From concept to delivery: a yeast-expressed recombinant protein-based COVID-19 vaccine technology suitable for global access.

INTRODUCTION: The development of a yeast-expressed recombinant protein-based vaccine technology co-developed with LMIC vaccine producers and suitable as a COVID-19 vaccine for global access is described. The proof-of-concept for developing a SARS-CoV-2 spike protein receptor-binding domain (RBD) antigen as a yeast-derived recombinant protein vaccine technology is described. AREAS COVERED: Genetic Engineering: The strategy is presented for the design and genetic modification used during cloning and expression in the yeast system. Process and Assay Development: A summary is presented of how a scalable, reproducible, and robust production process for the recombinant protein COVID-19 vaccine antigen was developed. Formulation and Pre-clinical Strategy: We report on the pre-clinical and formulation strategy used for the proof-of-concept evaluation of the SARS-CoV-2 RBD vaccine antigen. Technology Transfer and Partnerships: The process used for the technology transfer and co-development with LMIC vaccine producers is described. Clinical Development and Delivery: The approach used by LMIC developers to establish the industrial process, clinical development, and deployment is described. EXPERT OPINION: Highlighted is an alternative model for developing new vaccines for emerging infectious diseases of pandemic importance starting with an academic institution directly transferring their technology to LMIC vaccine producers without the involvement of multinational pharma companies.

A Novel Targeted RIG-I Receptor 5'Triphosphate Double Strain RNA-Based Adjuvant Significantly Improves the Immunogenicity of the SARS-CoV-2 Delta-Omicron Chimeric RBD-Dimer Recombinant Protein Vaccine.

The rapid mutation and spread of SARS-CoV-2 variants recently, especially through the emerging variants Omicron BA5, BF7, XBB and BQ1, necessitate the development of universal vaccines to provide broad spectrum protection against variants. For the SARS-CoV-2 universal recombinant protein vaccines, an effective approach is necessary to design broad-spectrum antigens and combine them with novel adjuvants that can induce high immunogenicity. In this study, we designed a novel targeted retinoic acid-inducible gene-I (RIG-I) receptor 5'triphosphate double strain RNA (5'PPP dsRNA)-based vaccine adjuvant (named AT149) and combined it with the SARS-CoV-2 Delta and Omicron chimeric RBD-dimer recombinant protein (D-O RBD) to immunize mice. The results showed that AT149 activated the P65 NF-κB signaling pathway, which subsequently activated the interferon signal pathway by targeting the RIG-I receptor. The D-O RBD + AT149 and D-O RBD + aluminum hydroxide adjuvant (Al) + AT149 groups showed elevated levels of neutralizing antibodies against the authentic Delta variant, and Omicron subvariants, BA1, BA5, and BF7, pseudovirus BQ1.1, and XBB compared with D-O RBD + Al and D-O RBD + Al + CpG7909/Poly (I:C) groups at 14 d after the second immunization, respectively. In addition, D-O RBD + AT149 and D-O RBD + Al + AT149 groups presented higher levels of the T-cell-secreted IFN-γ immune response. Overall, we designed a novel targeted RIG-I receptor 5'PPP dsRNA-based vaccine adjuvant to significantly improve the immunogenicity and broad spectrum of the SARS-CoV-2 recombinant protein vaccine.

Inducer-free recombinant protein production in Trichoderma reesei: secretory production of endogenous enzymes and heterologous nanobodies using glucose as the sole carbon source.

