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According to the CDC, both Pfizer and Moderna COVID‐19 vaccines contain nucleoside‐modified messenger RNA (mRNA) encoding the viral spike glycoprotein of severe acute respiratory syndrome caused by corona virus (SARS‐CoV‐2), administered via intramuscular injections. Despite their worldwide use, very little is known about how nucleoside modifications in mRNA sequences affect their breakdown, transcription and protein synthesis. It was hoped that resident and circulating immune cells attracted to the injection site make copies of the spike protein while the injected mRNA degrades within a few days. It was also originally estimated that recombinant spike proteins generated by mRNA vaccines would persist in the body for a few weeks. In reality, clinical studies now report that modified SARS‐CoV‐2 mRNA routinely persist up to a month from injection and can be detected in cardiac and skeletal muscle at sites of inflammation and fibrosis, while the recombinant spike protein may persist a little over half a year in blood. Vaccination with 1‐methylΨ (pseudouridine enriched) mRNA can elicit cellular immunity to peptide antigens produced by +1 ribosomal frameshifting in major histocompatibility complex‐diverse people. The translation of 1‐methylΨ mRNA using liquid chromatography tandem mass spectrometry identified nine peptides derived from the mRNA +1 frame. These products impact on off‐target host T cell immunity that include increased production of new B cell antigens with far reaching clinical consequences. As an example, a highly significant increase in heart muscle 18‐flourodeoxyglucose uptake was detected in vaccinated patients up to half a year (180 days). This review article focuses on medical biochemistry, proteomics and deutenomics principles that explain the persisting spike phenomenon in circulation with organ‐related functional damage even in asymptomatic individuals. Proline and hydroxyproline residues emerge as prominent deuterium (heavy hydrogen) binding sites in structural proteins with robust isotopic stability that resists not only enzymatic breakdown, but virtually all (non)‐enzymatic cleavage mechanisms known in chemistry. [ABSTRACT FROM AUTHOR]

Intracellular lipid droplets (LDs) can accumulate in response to inflammation, metabolic stresses, and other physiological/pathological processes. Herein, we investigated whether spike proteins of SARS-CoV-2 induce LDs in human peripheral blood mononuclear cells (PBMCs) and in pulmonary microvascular endothelial cells (HPMECs). PBMCs or HPMECs were incubated alone or with endotoxin-free recombinant variants of trimeric spike glycoproteins (Alpha, Beta, Delta, and Omicron, 12 µg/mL). Afterward, cells were stained with Oil Red O for LDs, cytokine release was determined through ELISA, and the gene expression was analyzed through real-time PCR using TaqMan assays. Our data show that spikes induce LDs in PBMCs but not in HPMECs. In line with this, in PBMCs, spike proteins lower the expression of genes involving lipid metabolism and LD formation, such as SREBF1, HMGCS1, LDLR, and CD36. On the other hand, PBMCs exposed to spikes for 6 or 18 h did not increase in IL-1β, IL-6, IL-8, MCP-1, and TNFα release or expression as compared to non-treated controls. Thus, spike-induced LD formation in PBMCs seems to not be related to cell inflammatory activation. Further detailed studies are warranted to investigate in which specific immune cells spikes induce LDs, and what are the pathophysiological mechanisms and consequences of this induction in vivo. [ABSTRACT FROM AUTHOR]

• S-268019-a is a recombinant spike protein vaccine adjuvanted with agatolimod sodium. • S-268019-a elicited immunogenicity and efficacy against SARS-CoV-2 in mice. • S-268019-a was safe but insufficiently immunogenic in humans. • Translating preclinical data to clinical studies is innately challenging. • S-268019-b formulation with a squalene-based adjuvant is in Phase 3 clinical trials. In early 2020, developing vaccines was an urgent need for preventing COVID-19 from a contingency perspective. S-268019-a is a recombinant protein-based vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), comprising a modified recombinant spike protein antigen adjuvanted with agatolimod sodium, a Toll-like receptor-9 agonist. In the preclinical phase, it was administered intramuscularly twice at a 2-week interval in 7-week-old mice. Immunogenicity was assessed, and the mice were challenged intranasally with mouse-adapted SARS-CoV-2 at 2 and 8 weeks, respectively, after the second immunization. After confirming the preclinical effect, a Phase 1/2, randomized, parallel-group clinical study was conducted in healthy adults (aged 20–64 years). All participants received 2 intramuscular injections at various combinations of the antigen and the adjuvant (S-910823/agatolimod sodium, in μg: 12.5/250, 25/250, 50/250, 25/500, 50/500, 100/500, 10/500, 100/100, 200/1000) or placebo (saline) in an equivalent volume at a 3-week interval and were followed up until Day 50 in this interim analysis. In the preclinical studies, S-268019-a was safe and elicited robust immunoglobulin G (IgG) and neutralizing antibody responses in mice. When challenged with SARS-CoV-2, all S-268019-a-treated mice survived and maintained weight until 10 days, whereas all placebo- or adjuvant-treated (without antigen) mice died within 6 days. In the Phase 1/2 trial, although S-268019-a was well tolerated in adult participants, was safe up to Day 50, and elicited robust anti-spike protein IgG antibodies, it did not elicit sufficient neutralizing antibody levels. The S-268019-a vaccine was not sufficiently immunogenic in Japanese adults despite robust immunogenicity and efficacy in mice. Our results exemplify the innate challenges in translating preclinical data in animals to clinical trials, and highlight the need for continued research to overcome such barriers. (jRCT2051200092) [ABSTRACT FROM AUTHOR]

