Domain Fusion Protein Research Articles

A novel subunit vaccine based on Fiber1/2 knob domain provides full protection against fowl adenovirus serotype 4 and induces stronger immune responses than a Fiber2 subunit vaccine

Outbreaks of hepatitis-hydropericardium syndrome (HHS) caused by fowl adenovirus serotype 4 (FAdV-4) have resulted in huge economic losses to the poultry industry in China since 2015. However, commercially available vaccines against the FAdV-4 infection remain scarce. In our study, subunit vaccine candidates derived from the bacterially expressed recombinant Fiber1 knob domain and Fiber2 knob domain fusion protein (termed as Fiber1/2 knob subunit vaccine) and Fiber2 protein (termed as Fiber2 subunit vaccine) of the FAdV-4 SDSX strain were developed. Immunogenicity evaluation showed that the Fiber1/2 knob subunit vaccine induced the production of antibodies at 7 d postvaccination (dpv), earlier than the Fiber2 subunit vaccine. Moreover, the neutralizing antibody level of the Fiber1/2 subunit vaccine group was higher than the Fiber2 subunit vaccine group, showing significant differences at 14, 21, and 28 dpv. Immune protection test results revealed that both Fiber1/2 knob subunit and Fiber2 subunit vaccines could protect chickens from death against FAdV-4 challenge, although the weight of chickens in the Fiber1/2 knob subunit vaccine group decreased less. Furthermore, analysis of plasma Glutamic oxaloacetic transaminase (AST) and blood glutamic pyruvic transaminase (ALT) levels suggested that the Fiber1/2 subunit vaccine can significantly inhibit liver damage caused by FAdV-4 infection and is more effective in blocking the pathogenicity of FAdV-4 in target organs. In addition, the Fiber1/2 knob subunit vaccine further reduced the viral load in different tissues and virus shedding in chickens than the Fiber2 subunit vaccine. Overall, the Fiber1/2 knob subunit vaccine was more effective than the Fiber2 subunit vaccine. These findings lay the foundation for the development of more effective FAdV-4 subunit vaccines.

‘Nunchuck’ proteins: Short flexible linkers resist proteolysis by facilitating motions in flanking domains to inhibit the approach of proteases

Peptides linking well-folded and non-interacting domains in fusion proteins can undergo proteolytic degradation. This leads to physical separation of the domains that were originally sought to be joined. In order to identify characteristics that determine linker degradation propensity, we selected a pair of thermostable, proteolytically-resistant domains, and joined them using five different linkers. We then assessed linker degradation propensities through size-exclusion chromatography, and denaturing and non-denaturing electrophoresis. The domains used were Coh2, an all-beta cohesin from C. thermocellum CipA, and BSX, a beta/alpha barrel xylanase from Bacillus sp. NG-27, while the linkers used were Rigid (3 repeats of N-EAAAK-C), Flexible (two repeats of N-SGGGG-C), Nat-full (42 residues of a Coh2-adjacent linker from CipA), Nat-half (a 21 residues-long derivative of Nat-full) and Nat-quarter (a 9 residues-long derivative of Nat-full). Both with proteolysis effected by proteases present in the environment, and with an exogenously-added protease (Subtilisin A), we found that Flexible underwent little or no degradation, whereas linkers of comparable length like Nat-quarter or Rigid underwent extensive degradation, as did longer linkers like Nat-Half and Nat-Full. Our analyses disfavor the likelihood of the sequence of Flexible being naturally resistant to proteolysis, and instead favor the explanation that the flexibility of Flexible facilitates movements of Coh2 relative to BSX which then serve to sterically prevent the approach of proteases. Thus, the construct incorporating Flexible appears to behave like a ‘nunchuck’ in which rods/spheres flanking a chain collide with approaching swords that are capable of severing the chain, to prevent severance.

Immunity-linked genes are stimulated by a membrane stress pathway linked to Golgi function and the ARF-1 GTPase.

Infection response and other immunity-linked genes (ILGs) were first named in Caenorhabditis elegans-based expression after pathogen challenge, but many are also up-regulated when lipid metabolism is perturbed. Why pathogen attack and metabolic changes both increase ILGs is unclear. We find that ILGs are activated when phosphatidylcholine (PC) levels change in membranes of secretory organelles in C. elegans. RNAi targeting of the ADP-ribosylation factor arf-1, which disrupts the Golgi and secretory function, also activates ILGs. Low PC limits ARF-1 function, suggesting a mechanism for ILG activation via lipid metabolism, as part of a membrane stress response acting outside the ER. RNAi of selected ILGs uncovered defects in the secretion of two GFP reporters and the accumulation of a pathogen-responsive complement C1r/C1s, Uegf, Bmp1 (CUB) domain fusion protein. Our data argue that up-regulation of some ILGs is a coordinated response to changes in trafficking and may act to counteract stress on secretory function.