BACKGROUND: The filamentous fungus Trichoderma reesei has been used as a host organism for the production of lignocellulosic biomass-degrading enzymes. Although this microorganism has high potential for protein production, it has not yet been widely used for heterologous recombinant protein production. Transcriptional induction of the cellulase genes is essential for high-level protein production in T. reesei; however, glucose represses this transcriptional induction. Therefore, cellulose is commonly used as a carbon source for providing its degraded sugars such as cellobiose, which act as inducers to activate the strong promoters of the major cellulase (cellobiohydrolase 1 and 2 (cbh1 and cbh2) genes. However, replacement of cbh1 and/or cbh2 with a gene encoding the protein of interest (POI) for high productivity and occupancy of recombinant proteins remarkably impairs the ability to release soluble inducers from cellulose, consequently reducing the production of POI. To overcome this challenge, we first used an inducer-free biomass-degrading enzyme expression system, previously developed to produce cellulases and hemicellulases using glucose as the sole carbon source, for recombinant protein production using T. reesei. RESULTS: We chose endogenous secretory enzymes and heterologous camelid small antibodies (nanobody) as model proteins. By using the inducer-free strain as a parent, replacement of cbh1 with genes encoding two intrinsic enzymes (aspartic protease and glucoamylase) and three different nanobodies (1ZVH, caplacizumab, and ozoralizumab) resulted in their high secretory productions using glucose medium without inducers such as cellulose. Based on signal sequences (carrier polypeptides) and protease inhibitors, additional replacement of cbh2 with the nanobody gene increased the percentage of POI to about 20% of total secreted proteins in T. reesei. This allowed the production of caplacizumab, a bivalent nanobody, to be increased to 9.49-fold (508 mg/L) compared to the initial inducer-free strain. CONCLUSIONS: In general, whereas the replacement of major cellulase genes leads to extreme decrease in the degradation capacity of cellulose, our inducer-free system enabled it and achieved high secretory production of POI with increased occupancy in glucose medium. This system would be a novel platform for heterologous recombinant protein production in T. reesei.

A rapid procedure to generate stably transfected HEK293 suspension cells for recombinant protein manufacturing: Yield improvements, bioreactor production and downstream processing.

The human cell line HEK293 is one of the preferred choices for manufacturing therapeutic proteins and viral vectors for human applications. Despite its increased use, it is still considered in disadvantage in production aspects compared to cell lines such as the CHO cell line. We provide here a simple workflow for the rapid generation of stably transfected HEK293 cells expressing an engineered variant of the SARS-CoV-2 Receptor Binding Domain (RBD) carrying a coupling domain for linkage to VLPs through a bacterial transpeptidase-sortase (SrtA). To generate stable suspension cells expressing the RBD-SrtA, a single two plasmids transfection was performed, with hygromycin selection. The suspension HEK293 were grown in adherent conditions, with 20% FBS supplementation. These transfection conditions increased cell survival, allowing the selection of stable cell pools, which was otherwise not possible with standard procedures in suspension. Six pools were isolated, expanded and successfully re-adapted to suspension with a gradual increase of serum-free media and agitation. The complete process lasted four weeks. Stable expression with viability over 98% was verified for over two months in culture, with cell passages every 4-5 days. With process intensification, RBD-SrtA yields reached 6.4 µg/mL and 13.4 µg/mL in fed-batch and perfusion-like cultures, respectively. RBD-SrtA was further produced in fed-batch stirred tank 1L-bioreactors, reaching 10-fold higher yields than perfusion flasks. The trimeric antigen displayed the conformational structure and functionality expected. This work provides a series of steps for stable cell pool development using suspension HEK293 cells aimed at the scalable production of recombinant proteins.

Development of an Indirect Enzyme-Linked Immunosorbent Assay Based on the Yeast-Expressed CO-26K-Equivalent Epitope-Containing Antigen for Detection of Serum Antibodies against Porcine Epidemic Diarrhea Virus.

Porcine epidemic diarrhea (PED) is a severe contagious intestinal disease caused by the porcine epidemic diarrhea virus (PEDV), which leads to high mortality in piglets. In this study, by analyzing a total of 53 full-length spike genes and COE domain regions of PEDVs, the conserved COE fragment of the spike protein from the dominant strain SC1402 was chosen as the target protein and expressed successfully in Pichia pastoris (P. pastoris). Furthermore, an indirect enzyme-linked immunosorbent assay (iELISA) based on the recombinant COE protein was developed for the detection of anti-PEDV antibodies in pig sera. The results showed that under the optimized conditions, the cut-off value of COE-based indirect ELISA (COE-iELISA) was determined to be 0.12. Taking the serum neutralization test as standard, the relative sensitivity of the COE-iELISA was 94.4% and specificity 92.6%. Meanwhile, no cross-reactivity to other porcine pathogens was noted with this assay. The intra-assay and inter-assay coefficients of variation were less than 7%. Moreover, 164 vaccinated serum samples test showed that overall agreement between COE-iELISA and the actual diagnosis result was up to 99.4%. More importantly, the developed iELISA exhibited a 95.08% agreement rate with the commercial ELISA kit (Kappa value = 0.88), which suggested that the expressed COE protein was an effective antigen in serologic tests and the established COE-iELISA is reliable for monitoring PEDV infection in pigs or vaccine effectiveness.