Abstract We have developed a method for the inexpensive, high-level expression of antigenic protein fragments of SARS-CoV-2 proteins in Escherichia coli. Our approach uses the thermophilic family 9 carbohydrate-binding module (CBM9) as an N-terminal carrier protein and affinity tag. The CBM9 module was joined to SARS-CoV-2 protein fragments via a flexible proline–threonine linker, which proved to be resistant to E. coli proteases. Two CBM9-spike protein fragment fusion proteins and one CBM9-nucleocapsid fragment fusion protein largely resisted protease degradation, while most of the CBM9 fusion proteins were degraded at some site in the SARS-CoV-2 protein fragment. All of the fusion proteins were highly expressed in E. coli and the CBM9-ID-H1 fusion protein was shown to yield 122 mg/L of purified product. Three purified CBM9-SARS-CoV-2 fusion proteins were tested and found to bind antibodies directed to the appropriate SARS-CoV-2 antigenic regions. The largest intact CBM9 fusion protein, CBM9-ID-H1, incorporates spike protein amino acids 540–588, which is a conserved region overlapping and C-terminal to the receptor binding domain that is widely recognized by human convalescent sera and contains a putative protective epitope.

Intracellular lipid droplets (LDs) can accumulate in response to inflammation, metabolic stresses, and other physiological/pathological processes. Herein, we investigated whether spike proteins of SARS-CoV-2 induce LDs in human peripheral blood mononuclear cells (PBMCs) and in pulmonary microvascular endothelial cells (HPMECs). PBMCs or HPMECs were incubated alone or with endotoxin-free recombinant variants of trimeric spike glycoproteins (Alpha, Beta, Delta, and Omicron, 12 µg/mL). Afterward, cells were stained with Oil Red O for LDs, cytokine release was determined through ELISA, and the gene expression was analyzed through real-time PCR using TaqMan assays. Our data show that spikes induce LDs in PBMCs but not in HPMECs. In line with this, in PBMCs, spike proteins lower the expression of genes involving lipid metabolism and LD formation, such as SREBF1, HMGCS1, LDLR, and CD36. On the other hand, PBMCs exposed to spikes for 6 or 18 h did not increase in IL-1β, IL-6, IL-8, MCP-1, and TNFα release or expression as compared to non-treated controls. Thus, spike-induced LD formation in PBMCs seems to not be related to cell inflammatory activation. Further detailed studies are warranted to investigate in which specific immune cells spikes induce LDs, and what are the pathophysiological mechanisms and consequences of this induction in vivo.

• Third COVID-19 vaccine dose (booster) enhances immune response. • Interim phase 2/3 data for booster ≥6 months after the 2nd dose in Japan are shown. • S-268019-b was noninferior to BNT162b2 in inducing neutralizing antibodies. • Sera boosted with either vaccine neutralized Delta and Omicron virus variants. • S-268019-b was safe, and results support its use as a booster in vaccinated adults. In this randomized, observer-blinded, phase 2/3 study, S-268019-b (n = 101), a recombinant spike protein vaccine, was analyzed for noninferiority versus BNT162b2 (n = 103), when given as a booster ≥6 months after 2-dose BNT162b2 regimen in Japanese adults without prior SARS-CoV-2 infection. Interim results showed noninferiority of S-268019-b versus BNT162b2 in co-primary endpoints for neutralizing antibodies on day 29: geometric mean titer (GMT) (124.97 versus 109.70; adjusted-GMT ratio [95% CI], 1.14 [0.94–1.39]; noninferiority P -value, <0.0001) and seroresponse rate (both 100%; noninferiority P -value, 0.0004). Both vaccines elicited anti-spike-protein immunoglobulin G antibodies, and produced T-cell response (n = 29/group) and neutralizing antibodies against Delta and Omicron pseudovirus and live virus variants (n = 24/group) in subgroups. Most participants reported low-grade reactogenicity on days 1–2, the most frequent being fatigue, fever, myalgia, and injection-site pain. No serious adverse events were reported. In conclusion, S-268019-b was safe and showed robust immunogenicity as a booster, supporting its use as COVID-19 booster vaccine. [ABSTRACT FROM AUTHOR]