627. Bioinformatic Approach to Design a Plasmodium falciparum PfRipr Multi-Epitope Vaccine Construct

Abstract Background Malaria infections remain an enormous contributor to global fatalities with an estimated death toll of 619,000 in 2021 and 77% of deaths being children aged under 5 years. Transmitted via mosquito, Plasmodium falciparum is the most lethal parasite of its genus but has evaded many treatment and vaccine efforts due to its complex life cycle and redundant invasion mechanisms. Epitope-based vaccines hold significant promise for malaria vaccine development due to their ease of development and ability to target dominant regions in antigenically variable pathogens. The recently characterized protein P. falciparum merozoite Rh5 interacting protein (PfRipr) is nonredundant, highly conserved, and essential for erythrocyte invasion, making it an ideal target for a bloodstage malaria vaccine. Merozoite Invasion Model of binding and insertion of PRCR complex to erythrocyte basigin in merozoite invasion. Adapted from Scally et al. 2022. Methods Using P. falciparum sequences collected from Burkina Faso and Uganda, we assessed the immunogenic potential of PfRipr epitopes with regard to T-cell receptor binding and B-cell recognition. T-cell receptor binding was predicted using NetMHCpan searching against MHC I and II alleles with high regional frequencies. Using an in-silico 3D model of PfRipr predicted via AlphaFold, tertiary structures of all PfRipr sample sequences were predicted via SWISS-MODEL then analyzed by ElliPro to identify linear and discontinuous B-cell epitopes. Putative epitopes were filtered using allele coverage, conservation, antigenicity, and allergenicity. B-Cell Epitope Prediction Protocol for B-cell epitope prediction using AlphaFold for template modeling, Swiss-Model for homology-based modeling of sample sequences, and ElliPro for continuous and discontinuous epitope prediction.​ Epitope Sorting and Filtering Protocol for sorting T-cell epitopes using Average Allele Coverage (percentage of alleles in which the protein sequence was identified as an epitope) and B-cell epitopes using ElliPro Score (Protrusion Index representing surface accessibility of the epitope).​ Results Between the two datasets, there were 19 matching epitopes with 7 MHC I, 9 MHC II, 1 linear, and 2 discontinuous. These epitopes were used to design a multi-epitope-based bloodstage vaccine construct against P. falciparum. MHC Epitopes Partial list of MHC epitopes found in Burkina Faso and Uganda datasets. B-Cell Epitopes B-cell epitopes found in Burkina Faso and Uganda datasets. PfRipr 3D Structure with B-cell Epitopes 3D model of PfRipr predicted via AlphaFold with labeled B-cell epitopes Conclusion To validate their predicted immunogenicity, epitopes can be further investigated using in silico protein stabilization and docking simulations, in vitro methods such as HLA stabilization or T-cell activation assays, and in vivo methods using transgenic mouse models. The pipeline of immunoinformatic analyses formulated in this project can be further applied to P. falciparum sequence datasets collected from other malaria endemic regions to develop vaccines effective against circulating strains. Multi-Epitope Vaccine Construct Schematic representation of hypothetical multi-epitope vaccine construct. FliC is added to the N-terminus to stimulate and amplify immune response. The EAAAK linker is used to separate the bifunctional fusion protein domains. AAY linkers are used to amplify MHC I and II epitopes and prevent junctional epitope binding. GPGPG linkers between B-cell epitopes function to minimize junctional epitopes and retain conformational-dependent immunogenicity. A six amino acid Histidine (6H) chain was added to the C-terminus to aid during lab purification processes. Disclosures All Authors: No reported disclosures

In Silico Prediction of Plasmodium falciparum PfRipr Epitopes as Vaccine Candidates