Advances in developing ACE2 derivatives against SARS-CoV-2.

Extensive immune evasion of SARS-CoV-2 rendered therapeutic antibodies ineffective in the COVID-19 pandemic. Propagating SARS-CoV-2 variants are characterised by immune evasion capacity through key amino acid mutations, but can still bind human angiotensin-converting enzyme 2 (ACE2) through the spike protein and are, thus, sensitive to ACE2-mimicking decoys as inhibitors. In this Review, we examine advances in the development of ACE2 derivatives from the past 3 years, including the recombinant ACE2 proteins, ACE2-loaded extracellular vesicles, ACE2-mimicking antibodies, and peptide or mini-protein mimetics of ACE2. Several ACE2 derivatives are granted potent neutralisation efficacy against SARS-CoV-2 variants that rival or surpass endogenous antibodies by various auxiliary techniques such as chemical modification and practical recombinant design. The derivatives also represent enhanced production efficiency and improved bioavailability. In addition to these derivatives of ACE2, new effective therapeutics against SARS-CoV-2 variants are expected to be developed.

Structure-based design of oligomeric receptor-binding domain (RBD) recombinant proteins as potent vaccine candidates against SARS-CoV-2.

The receptor-binding domain (RBD) of SARS-CoV-2 S protein is proved to be the major target of neutralizing antibodies. However, on the S protein, only a portion of epitopes in RBD can be effectively displayed with dynamic changes in spatial conformations. Using RBD fragment as antigen can better expose the neutralizing epitopes, but the immunogenicity of RBD monomer is suboptimal. Multimeric display of RBD molecules is a feasible strategy to optimize RBD-based vaccines. In this study, RBD single-chain dimer derived from Wuhan-Hu-1 was fused with a trimerization motif, and a cysteine was also introduced at the C-terminus. The resultant recombinant protein 2RBDpLC was expressed in Sf9 cells using a baculovirus expression system. Reducing/non-reducing PAGE, size-exclusion chromatography and in silico structure prediction indicated that 2RBDpLC polymerized and possibly formed RBD dodecamers through trimerization motif and intermolecular disulfide bonds. In mice, 2RBDpLC induced higher levels of RBD-specific and neutralizing antibody responses than RBD dimer, RBD trimer and prefusion-stabilized S protein (S2P). In addition, cross-neutralizing antibodies against Delta and Omicron VOC were also detected in the immune sera. Our results demonstrate that 2RBDpLC is a promising vaccine candidate, and the method of constructing dodecamers may be an effective strategy for designing RBD-based vaccines.

Process development and characterization of recombinant nucleocapsid protein for its application on COVID-19 diagnosis.

COVID-19 pandemic was caused by the severe acute respiratory syndrome coronavirus 2 (Sars-CoV-2). The nucleocapsid (N) protein from Sars-CoV-2 is a highly immunogenic antigen and responsible for genome packing. Serological assays are important tools to detect previous exposure to SARS-CoV-2, complement epidemiological studies, vaccine evaluation and also in COVID-19 surveillance. SARS-CoV-2 N (r2N) protein was produced in Escherichia coli, characterized, and the immunological performance was evaluated by enzyme-linked immunosorbent assay (ELISA) and beads-based array immunoassay. r2N protein oligomers were evidenced when it is associated to nucleic acid. Benzonase treatment reduced host nucleic acid associated to r2N protein, but crosslinking assay still demonstrates the presence of higher-order oligomers. Nevertheless, after RNase treatment the higher-order oligomers reduced, and dimer form increased, suggesting RNA contributes to the oligomer formation. Structural analysis revealed nucleic acid did not interfere with the thermal stability of the recombinant protein. Interestingly, nucleic acid was able to prevent r2N protein aggregation even with increasing temperature while the protein benzonase treated begin aggregation process above 55 °C. In immunological characterization, ELISA performed with 233 serum samples presented a sensitivity of 97.44% (95% Confidence Interval, CI, 91.04%, 99.69%) and a specificity of 98.71% (95% CI, 95.42%, 99.84%) while beads-based array immunoassay carried out with 217 samples showed 100% sensitivity and 98.6% specificity. The results exhibited an excellent immunological performance of r2N protein in serologic assays showing that, even in presence of nucleic acid, it can be used as a component of an immunoassay for the sensitive and specific detection of SARS-CoV-2 antibodies.