We have developed a method for the inexpensive, high-level expression of antigenic protein fragments of SARS-CoV-2 proteins in Escherichia coli. Our approach uses the thermophilic family 9 carbohydrate-binding module (CBM9) as an N-terminal carrier protein and affinity tag. The CBM9 module was joined to SARS-CoV-2 protein fragments via a flexible proline–threonine linker, which proved to be resistant to E. coli proteases. Two CBM9-spike protein fragment fusion proteins and one CBM9-nucleocapsid fragment fusion protein largely resisted protease degradation, while most of the CBM9 fusion proteins were degraded at some site in the SARS-CoV-2 protein fragment. All of the fusion proteins were highly expressed in E. coli and the CBM9-ID-H1 fusion protein was shown to yield 122 mg/L of purified product. Three purified CBM9-SARS-CoV-2 fusion proteins were tested and found to bind antibodies directed to the appropriate SARS-CoV-2 antigenic regions. The largest intact CBM9 fusion protein, CBM9-ID-H1, incorporates spike protein amino acids 540–588, which is a conserved region overlapping and C-terminal to the receptor binding domain that is widely recognized by human convalescent sera and contains a putative protective epitope. [ABSTRACT FROM AUTHOR]

The avian coronavirus infectious bronchitis virus (IBV) S1 subunit of the spike (S) glycoprotein mediates viral attachment to host cells and the S2 subunit is responsible for membrane fusion. Using IBV Arkansas-type (Ark) S protein histochemistry, we show that extension of S1 with the S2 ectodomain improves binding to chicken tissues. Although the S1 subunit is the major inducer of neutralizing antibodies, vaccination with S1 protein has been shown to confer inadequate protection against challenge. The demonstrated contribution of S2 ectodomain to binding to chicken tissues suggests that vaccination with the ectodomain might improve protection compared to vaccination with S1 alone. Therefore, we immunized chickens with recombinant trimeric soluble IBV Ark-type S1 or S-ectodomain protein produced from codon-optimized constructs in mammalian cells. Chickens were primed at 12 days of age with water-in-oil emulsified S1 or S-ectodomain proteins, and then boosted 21 days later. Challenge was performed with virulent Ark IBV 21 days after boost. Chickens immunized with recombinant S-ectodomain protein showed statistically significantly ( P < 0.05) reduced viral loads 5 days post-challenge in both tears and tracheas compared to chickens immunized with recombinant S1 protein. Consistent with viral loads, significantly reduced ( P < 0.05) tracheal mucosal thickness and tracheal lesion scores revealed that recombinant S-ectodomain protein provided improved protection of tracheal integrity compared to S1 protein. These results indicate that the S2 domain has an important role in inducing protective immunity. Thus, including the S2 domain with S1 might be promising for better viral vectored and/or subunit vaccine strategies. [ABSTRACT FROM AUTHOR]

Objective: To assemble infectious bronchitis virus (IBV)-like particles bearing the recombinant spike protein and investigate the humoral immune responses in chickens. Results: IBV virus-like particles (VLPs) were generated through the co-infection with three recombinant baculoviruses separately encoding M, E or the recombinant S genes. The recombinant S protein was sufficiently flexible to retain the ability to self-assemble into VLPs. The size and morphology of the VLPs were similar to authentic IBV particles. In addition, the immunogenicity of IBV VLPs had been investigated. The results demonstrated that the efficiency of the newly generated VLPs was comparable to that of the inactivated M41 viruses in eliciting IBV-specific antibodies and neutralizing antibodies in chickens via subcutaneous inoculation. Conclusions: This work provides basic information for the mechanism of IBV VLP formation and develops a platform for further designing IBV VLP-based vaccines against IBV or other viruses. [ABSTRACT FROM AUTHOR]