Abstract Background The global malarial death toll in 2020 was ~627,000, 77% of which occurred in children <5 years of age. Plasmodium falciparum is the most lethal parasite of its genus but has evaded vaccine development due to its complex life cycle and redundant invasion mechanisms. Epitope-based vaccines hold significant promise due to their ability to target dominant and/or conserved regions in highly antigenically variable pathogens, including malaria. The recently characterized P. falciparum merozoite Rh5 interacting protein (PfRipr) is nonredundant, highly conserved, and essential for erythrocyte invasion, making it an ideal target for a bloodstage malaria vaccine. (Figure 1) We sought to analyze P. falciparum sequences from highly endemic areas using bioinformatic approaches that identify candidate PfRipr epitope-based bloodstage vaccine antigens for inclusion in a multiantigen vaccine. Figure 1: Model of binding and insertion of PRCR complex to erythrocyte basigin in merozoite invasion. Adapted from Scally et al. 2022. (https://doi.org/10.1038/s41564-022-01261-2 , https://creativecommons.org/licenses/by/4.0/) Methods Using two P. falciparum sequence datasets from Burkina Faso and Uganda, we assessed the immunogenic potential of PfRipr epitopes for T-cell receptor binding and B-cell recognition. T-cell receptor binding was predicted using NetMHCpan and NetMHCIIpan searching against MHC I and II alleles with high regional frequencies. Using an in-silico 3D model of PfRipr predicted via AlphaFold, tertiary structures of all PfRipr sample sequences were predicted via SWISS-MODEL then analyzed by ElliPro to identify linear and discontinuous B-cell epitopes. (Figure 2) Putative epitopes were filtered using binding thresholds, allele coverage, conservation, antigenicity, and allergenicity. (Figure 3) Figure 2: Protocol for B-cell epitope prediction using AlphaFold for template modeling, Swiss-Model for homology-based modeling of sample sequences, and ElliPro for continuous and discontinuous epitope prediction. Figure 3: Protocol for sorting T-cell epitopes using Average Allele Coverage (percentage of alleles in which the protein sequence was identified as an epitope) and B-cell epitopes using ElliPro Score (Protrusion Index representing surface accessibility of the epitope). Results The Burkina Faso PfRipr dataset predicted 1729 unique T-cell and 51 unique B-cell epitopes, and 28 T-cell and 4 B-cell epitopes remained post-filtration. Epitope prediction using the Ugandan dataset predicted 576 unique T-cell and 53 unique B-cell epitopes, and 57 T-cell and 7 B-cell epitopes remained post-filtration. Between the two datasets, there were 19 matching epitopes with 7 MHC I, 9 MHC II, 1 linear, and 2 discontinuous. (Tables 1-2, Figure 4) These epitopes were used to design a multi-epitope-based vaccine construct with a Flagellin adjuvant and peptide linkers to separate functional regions for immune processing. (Figure 5) Figure 4: 3D model of PfRipr with labeled B-cell epitopes. Predicted tertiary structure adapted from AlphaFold database. (https://doi.org/10.1038/s41586-021-03819-2, https://doi.org/10.1093/nar/gkab1061, https://creativecommons.org/licenses/by/4.0/) Figure 5: Schematic representation of hypothetical multi-epitope vaccine construct. FliC is added to the N-terminus to stimulate and amplify immune response. The EAAAK linker is used to separate the bifunctional fusion protein domains. AAY linkers are used to amplify MHC I and II epitopes and prevent junctional epitope binding. GPGPG linkers between B-cell epitopes function to minimize junctional epitopes and retain conformational-dependent immunogenicity. A six amino acid Histidine (6H) chain was added to the C-terminus to aid during lab purification processes. Conclusions Immunoinformatic tools employed in sequence identified candidate PfRipr epitopes used to design a bloodstage vaccine construct against P. falciparum. To validate their predicted immunogenicity, epitopes can be further investigated using in silico protein stabilization and docking simulations, in vitro methods such as HLA stabilization or T-cell activation assays, and in vivo methods using transgenic mouse models. The pipeline of immunoinformatic analyses formulated in this project can be further applied to a larger database of diverse P. falciparum sequences collected from other malaria endemic regions to develop vaccines effective against circulating strains.

An engineered N-acyltransferase-LOV2 domain fusion protein enables light-inducible allosteric control of enzymatic activity

Transferases are ubiquitous across all known life. While much work has been done to understand and describe these essential enzymes, there have been minimal efforts to exert tight and reversible control over their activity for various biotechnological applications. Here, we apply a rational, computation-guided methodology to design and test a transferase-class enzyme allosterically regulated by light-oxygen-voltage 2 sensing domain. We utilize computational techniques to determine the intrinsic allosteric networks within N-acyltransferase (Orf11/∗Dbv8) and identify potential allosteric sites on the protein's surface. We insert light-oxygen-voltage 2 sensing domain at the predicted allosteric site, exerting reversible control over enzymatic activity. We demonstrate blue-light regulation of N-acyltransferase (Orf11/∗Dbv8) function. Our study for the first time demonstrates optogenetic regulation of a transferase-class enzyme as a proof-of-concept for controllable transferase design. This successful design opens the door for many future applications in metabolic engineering and cellular programming.