[Preparation and immungenicity of recombinant protein containing intramolecular adjuvant in SARS-CoV-2 RBD domain].

A fusion protein containing a tetanus toxin peptide, a tuftsin peptide and a SARS-CoV-2S protein receptor-binding domain (RBD) was prepared to investigate the effect of intramolecular adjuvant on humoral and cellular immunity of RBD protein. The tetanus toxin peptide, tuftsin peptide and S protein RBD region were connected by a flexible polypeptide, and a recombinant vector was constructed after codon optimization. The recombinant S-TT-tuftsin protein was prepared by prokaryotic expression and purification. BALB/c mice were immunized after mixed with aluminum adjuvant, and the humoral and cellular immune effects were evaluated. The recombinant S-TT-tuftsin protein was expressed as an inclusion body, and was purified by ion exchange chromatography and renaturated by gradient dialysis. The renaturated protein was identified by Dot blotting and reacted with serum of descendants immunized with SARS-CoV-2 subunit vaccine. The results showed that the antibody level reached a plateau after 35 days of immunization, and the serum antibody ELISA titer of mice immunized with recombinant protein containing intramolecular adjuvant was up to 1:66 240, which was significantly higher than that of mice immunized with S-RBD protein (P < 0.05). At the same time, the recombinant protein containing intramolecular adjuvant stimulated mice to produce a stronger lymphocyte proliferation ability. The stimulation index was 4.71±0.15, which was significantly different from that of the S-RBD protein (1.83±0.09) (P < 0.000 1). Intramolecular adjuvant tetanus toxin peptide and tuftsin peptide significantly enhanced the humoral and cellular immune effect of the SARS-CoV-2 S protein RBD domain, which provideda theoretical basis for the development of subunit vaccines for SARS-CoV-2 and other viruses.

Recombinant Protein Vaccines against Human Betacoronaviruses: Strategies, Approaches and Progress.

Betacoronaviruses have already troubled humanity more than once. In 2002-2003 and 2012, the SARS-CoV and MERS-CoV, respectively, caused outbreaks of respiratory syndromes with a fatal outcome. The spread of the SARS-CoV-2 coronavirus has become a pandemic. These three coronaviruses belong to the genus Betacoronavirus and have a zoonotic origin. The emergence of new coronavirus infections in the future cannot be ruled out, and vaccination is the main way to prevent the spread of the infection. Previous experience in the development of vaccines against SARS and MERS has helped to develop a number of vaccines against SARS-CoV-2 in a fairly short time. Among them, there are quite a few recombinant protein vaccines, which seem to be very promising in terms of safety, minimization of side effects, storage and transportation conditions. The problem of developing a universal betacoronavirus vaccine is also still relevant. Here, we summarize the information on the designing of vaccines based on recombinant proteins against highly pathogenic human betacoronaviruses SARS-CoV, MERS-CoV and SARS-CoV-2.

Assessment of Immunogenic and Antigenic Properties of Recombinant Nucleocapsid Proteins of Five SARS-CoV-2 Variants in a Mouse Model.

COVID-19 cases caused by new variants of highly mutable SARS-CoV-2 continue to be identified worldwide. Effective control of the spread of new variants can be achieved through targeting of conserved viral epitopes. In this regard, the SARS-CoV-2 nucleocapsid (N) protein, which is much more conserved than the evolutionarily influenced spike protein (S), is a suitable antigen. The recombinant N protein can be considered not only as a screening antigen but also as a basis for the development of next-generation COVID-19 vaccines, but little is known about induction of antibodies against the N protein via different SARS-CoV-2 variants. In addition, it is important to understand how antibodies produced against the antigen of one variant can react with the N proteins of other variants. Here, we used recombinant N proteins from five SARS-CoV-2 strains to investigate their immunogenicity and antigenicity in a mouse model and to obtain and characterize a panel of hybridoma-derived monoclonal anti-N antibodies. We also analyzed the variable epitopes of the N protein that are potentially involved in differential recognition of antiviral antibodies. These results will further deepen our knowledge of the cross-reactivity of the humoral immune response in COVID-19.

