (MLV) pseudoviruses (PVs) pseudotyped with the S proteins of the Wuhan strain and the new variants to show that this interaction is sufficient for the viral cell entry inhibition by ARB. Finally, our experiments reveal that the ARB interaction leads to a significant destabilization and eventual lysosomal degradation of the S protein in cells. Collectively, our results identify ARB as the first clinically approved small molecule drug binder of the SARS-CoV-2 S protein and place ARB among the more promising drug candidates for COVID-19.

Functional importance of the D614G mutation in the SARS-CoV-2 spike protein.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped virus which binds its cellular receptor angiotensin-converting enzyme 2 (ACE2) and enters hosts cells through the action of its spike (S) glycoprotein displayed on the surface of the virion. Compared to the reference strain of SARS-CoV-2, the majority of currently circulating isolates possess an S protein variant characterized by an aspartic acid-to-glycine substitution at amino acid position 614 (D614G). Residue 614 lies outside the receptor binding domain (RBD) and the mutation does not alter the affinity of monomeric S protein for ACE2. However, S(G614), compared to S(D614), mediates more efficient ACE2-mediated transduction of cells by S-pseudotyped vectors and more efficient infection of cells and animals by live SARS-CoV-2. This review summarizes and synthesizes the epidemiological and functional observations of the D614G spike mutation, with focus on the biochemical and cell-biological impact of this mutation and its consequences for S protein function. We further discuss the significance of these recent findings in the context of the current global pandemic.

AAV vectors engineered to target insulin receptor greatly enhance intramuscular gene delivery.

Adeno-associated virus (AAV) is one of the most commonly used vectors for gene therapy, and the applications for AAV-delivered therapies are numerous. However, the current state of technology is limited by the low efficiency with which most AAV vectors transduce skeletal muscle tissue. We demonstrate that vector efficiency can be enhanced by modifying the AAV capsid with a peptide that binds a receptor highly expressed in muscle tissue. When an insulin-mimetic peptide, S519, previously characterized for its high affinity to insulin receptor (IR), was inserted into the capsid, the AAV9 transduction efficiency of IR-expressing cell lines as well as differentiated primary human muscle cells was dramatically enhanced. This vector also exhibited efficient transduction of mouse muscle in vivo, resulting in up to 18-fold enhancement over AAV9. Owing to its superior transduction efficiency in skeletal muscle, we named this vector "enhanced AAV9" (eAAV9). We also found that the modification enhanced the transduction efficiency of several other AAV serotypes. Together, these data show that AAV transduction of skeletal muscle can be improved by targeting IR. They also show the broad utility of this modular strategy and suggest that it could also be applied to next-generation vectors that have yet to be engineered.

SARS-CoV-2 spike-protein D614G mutation increases virion spike density and infectivity.

SARS-CoV-2 variants with spike (S)-protein D614G mutations now predominate globally. We therefore compare the properties of the mutated S protein (SG614) with the original (SD614). We report here pseudoviruses carrying SG614 enter ACE2-expressing cells more efficiently than those with SD614. This increased entry correlates with less S1-domain shedding and higher S-protein incorporation into the virion. Similar results are obtained with virus-like particles produced with SARS-CoV-2 M, N, E, and S proteins. However, D614G does not alter S-protein binding to ACE2 or neutralization sensitivity of pseudoviruses. Thus, D614G may increase infectivity by assembling more functional S protein into the virion.

Phosphatidylethanolamine and Phosphatidylserine Synergize To Enhance GAS6/AXL-Mediated Virus Infection and Efferocytosis.

Phosphatidylserine (PS) receptors mediate clearance of apoptotic cells-efferocytosis-by recognizing the PS exposed on those cells. They also mediate the entry of enveloped viruses by binding PS in the virion membrane. Here, we show that phosphatidylethanolamine (PE) synergizes with PS to enhance PS receptor-mediated efferocytosis and virus entry. The presence of PE on the same surface as PS dramatically enhances recognition of PS by PS-binding proteins such as GAS6, PROS, and TIM1. Liposomes containing both PE and PS bound to GAS6 and were engulfed by AXL-expressing cells much more efficiently than those containing PS alone. Further, infection of AXL-expressing cells by infectious Zika virus or Ebola, Chikungunya, or eastern equine encephalitis pseudoviruses was inhibited with greater efficiency by the liposomes containing both PS and PE compared to a mixture of liposomes separately composed of PS and PE. These data demonstrate that simultaneous recognition of PE and PS maximizes PS receptor-mediated virus entry and efferocytosis and underscore the important contribution of PE in these major biological processes.IMPORTANCE Phosphatidylserine (PS) and phosphatidylethanolamine (PE) are usually sequestered to the inner leaflet of the plasma membrane of the healthy eukaryotic cells. During apoptosis, these phospholipids move to the cell's outer leaflet where they are recognized by so-called PS receptors on surveilling phagocytes. Several pathogenic families of enveloped viruses hijack these PS receptors to gain entry into their target cells. Here, we show that efficiency of these processes is enhanced, namely, PE synergizes with PS to promote PS receptor-mediated virus infection and clearance of apoptotic cells. These findings deepen our understanding of how these fundamental biological processes are executed.