Production of recombinant human tumor necrosis factor receptor- IgG1 Fc domain fusion protein expressed by genetically CHO- DG44 cells

BACKGROUND: TNF-α (Tumor necrosis factor-alpha) plays a vital role in the human immune system. However, abnormal activity and overexpression of TNF-α are some of the causes of allergic and autoimmune diseases. Inhibiting the activity of this molecule is one of the novel pathologies for these diseases. The development of this recombinant protein is expected to reduce the financial burden of treating inflammatory rheumatic diseases. OBJECTIVE: The study’s objective was to generate and evaluate the biological activity of TNFR-Fc, construct of fusions an extracellular part TNF-α receptor (p75) and an Fc fragment of human immunoglobulin G1, expressed from the CHO-DG44 cell system. METHODS: The recombinant TNFR- Fc plasmid was constructed and identified by PCR, restriction enzyme digestion, and sequencing. A stable cell line for expression of TNFR-Fc was selected by limiting dilution cloning. Structural characterization, the binding affinity of TNFR-Fc to TNF-α, the neutralizing the cytotoxic activity- induced TNF-α, and the TNF-α-induced apoptosis suppression of TNFR- Fc were analyzed by SDS/PAGE Western blotting, ELISA, WST assay, Immunofluorescence, and flow cytometry. RESULTS: Preliminary analysis of the structural characteristics showed that TNFR-Fc is a low- glycosylated protein and perhaps in dimeric form. Furthermore, the recombinant TNFR-Fc can interact with its ligand TNF-α with a dissociation constant Kd 0.25±0.03μM equivalent to that of the original drug, Etanercept. We also demonstrated that TNFR-Fc expressed from CHO-DG44 was able to neutralize TNF-α- induced cytotoxic activity and inhibited p53-related apoptosis in vitro, similar to Etanercept. CONCLUSIONS: These data collectively suggested that TNFR-Fc potently blocks TNF-α, which could be a novel therapeutic strategy for cytokine-driven diseases.

Development and characterization of influenza M2 ectodomain and/or hemagglutinin stalk-based dendritic cell-targeting vaccines.

A universal influenza vaccine is required for broad protection against influenza infection. Here, we revealed the efficacy of novel influenza vaccine candidates based on Ebola glycoprotein dendritic cell (DC)-targeting domain (EΔM) fusion protein technology. The four copies of ectodomain matrix protein of influenza (tM2e) or M2e hemagglutinin stalk (HA stalk) peptides (HM2e) were fused with EΔM to generate EΔM-tM2e or EΔM-HM2e, respectively. We demonstrated that EΔM-HM2e- or EΔM-tM2e-pseudotyped viral particles can efficiently target DC/macrophages in vitro and induced significantly high titers of anti-HA and/or anti-M2e antibodies in mice. Significantly, the recombinant vesicular stomatitis virus (rVSV)-EΔM-tM2e and rVSV-EΔM-HM2e vaccines mediated rapid and potent induction of M2 or/and HA antibodies in mice sera and mucosa. Importantly, vaccination of rVSV-EΔM-tM2e or rVSV-EΔM-HM2e protected mice from influenza H1N1 and H3N2 challenges. Taken together, our study suggests that rVSV-EΔM-tM2e and rVSV-EΔM-HM2e are promising candidates that may lead to the development of a universal vaccine against different influenza strains.

Abstract 5067: Identify the combination efficacy and immune regulation of Abraxane combines with human IL-15 albumin binding domain fusion protein on pancreatic ductal adenocarcinoma animal model

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the hardest medical challenge in the world. Nab-paclitaxel is significantly more effective chemotherapy drugs than paclitaxel formulated as Cremophor EL. In this study, we reported the immune effect of Abraxane to combine with our new design recombinant human IL-15 fused with albumin binding domain (hIL-15-ABD) on subcutaneously Panc02 and orthotopically KPC PDAC animal models. In our results, the combination treatment greatly reduced the subcutaneous tumor growth in Panc02 and KPC bearing models, compared with vehicle group. Furthermore, the combinational therapy successfully buildup the anticancer immunity microenvironment (TME) through increasing the activation of effector cells, such as of NK cells (CD3-/CD49b+/NK1.1+) and CD8+ T (CD8+/IFN-γ+/IL-2+) cells. Moreover, combination therapy may also decrease the accumulation of immunosuppressvie cells such as MDSCs (CD11b+/Gr-1+) and Tregs (CD4+/CD25+/Foxp3+). The combination of Abraxane and hIL-15-ABD were also reduced the NF-κB-mediated immunosuppressive markers, including IDO, Foxp3 and VEGF. In conclusion, Abraxane combined with hIL-15-ABD enhanced immue effector cells involved in both innate and adaptive immunities, reduced the TME’s immunosuppressive cells, and displayed superior inhibitory effect on both subcutaneous and orthotopic syngeneic murine pancreatic ductal adenocarcinoma models than that of either monotherapy. These results suggest that combinational therapy of Abraxane and hIL-15-ABD could be a promising treatment stratedgy for PDAC. Citation Format: Po-Fu Yueh, Fei-Ting Hsu, Keng-Li Lan. Identify the combination efficacy and immune regulation of Abraxane combines with human IL-15 albumin binding domain fusion protein on pancreatic ductal adenocarcinoma animal model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5067.