Mucosal immune responses induced by oral administration of recombinant Lactococcus lactis expressing the S1 protein of PDCoV.

Porcine deltacoronavirus is an evolving coronavirus that primarily infects the intestine and may lead to intestinal disease in piglets. Up to now, no commercial vaccination is readily accessible to protect against the spread of PDCoV. Lactococcus lactis has been shown to have good immune efficacy and safety and can be used as a genetically engineered vaccine to deliver antigens. In this research, we utilized L. lactis NZ9000 to provide the S1 protein orally and improved the delivery efficiency by connecting the M cell targeting ligand Co1 with the S1 protein of PDCoV in tandem to obtain the recombinant protein S1-Co1. We successfully constructed two recombinant strains capable of expressing PDCoV-S1 and PDCoV-S1-Co1 proteins (i.e., L. lactis NZ9000-S1 and L. lactis NZ9000-S1-Co1), and their immunogenic capacity was evaluated in mice. Our study shows that Lactococcus is an advantageous bacterial live vector vaccine and is anticipated as a potential PDCoV vaccination option.

Interim analysis from a phase 2 randomized trial of EuCorVac-19: a recombinant protein SARS-CoV-2 RBD nanoliposome vaccine.

BACKGROUND: Numerous vaccine strategies are being advanced to control SARS-CoV-2, the cause of the COVID-19 pandemic. EuCorVac-19 (ECV19) is a recombinant protein nanoparticle vaccine that displays the SARS-CoV-2 receptor-binding domain (RBD) on immunogenic nanoliposomes. METHODS: Initial study of a phase 2 randomized, observer-blind, placebo-controlled trial to assess the immunogenicity, safety, and tolerance of ECV19 was carried out between July and October 2021. Two hundred twenty-nine participants were enrolled at 5 hospital sites in South Korea. Healthy adults aged 19-75 without prior known exposure to COVID-19 were vaccinated intramuscularly on day 0 and day 21. Of the participants who received two vaccine doses according to protocol, 100 received high-dose ECV19 (20 µg RBD), 96 received low-dose ECV19 (10 µg RBD), and 27 received placebo. Local and systemic adverse events were monitored. Serum was assessed on days 0, 21, and 42 for immunogenicity analysis by ELISA and neutralizing antibody response by focus reduction neutralization test (FRNT). RESULTS: Low-grade injection site tenderness and pain were observed in most participants. Solicited systemic adverse events were less frequent, and mostly involved low-grade fatigue/malaise, myalgia, and headache. No clinical laboratory abnormalities were observed. Adverse events did not increase with the second injection and no serious adverse events were solicited by ECV19. On day 42, Spike IgG geometric mean ELISA titers were 0.8, 211, and 590 Spike binding antibody units (BAU/mL) for placebo, low-dose and high-dose ECV19, respectively (p < 0.001 between groups). Neutralizing antibodies levels of the low-dose and high-dose ECV19 groups had FRNT50 geometric mean values of 129 and 316, respectively. Boosting responses and dose responses were observed. Antibodies against the RBD correlated with antibodies against the Spike and with virus neutralization. CONCLUSIONS: ECV19 was generally well-tolerated and induced antibodies in a dose-dependent manner that neutralized SARS-CoV-2. The unique liposome display approach of ECV19, which lacks any immunogenic protein components besides the antigen itself, coupled with the lack of increased adverse events during boosting suggest the vaccine platform may be amenable to multiple boosting regimes in the future. Taken together, these findings motivate further investigation of ECV19 in larger scale clinical testing that is underway. TRIAL REGISTRATION: The trial was registered at ClinicalTrials.gov as # NCT04783311.