The D614G mutation in the SARS-CoV-2 spike protein reduces S1 shedding and increases infectivity.

SARS coronavirus 2 (SARS-CoV-2) isolates encoding a D614G mutation in the viral spike (S) protein predominate over time in locales where it is found, implying that this change enhances viral transmission. We therefore compared the functional properties of the S proteins with aspartic acid (S D614 ) and glycine (S G614 ) at residue 614. We observed that retroviruses pseudotyped with S G614 infected ACE2-expressing cells markedly more efficiently than those with S D614 . This greater infectivity was correlated with less S1 shedding and greater incorporation of the S protein into the pseudovirion. Similar results were obtained using the virus-like particles produced with SARS-CoV-2 M, N, E, and S proteins. However, S G614 did not bind ACE2 more efficiently than S D614 , and the pseudoviruses containing these S proteins were neutralized with comparable efficiencies by convalescent plasma. These results show S G614 is more stable than S D614 , consistent with epidemiological data suggesting that viruses with S G614 transmit more efficiently.

Zika Virus-Immune Plasmas from Symptomatic and Asymptomatic Individuals Enhance Zika Pathogenesis in Adult and Pregnant Mice.

Preexisting immunity against dengue virus or West Nile virus was previously reported to mediate antibody-dependent enhancement (ADE) of Zika virus (ZIKV) infection in a mouse model. We show here that ZIKV-immune plasma samples from both symptomatic and asymptomatic individuals mediated ZIKV ADE of infection in vitro and in mice. In a lethal infection model with a viral inoculum 10 times higher, both ADE and protection were observed, depending on the amount of infused immune plasma. In a vertical-transmission model, ZIKV-immune plasma infused to timed pregnant mice increased fetal demise and decreased the body weight of surviving fetuses. Depletion of IgG from an immune plasma abolished ADE of infection, and the presence of purified IgG alone mediated ADE of infection. Higher viral loads and proinflammatory cytokines were detected in mice treated with ZIKV-immune plasma samples compared to those receiving control plasma. Together, these data show that passive immunization with homotypic ZIKV antibodies, depending on the concentration, could either worsen or limit a subsequent ZIKV infection.IMPORTANCE Antibody-dependent enhancement (ADE) of virus infection is common to many viruses and is problematic when plasma antibody levels decline to subneutralizing concentrations. ADE of infection is especially important among flaviviruses, many of which are the cause of global health problems. Recently, human plasma samples immune to heterologous flaviviruses were shown to promote Zika virus (ZIKV) infection. Here we showed in immunocompromised mouse models that homologous immune plasma samples protect mice from subsequent infection at high antibody concentrations but that they mediate ADE of infection and increase ZIKV pathogenesis in adult mice and fetal demise during pregnancy at low concentrations.

Myxoma virus M013 protein antagonizes NF-κB and inflammasome pathways via distinct structural motifs.

Among the repertoire of immunoregulatory proteins encoded by myxoma virus, M013 is a viral homologue of the viral pyrin domain-only protein (vPOP) family. In myeloid cells, M013 protein has been shown to inhibit both the inflammasome and NF-κB signaling pathways by direct binding to ASC1 and NF-κB1, respectively. In this study, a three-dimensional homology model of the M013 pyrin domain (PYD) was built based on similarities to known PYD structures. A distinctive feature of the deduced surface electrostatic map of the M013 PYD is the presence of a negatively region consisting of numerous aspartate and glutamate residues in close proximity. Single-site mutations of aspartate and glutamate residues reveal their role in interactions with ASC-1. The biological significance of charge complementarity in the M013-ASC-1 interaction was further confirmed by functional assays of caspase-1 activation and subsequent secretion of cytokines. M013 also has a unique 33-residue C-terminal tail that follows the N-terminal PYD, and it is enriched in positively charged residues. Deletion of the tail of M013 significantly inhibited the interactions between M013 and NF-κB1, thus compromising the ability of the viral protein to suppress the secretion of pro-inflammatory cytokines. These results demonstrate that vPOP M013 exploits distinct structural motifs to regulate both the inflammasome and NF-κB pathways.

The Xenobiotic Transporter Mdr1 Enforces T Cell Homeostasis in the Presence of Intestinal Bile Acids.

CD4+ T cells are tightly regulated by microbiota in the intestine, but whether intestinal T cells interface with host-derived metabolites is less clear. Here, we show that CD4+ T effector (Teff) cells upregulated the xenobiotic transporter, Mdr1, in the ileum to maintain homeostasis in the presence of bile acids. Whereas wild-type Teff cells upregulated Mdr1 in the ileum, those lacking Mdr1 displayed mucosal dysfunction and induced Crohn's disease-like ileitis following transfer into Rag1-/- hosts. Mdr1 mitigated oxidative stress and enforced homeostasis in Teff cells exposed to conjugated bile acids (CBAs), a class of liver-derived emulsifying agents that actively circulate through the ileal mucosa. Blocking ileal CBA reabsorption in transferred Rag1-/- mice restored Mdr1-deficient Teff cell homeostasis and attenuated ileitis. Further, a subset of ileal Crohn's disease patients displayed MDR1 loss of function. Together, these results suggest that coordinated interaction between mucosal Teff cells and CBAs in the ileum regulate intestinal immune homeostasis.