Biochemical and structural characterizations of thioredoxin reductase selenoproteins of the parasitic filarial nematodes Brugia malayi and Onchocerca volvulus

Enzymes in the thiol redox systems of microbial pathogens are promising targets for drug development. In this study we characterized the thioredoxin reductase (TrxR) selenoproteins from Brugia malayi and Onchocerca volvulus, filarial nematode parasites and causative agents of lymphatic filariasis and onchocerciasis, respectively. The two filarial enzymes showed similar turnover numbers and affinities for different thioredoxin (Trx) proteins, but with a clear preference for the autologous Trx. Human TrxR1 (hTrxR1) had a high and similar specific activity versus the human and filarial Trxs, suggesting that, in vivo, hTrxR1 could possibly be the reducing agent of parasite Trxs once they are released into the host. Both filarial TrxRs were efficiently inhibited by auranofin and by a recently described inhibitor of human TrxR1 (TRi-1), but not as efficiently by the alternative compound TRi-2. The enzyme from B. malayi was structurally characterized also in complex with NADPH and auranofin, producing the first crystallographic structure of a nematode TrxR. The protein represents an unusual fusion of a mammalian-type TrxR protein architecture with an N-terminal glutaredoxin-like (Grx) domain lacking typical Grx motifs. Unlike thioredoxin glutathione reductases (TGRs) found in platyhelminths and mammals, which are also Grx–TrxR domain fusion proteins, the TrxRs from the filarial nematodes lacked glutathione disulfide reductase and Grx activities. The structural determinations revealed that the Grx domain of TrxR from B. malayi contains a cysteine (C22), conserved in TrxRs from clade IIIc nematodes, that directly interacts with the C-terminal cysteine-selenocysteine motif of the homo-dimeric subunit. Interestingly, despite this finding we found that altering C22 by mutation to serine did not affect enzyme catalysis. Thus, although the function of the Grx domain in these filarial TrxRs remains to be determined, the results obtained provide insights on key properties of this important family of selenoprotein flavoenzymes that are potential drug targets for treatment of filariasis.

Characterization of the binding of cytosolic phospholipase A2 alpha and NOX2 NADPH oxidase in mouse macrophages.

Previous studies have demonstrated that cytosolic phospholipase A2α (cPLA2α) is required for NOX2 NADPH oxidase activation in human and mouse phagocytes. Moreover, upon stimulation, cPLA2α translocates to the plasma membranes by binding to the assembled oxidase, forming a complex between its C2 domain and the PX domain of the cytosolic oxidase factor, p47phox in human phagocytes. Intravenous administration of antisense against cPLA2α that significantly inhibited its expression in mouse peritoneal neutrophils and macrophages also inhibited superoxide production, in contrast to cPLA2α knockout mice that showed normal superoxide production. The present study aimed to determine whether there is a binding between cPLA2α-C2 domain and p47phox-PX in mouse macrophages, to further support the role of cPLA2α in oxidase regulation also in mouse phagocytes. A significant binding of mouse GST-p47phox-PX domain fusion protein and cPLA2α in stimulated mouse phagocyte membranes was demonstrated by pull-down experiments, although lower than that detected by the human p47phox-PX domain. Substituting the amino acids Phe98, Asn99, and Gly100 to Cys98, Ser99, and Thr100 in the mouse p47phox-PX domain (present in the human p47phox-PX domain) caused strong binding that was similar to that detected by the human p47phox-PX domain CONCLUSIONS: The binding between cPLA2α-C2 and p47phox-PX domains exists in mouse macrophages and is not unique to human phagocytes. The binding between the two proteins is lower in the mice, probably due to the absence of amino acids Cys98, Ser 99, and Thr100in the p47phox-PX domain that facilitate the binding to cPLA2α.

SARS-CoV-2 receptor binding domain fusion protein efficiently neutralizes virus infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. Currently, as dangerous mutations emerge, there is an increased demand for specific treatments for SARS-CoV-2 infected patients. The spike glycoprotein on the virus envelope binds to the angiotensin converting enzyme 2 (ACE2) on host cells through its receptor binding domain (RBD) to mediate virus entry. Thus, blocking this interaction may inhibit viral entry and consequently stop infection. Here, we generated fusion proteins composed of the extracellular portions of ACE2 and RBD fused to the Fc portion of human IgG1 (ACE2-Ig and RBD-Ig, respectively). We demonstrate that ACE2-Ig is enzymatically active and that it can be recognized by the SARS-CoV-2 RBD, independently of its enzymatic activity. We further show that RBD-Ig efficiently inhibits in-vivo SARS-CoV-2 infection better than ACE2-Ig. Mechanistically, we show that anti-spike antibody generation, ACE2 enzymatic activity, and ACE2 surface expression were not affected by RBD-Ig. Finally, we show that RBD-Ig is more efficient than ACE2-Ig at neutralizing high virus titers. We thus propose that RBD-Ig physically blocks virus infection by binding to ACE2 and that RBD-Ig should be used for the treatment of SARS-CoV-2-infected patients.