Expression and Immunogenicity of SARS-CoV-2 Virus-Like Particles based on Recombinant Truncated HEV-3 ORF2 Capsid Protein.

COVID-19 is an emerging disease that poses a severe threat to global public health. As such, there is an urgent demand for vaccines against SARS-CoV-2, the virus that causes COVID-19. Here, we describe a virus-like nanoparticle candidate vaccine against SARS-CoV-2 produced by an E. coli expression system. The fusion protein of a truncated ORF2-encoded protein of aa 439~608 (p170) from hepatitis E virus CCJD-517 and the receptor-binding domain of the spike protein from SARS-CoV-2 were expressed, purified and characterized. The antigenicity and immunogenicity of p170-RBD were evaluated in vitro and in Kunming mice. Our investigation revealed that p170-RBD self-assembled into approximately 24 nm virus-like particles, which could bind to serum from vaccinated people (p < 0.001) and receptors on cells. Immunization with p170-RBD induced the titer of IgG antibody vaccine increased from 14 days post-immunization and was significantly enhanced after a booster immunization at 28 dpi, ultimately reaching a peak level on 42 dpi with a titer of 4.97 log10. Pseudovirus neutralization tests showed that the candidate vaccine induced a strong neutralizing antibody response in mice. In this research, we demonstrated that p170-RBD possesses strong antigenicity and immunogenicity and could be a potential candidate for use in future SARS-CoV-2 vaccine development.

The transient expression of recombinant proteins in plant cell packs facilitates stable isotope labelling for NMR spectroscopy.

Nuclear magnetic resonance (NMR) spectroscopy can be used to determine the structure, dynamics and interactions of proteins. However, protein NMR requires stable isotope labelling for signal detection. The cells used for the production of recombinant proteins must therefore be grown in medium containing isotopically labelled substrates. Stable isotope labelling is well established in Escherichia coli, but bacteria are only suitable for the production of simple proteins without post-translational modifications. More complex proteins require eukaryotic production hosts, but their growth can be impaired by labelled media, thus reducing product yields and increasing costs. To address this limitation, we used media supplemented with isotope-labelled substrates to cultivate the tobacco-derived cell line BY-2, which was then cast into plant cell packs (PCPs) for the transient expression of a labelled version of the model protein GB1. Mass spectrometry confirmed the feasibility of isotope labelling with 15 N and 2 H using this approach. The resulting NMR spectrum featured a signal dispersion comparable to recombinant GB1 produced in E. coli. PCPs therefore offer a rapid and cost-efficient alternative for the production of isotope-labelled proteins for NMR analysis, especially suitable for complex proteins that cannot be produced in microbial systems.

Identification and characterization of a novel cell binding and cross-reactive region on spike protein of SARS-CoV-2.

Given that COVID-19 continues to wreak havoc around the world, it is imperative to search for a conserved region involved in viral infection so that effective vaccines can be developed to prevent the virus from rapid mutations. We have established a twelve-fragment library of recombinant proteins covering the entire region of spike protein of both SARS-CoV-2 and SARS-CoV from Escherichia coli. IgGs from murine antisera specifically against 6 spike protein fragments of SARS-CoV-2 were produced, purified, and characterized. We found that one specific IgG against the fusion process region, named COVID19-SF5, serologically cross-reacted with all twelve S-protein fragments. COVID19-SF5, with amino acid sequences from 880 to 1084, specifically bound to VERO-E6 and BEAS-2B cells, with K_d values of 449.1 ± 21.41 and 381.9 ± 31.53 nM, and IC₅₀ values of 761.2 ± 28.2 nM and 862.4 ± 32.1 nM, respectively. In addition, COVID19-SF5 greatly enhanced binding of the full-length CHO cell-derived spike protein to the host cells in a concentration-dependent manner. Furthermore, COVID19-SF5 and its IgGs inhibited the infection of the host cells by pseudovirus. The combined data from our studies reveal that COVID19-SF5, a novel cell-binding fragment, may contain a common region(s) for mediating viral binding during infection. Our studies also provide valuable insights into how virus variants may evade host immune recognition. Significantly, the observation that the IgGs against COVID19-SF5 possesses cross reactivity to all other fragments of S protein, suggesting that it is possible to develop universal neutralizing monoclonal antibodies to curb rapid mutations of COVID-19.

