A Recombinant Protein SARS-CoV-2 Candidate Vaccine Elicits High-titer Neutralizing Antibodies in Macaques.

Background: Vaccines that generate robust and long-lived protective immunity against SARS-CoV-2 infection are urgently required. Methods: We assessed the potential of vaccine candidates based on the SARS-CoV-2 spike in cynomolgus macaques (M. fascicularis) by examining their ability to generate spike binding antibodies with neutralizing activity. Antigens were derived from two distinct regions of the spike S1 subunit, either the N-terminal domain or an extended C-terminal domain containing the receptor-binding domain and were fused to the human IgG1 Fc domain. Three groups of 2 animals each were immunized with either antigen, alone or in combination. The development of antibody responses was evaluated through 20 weeks post-immunization. Results: A robust IgG response to the spike protein was detected as early as 2 weeks after immunization with either protein and maintained for over 20 weeks. Sera from animals immunized with antigens derived from the RBD were able to prevent binding of soluble spike proteins to the ACE2 receptor, shown by in vitro binding assays, while sera from animals immunized with the N-terminal domain alone lacked this activity. Crucially, sera from animals immunized with the extended receptor binding domain but not the N-terminal domain had potent neutralizing activity against SARS-CoV-2 pseudotyped virus, with titers in excess of 10,000, greatly exceeding that typically found in convalescent humans. Neutralizing activity persisted for more than 20 weeks. Conclusions: These data support the utility of spike subunit-based antigens as a vaccine for use in humans.

"A recombinant protein SARS-CoV-2 candidate vaccine elicits high-titer neutralizing antibodies in macaques."

Vaccines that generate robust and long-lived protective immunity against SARS-CoV-2 infection are urgently required. We assessed the potential of vaccine candidates based on the SARS-CoV-2 spike in cynomolgus macaques (M. fascicularis) by examining their ability to generate spike binding antibodies with neutralizing activity. Antigens were derived from two distinct regions of the spike S1 subunit, either the N-terminal domain (NTD) or an extended C-terminal domain containing the receptor-binding domain (RBD) and were fused to the human IgG1 Fc domain. Three groups of 2 animals each were immunized with either antigen, alone or in combination. The development of antibody responses was evaluated through 20 weeks post-immunization. A robust IgG response to the spike protein was detected as early as 2 weeks after immunization with either protein and maintained for over 20 weeks. Sera from animals immunized with antigens derived from the RBD were able to prevent binding of soluble spike proteins to the ACE2 receptor, shown by in vitro binding assays, while sera from animals immunized with the NTD alone lacked this activity. Crucially, sera from animals immunized with the RBD but not the NTD had potent neutralizing activity against SARS-CoV-2 pseudotyped virus, with titers in excess of 10,000, greatly exceeding that typically found in convalescent humans. Neutralizing activity persisted for more than 20 weeks. These data support the utility of spike subunit-based antigens as a vaccine for use in humans.

Vitamin D is not required for adaptive immunity to listeria.

Although ex vivo research suggests that vitamin D may play a role in innate and adaptive immunity, clear in vivo evidence is lacking. We have tested whether severe vitamin D deficiency alters the ability of mice to resist infection by Listeria. Our results show that vitamin D deficiency does not affect the LD50 of naïve mice in response to Listeria. To study the adaptive immune response, the LD50 for Listeria-immunized mice was determined for vitamin D-deficient and vitamin D-sufficient mice. Although the LD50 clearly increased by immunization with inactivated Listeria, there was no effect of vitamin D deficiency on survival of mice infected with wild-type Listeria. Thus, in this model of adaptive immunity, we could find no evidence of a role for vitamin D.

Mitochondrial uncouplers inhibit clathrin-mediated endocytosis largely through cytoplasmic acidification.

ATP production requires the establishment of an electrochemical proton gradient across the inner mitochondrial membrane. Mitochondrial uncouplers dissipate this proton gradient and disrupt numerous cellular processes, including vesicular trafficking, mainly through energy depletion. Here we show that Endosidin9 (ES9), a novel mitochondrial uncoupler, is a potent inhibitor of clathrin-mediated endocytosis (CME) in different systems and that ES9 induces inhibition of CME not because of its effect on cellular ATP, but rather due to its protonophore activity that leads to cytoplasm acidification. We show that the known tyrosine kinase inhibitor tyrphostinA23, which is routinely used to block CME, displays similar properties, thus questioning its use as a specific inhibitor of cargo recognition by the AP-2 adaptor complex via tyrosine motif-based endocytosis signals. Furthermore, we show that cytoplasm acidification dramatically affects the dynamics and recruitment of clathrin and associated adaptors, and leads to reduction of phosphatidylinositol 4,5-biphosphate from the plasma membrane.

dsdA Does Not Affect Colonization of the Murine Urinary Tract by Escherichia coli CFT073.