Announcement of the 2022 winners of the Spychala and Women in Science Awards

Announcement of the 2022 winners of the Spychala and Women in Science Awards

Abstract 1740: Engineered variant domain fusion proteins provide checkpoint inhibition and tumor antigen dependent CD28 costimulation resulting in potent anti-tumor immunity

Abstract Introduction: Checkpoint inhibition (CPI) has been shown to be an effective anti-tumor therapy, but CPI alone is frequently insufficient to control tumor growth, and costimulatory signals may also be required to produce clinically significant anti-tumor responses. PD1-PDL1 are established CPI targets and TMIGD2 is an inhibitory receptor expressed on T cells that is engaged by its cognate ligand HHLA2 on tumor cells, leading to inhibition of T cell responses. Novel biologics combining CD28 costimulation and CPI may yield promising tumor antigen-specific therapeutic candidates. Methods: Variants of CD86 with increased CD28 affinity were engineered using our directed evolution platform. PD1 and TMIGD2 variants were also engineered for increased affinity to PDL1 and HHLA2, respectively. Fusion proteins were generated including either PD1 or TMIGD2 domains, an effectorless Fc domain, and an engineered CD86 domain to generate proteins to provide target-dependent costimulation (TDC) and evaluated in multiple in vitro T cell stimulation assays. Costimulatory effects were confirmed by evaluating T cell proliferation, cytokine release, and tumor cell killing. Fusion proteins were also tested in vivo in a mouse tumor model using MC38 cells expressing human PDL1 or HHLA2, and in a humanized tumor model where an HPV+ squamous cell carcinoma cell line expressing either PDL1 or HHLA2 was implanted into NSG mice. Tumor-bearing NSG mice were given primary human T cells expressing an HPV peptide-specific TCR, with or without the corresponding fusion protein, and tumor growth was measured over time. Results: PD1-CD86 and TMIGD2-CD86 TDC proteins enhanced T cell costimulation in multiple in vitro T cell response assays, and costimulation was dependent on target cell lines expressing PDL1 or HHLA2, respectively. There was no effect on T cell responses if the target cells did not express the relevant target protein. PD1 and TMIGD2 domains alone had little effect on T cell responses, indicating these TDC proteins were driving enhanced responses. PD1-CD86 and TMIGD2-CD86 TDC proteins enhanced anti-tumor responses in vivo in a syngeneic MC38 implantation model only when MC38 cells expressed human PDL1 or HHLA2, respectively. Both proteins also enhanced antitumor responses in a humanized tumor implantation system using an HPV+ tumor cell line expressing the corresponding target antigen in the presence of human T cells expressing an HPV-specific TCR, but failed to control tumor growth when tumor cells lacked expression of the target protein. Conclusions: Tumor antigen-specific antitumor therapy can be achieved with fusion proteins that combine engineered CPI and CD28 costimulatory domains. Such novel biologics may provide promising approaches to enhancing the efficacy of CPI monotherapies and to address checkpoint inhibitor-resistant tumors. Citation Format: Steven D. Levin, Mark F. Maurer, Chelsea Gudgeon, Siddarth Chandrasekaran, Daniel Ardourel, Daniel Demonte, Joseph Kuijper, Martin Wolfson, Logan Garrett, Kayla N. Kleist, Sherri Mudri, Hieu Nguyen, Michelle Seaberg, Rachel Wang, Jing Yang, Katherine E. Lewis, Stacey R. Dillon, Mark Rixon, Stanford L. Peng. Engineered variant domain fusion proteins provide checkpoint inhibition and tumor antigen dependent CD28 costimulation resulting in potent anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1740.