Steric accessibility of the N-terminus improves the titer and quality of recombinant proteins secreted from Komagataella phaffii.

BACKGROUND: Komagataella phaffii is a commonly used alternative host for manufacturing therapeutic proteins, in part because of its ability to secrete recombinant proteins into the extracellular space. Incorrect processing of secreted proteins by cells can, however, cause non-functional product-related variants, which are expensive to remove in purification and lower overall process yields. The secretion signal peptide, attached to the N-terminus of the recombinant protein, is a major determinant of the quality of the protein sequence and yield. In K. phaffii, the signal peptide from the Saccharomyces cerevisiae alpha mating factor often yields the highest secreted titer of recombinant proteins, but the quality of secreted protein can vary highly. RESULTS: We determined that an aggregated product-related variant of the SARS-CoV-2 receptor binding domain is caused by N-terminal extension from incomplete cleavage of the signal peptide. We eliminated this variant and improved secreted protein titer up to 76% by extension of the N-terminus with a short, functional peptide moiety or with the EAEA residues from the native signal peptide. We then applied this strategy to three other recombinant subunit vaccine antigens and observed consistent elimination of the same aggregated product-related variant. Finally, we demonstrated that this benefit in quality and secreted titer can be achieved with addition of a single amino acid to the N-terminus of the recombinant protein. CONCLUSIONS: Our observations suggest that steric hindrance of proteases in the Golgi that cleave the signal peptide can cause unwanted N-terminal extension and related product variants. We demonstrated that this phenomenon occurs for multiple recombinant proteins, and can be addressed by minimal modification of the N-terminus to improve steric accessibility. This strategy may enable consistent secretion of a broad range of recombinant proteins with the highly productive alpha mating factor secretion signal peptide.

A Novel Single-Stranded RNA-Based Adjuvant Improves the Immunogenicity of the SARS-CoV-2 Recombinant Protein Vaccine.

The research and development (R&D) of novel adjuvants is an effective measure for improving the immunogenicity of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) recombinant protein vaccine. Toward this end, we designed a novel single-stranded RNA-based adjuvant, L2, from the SARS-CoV-2 prototype genome. L2 could initiate retinoic acid-inducible gene-I signaling pathways to effectively activate the innate immunity. ZF2001, an aluminum hydroxide (Al) adjuvanted SARS-CoV-2 recombinant receptor binding domain (RBD) subunit vaccine with emergency use authorization in China, was used for comparison. L2, with adjuvant compatibility with RBD, elevated the antibody response to a level more than that achieved with Al, CpG 7909, or poly(I:C) as adjuvants in mice. L2 plus Al with composite adjuvant compatibility with RBD markedly improved the immunogenicity of ZF2001; in particular, neutralizing antibody titers increased by about 44-fold for Omicron, and the combination also induced higher levels of antibodies than CpG 7909/poly(I:C) plus Al in mice. Moreover, L2 and L2 plus Al effectively improved the Th1 immune response, rather than the Th2 immune response. Taken together, L2, used as an adjuvant, enhanced the immune response of the SARS-CoV-2 recombinant RBD protein vaccine in mice. These findings should provide a basis for the R&D of novel RNA-based adjuvants.

Systematic strategies for developing phage resistant Escherichia coli strains.

Phages are regarded as powerful antagonists of bacteria, especially in industrial fermentation processes involving bacteria. While bacteria have developed various defense mechanisms, most of which are effective against a narrow range of phages and consequently exert limited protection from phage infection. Here, we report a strategy for developing phage-resistant Escherichia coli strains through the simultaneous genomic integration of a DNA phosphorothioation-based Ssp defense module and mutations of components essential for the phage life cycle. The engineered E. coli strains show strong resistance against diverse phages tested without affecting cell growth. Additionally, the resultant engineered phage-resistant strains maintain the capabilities of producing example recombinant proteins, D-amino acid oxidase and coronavirus-encoded nonstructural protein nsp8, even under high levels of phage cocktail challenge. The strategy reported here will be useful for developing engineered E. coli strains with improved phage resistance for various industrial fermentation processes for producing recombinant proteins and chemicals of interest.