The urinary tract environment provides many conditions that deter colonization by microorganisms. D-serine is thought to be one of these stressors and is present at high concentrations in urine. D-serine interferes with L-serine and pantothenate metabolism and is bacteriostatic to many species. Uropathogenic Escherichia coli commonly possess the dsdCXA genetic locus, which allows them to use D-serine as a sole carbon, nitrogen, and energy source. It was previously reported that in the model UPEC strain CFT073, a dsdA mutant outcompetes wild type in the murine model of urinary tract infection. This "hypercolonization" was used to propose a model whereby UPEC strains sense D-serine in the urinary tract and subsequently up-regulate genes necessary for pathogenesis. Here, we show that inactivation of dsdA does not lead to hypercolonization. We suggest that this previously observed effect is due to an unrecognized secondary mutation in rpoS and that some D-serine specific effects described in other studies may be affected by the rpoS status of the strains used. Inactivation of dsdA in the original clinical isolate of CFT073 gives CFT073 ΔdsdA a growth defect in human urine and renders it unable to grow on minimal medium containing D-serine as the sole carbon source. However, CFT073 ΔdsdA is able to colonize the urinary tracts of CBA/J mice indistinguishably from wild type. These findings indicate that D-serine catabolism, though it may play role(s) during urinary tract infection, does not affect the ability of uropathogenic E. coli to colonize the murine urinary tract.

The VASCULATURE COMPLEXITY AND CONNECTIVITY gene encodes a plant-specific protein required for embryo provasculature development.

The molecular mechanisms by which vascular tissues acquire their identities are largely unknown. Here, we report on the identification and characterization of VASCULATURE COMPLEXITY AND CONNECTIVITY (VCC), a member of a 15-member, plant-specific gene family in Arabidopsis (Arabidopsis thaliana) that encodes proteins of unknown function with four predicted transmembrane domains. Homozygous vcc mutants displayed cotyledon vein networks of reduced complexity and disconnected veins. Similar disconnections or gaps were observed in the provasculature of vcc embryos, indicating that defects in vein connectivity appear early in mutant embryo development. Consistently, the overexpression of VCC leads to an unusually high proportion of cotyledons with high-complexity vein networks. Neither auxin distribution nor the polar localization of the auxin efflux carrier were affected in vcc mutant embryos. Expression of VCC was detected in developing embryos and procambial, cambial, and vascular cells of cotyledons, leaves, roots, hypocotyls, and anthers. To evaluate possible genetic interactions with other genes that control vasculature patterning in embryos, we generated a double mutant for VCC and OCTOPUS (OPS). The vcc ops double mutant embryos showed a complete loss of high-complexity vascular networks in cotyledons and a drastic increase in both provascular and vascular disconnections. In addition, VCC and OPS interact physically, suggesting that VCC and OPS are part of a complex that controls cotyledon vascular complexity.

Budding and braking news about clathrin-mediated endocytosis.

Clathrin-mediated endocytosis (CME) is the predominate mechanism of endocytosis in eukaryotes, but an understanding of this mechanism in plants has lagged behind yeast and mammalian systems. The generation of Arabidopsis mutant libraries, and the development of the molecular tools and equipment necessary to characterize these plant lines has led to an astonishing number of new insights into the mechanisms of membrane trafficking in plants. Over the past few years progress has been made on identifying, and in some instances confirming, the core components of CME in plants. This review focuses on the recent progress made in the understanding of the mechanism and regulation of CME in plants.

Characterization of Escherichia coli D-cycloserine transport and resistant mutants.

d-Cycloserine (DCS) is a broad-spectrum antibiotic that inhibits d-alanine ligase and alanine racemase activity. When Escherichia coli K-12 or CFT073 is grown in minimal glucose or glycerol medium, CycA transports DCS into the cell. E. coli K-12 cycA and CFT073 cycA mutant strains display increased DCS resistance when grown in minimal medium. However, the cycA mutants exhibit no change in DCS sensitivity compared to their parental strains when grown in LB (CFT073 and K-12) or human urine (CFT073 only). These data suggest that cycA does not participate in DCS sensitivity when strains are grown in a non-minimal medium. The small RNA GvcB acts as a negative regulator of E. coli K-12 cycA expression when grown in LB. Three E. coli K-12 gcvB mutant strains failed to demonstrate a change in DCS sensitivity when grown in LB. This further suggests a limited role for cycA in DCS sensitivity. To aid in the identification of E. coli genes involved in DCS sensitivity when grown on complex media, the Keio K-12 mutant collection was screened for DCS-resistant strains. dadA, pnp, ubiE, ubiF, ubiG, ubiH, and ubiX mutant strains showed elevated DCS resistance. The phenotypes associated with these mutants were used to further define three previously characterized E. coli DCS-resistant strains (χ316, χ444, and χ453) isolated by Curtiss and colleagues (R. Curtiss, III, L. J. Charamella, C. M. Berg, and P. E. Harris, J. Bacteriol. 90:1238-1250, 1965). A dadA mutation was identified in both χ444 and χ453. In addition, results are presented that indicate for the first time that DCS can antagonize d-amino acid dehydrogenase (DadA) activity.