Design BH3 domain fusion protein as targeting pro-apoptotic self-assembling nanoparticles

Cancer is a serious global health issue, and apoptosis is a logical and practical cancer therapeutic strategy. Apoptosis responses to internal and external signals. Both BH3 domain in the pro-apoptotic proteins and truncated BH3 domain can stimulate cell apoptosis. However, the faults of peptides in systemic administration restrict the applications of truncated BH3 domain. Ferritin, as an attractive nanoparticle with the capacity of self-assemble to unique hollow spherical structure, could display truncated BH3 domain an N-terminal. Thus, in this study, we designed a pro-apoptosis self-assembling protein nanoparticle by BH3 domain fusion at N-terminal of ferritin. We evaluated the size, cytotoxicity and pro-apoptosis effect of these nanoparticles. The results showed that RGD-BH3-HFn, BH3-HFn and HFn had uniformly spherical structure with sizes at 26.08 ± 0.11 nm, 22.07 ± 0.67 nm, and 16.81 ± 0.88 nm, respectively; RGD-BH3-HFn has stronger cytotoxicity against tumor cells than BH3-HFn and HFn. The total apoptosis ratios (including necrosis) of C6 cells induced by RGD-BH3-HFn, BH3-HFn, and HFn proteins were 15.24%, 10.13% and 2.14%, respectively; those of bEnd.3 cells were 15.47%, 7.33% and 1.70%, respectively; while the total apoptosis rate (including necrosis) of MCF-7 cells were 3.24%, 4.9% and − 1.68%, respectively. The results suggested self-assembling RGD-BH3-HFn could target to C6 cells and bEnd.3 cells, and enhance tumor cells apoptosis, its apoptosis effect against C6 cells was 7.11-fold that of HFn, and apoptosis effect against bEnd.3 cells was 9.08-fold that of HFn. These results indicated BH3 domain can be designed as targeting pro-apoptotic nanoparticles.

SARS-CoV-2 Envelope (E) protein interacts with PDZ-domain-2 of host tight junction protein ZO1.

Newly emerged SARS-CoV-2 is the cause of an ongoing global pandemic leading to severe respiratory disease in humans. SARS-CoV-2 targets epithelial cells in the respiratory tract and lungs, which can lead to amplified chloride secretion and increased leak across epithelial barriers, contributing to severe pneumonia and consolidation of the lungs as seen in many COVID-19 patients. There is an urgent need for a better understanding of the molecular aspects that contribute to SARS-CoV-2-induced pathogenesis and for the development of approaches to mitigate these damaging pathologies. The multifunctional SARS-CoV-2 Envelope (E) protein contributes to virus assembly/egress, and as a membrane protein, also possesses viroporin channel properties that may contribute to epithelial barrier damage, pathogenesis, and disease severity. The extreme C-terminal (ECT) sequence of E also contains a putative PDZ-domain binding motif (PBM), similar to that identified in the E protein of SARS-CoV-1. Here, we screened an array of GST-PDZ domain fusion proteins using either a biotin-labeled WT or mutant ECT peptide from the SARS-CoV-2 E protein. Notably, we identified a singular specific interaction between the WT E peptide and the second PDZ domain of human Zona Occludens-1 (ZO1), one of the key regulators of TJ formation/integrity in all epithelial tissues. We used homogenous time resolve fluorescence (HTRF) as a second complementary approach to further validate this novel modular E-ZO1 interaction. We postulate that SARS-CoV-2 E interacts with ZO1 in infected epithelial cells, and this interaction may contribute, in part, to tight junction damage and epithelial barrier compromise in these cell layers leading to enhanced virus spread and severe dysfunction that leads to morbidity. Prophylactic/therapeutic intervention targeting this virus-host interaction may effectively reduce airway and/or gastrointestinal barrier damage and mitigate virus spread.

An Oncolytic Virus Expressing IL15/IL15Rα Combined with Off-the-Shelf EGFR-CAR NK Cells Targets Glioblastoma.

IL15 is a pleiotropic cytokine with multiple roles that improve immune responses to tumor cells. Oncolytic viruses (OV) specifically lyse tumors and activate immune responses. Systemic administration of IL15 or its complex with the IL15Rα and chimeric antigen receptor (CAR) natural killer (NK) cells are currently being tested in the clinic. Here, we generated a herpes simplex 1-based OV-expressing human IL15/IL15Rα sushi domain fusion protein (named OV-IL15C), as well as off-the-shelf EGFR-CAR NK cells, and studied their monotherapy and combination efficacy in vitro and in multiple glioblastoma (GBM) mouse models. In vitro, soluble IL15/IL15Rα complex was secreted from OV-IL15C-infected GBM cells, which promoted GBM cytotoxicity and improved survival of NK and CD8+ T cells. Frozen, readily available off-the-shelf EGFR-CAR NK cells showed enhanced killing of tumor cells compared with empty vector-transduced NK cells. In vivo, OV-IL15C significantly inhibited tumor growth and prolonged survival of GBM-bearing mice in the presence of CD8+ T cells compared with parental OV. OV-IL15C plus EGFR-CAR NK cells synergistically suppressed tumor growth and significantly improved survival compared with either monotherapy, correlating with increased intracranial infiltration and activation of NK and CD8+ T cells and elevated persistence of CAR NK cells in an immunocompetent model. Collectively, OV-IL15C and off-the-shelf EGFR-CAR NK cells represent promising therapeutic strategies for GBM treatment to improve the clinical management of this devastating disease. SIGNIFICANCE: The combination of an oncolytic virus expressing the IL15/IL15Rα complex and frozen, ready-to-use EGFR-CAR NK cells elicits strong antitumor responses in glioblastoma.

Accelerated delivery of dsRNA in lepidopteran midgut cells by a Galanthus nivalis lectin (GNA)-dsRNA-binding domain fusion protein

Lepidopteran insects are highly refractory to oral RNA interference (RNAi). Degradation, impaired cellular uptake and intracellular transport of double-stranded RNA (dsRNA) are considered the major factors responsible for the reduced RNAi efficiency in these insects. In this study, the potential of lectins to improve dsRNA delivery and RNAi efficacy was evaluated. First, a fusion protein consisting of the Galanthus nivalis agglutinin (GNA) and a dsRNA binding domain was developed, further referred to as GNA:dsRBD (GNAF). Then, its ability to increase dsRNA uptake and transfection efficiency in lepidopteran midgut cells was evaluated, as well as its ability to protect and promote the RNAi response in the beet armyworm Spodoptera exigua. Confocal microscopy analysis showed that GNAF-complexed dsRNA was internalized faster in Choristoneura fumiferana midgut CF1 cells (1 min) compared to naked dsRNA (>1 h). The faster uptake was also correlated with an increased RNAi efficiency in these CF1 cells. In vivo feeding bioassays with GNAF-complexed dsRNA led to an increased mortality in S. exigua compared to the controls. By targeting the essential gene V-ATPase A, we observed that the mortality increased to 48% in the GNAF-dsRNA treatment compared to only 8.3% and 6.6% in the control treatments with the naked dsRNA and the GNAF, respectively.

NAC-Like Gene GIBBERELLIN SUPPRESSING FACTOR Regulates the Gibberellin Metabolic Pathway in Response to Cold and Drought Stresses in Arabidopsis

To investigate the functions of NAC-like genes, we reported the characterization and functional analysis of one Arabidopsis NAC-like gene which showed a novel function in the regulation of gibberellin biosynthesis and named as GIBBERELLIN SUPPRESSING FACTOR (GSF). GSF acts as a transcriptional activator and has transactivation capacity based on yeast transcription activity assays. YFP + GSF-TM (lacking a transmembrane domain) fusion proteins accumulated in the nuclei, while the YFP + GSF fusion proteins only accumulated in the ER membrane and were absent from the nuclei. These results revealed that GSF requires processing and release from the ER and transportation into the nucleus to perform its function. The ectopic expression of GSF-TM caused a dwarfism phenotype, which was correlated with the upregulation of the gibberellin (GA) deactivation genes GA2-oxidases 2/6 (GA2ox2/6) and the downregulation of the GA biosynthetic genes GA20-oxidases 1–4 (GA20ox1-4). The external application of GA rescued the dwarfism in the 35 S::GSF-TM plants, indicating that GSF affects GA biosynthesis, rather than the GA signaling pathway. Further analysis indicated that the upregulation of GA2ox2/6 is a key factor for the GSF function to regulate the GA level, since 35 S::GA20ox1 could not rescue the dwarfism in the 35 S::GSF-TM plants. Cold treatment induced the processing of the YFP + GSF fusion proteins from the ER membrane and their entry into the nuclei, which is correlated with the cold-induced upregulation of GA2oxs. In addition, the expression of GA2oxs was induced by drought, and the 35 S::GSF-TM plants showed drought tolerance compared to the wild-type plants. Our data suggest a role for GSF in response to abiotic stresses, such as cold and drought, by suppressing the biosynthesis of GA in Arabidopsis.

Chitin degradation enzyme-responsive system for controlled release of fibroblast growth factor-2

Chitin is widely found in fungal cell walls and arthropod exoskeletons, and is used as a biomedical material. However, chitin is not water-soluble, restricting its use for controlled release materials. We found that water-soluble chitosan can be acetylated to produce a chitin equivalent, or chitin gel. Chitin gel, produced by mixing chitosan solution with acetic anhydride, can be degraded by lysozyme and fetal bovine serum, so could provide an ideal means for controlled release in biological systems. We tested a combination of chitin gel with a chitin binding domain (CBD) fusion protein as a controlled release system regulated by chitin degradation. A fusion protein consisting of fibroblast growth factor 2 (FGF-2) fused to CBD bound the chitin gel, and was released time-dependently rather than as an initial burst during lysozyme degradation, suggesting that this system could provide a means for controlled drug release in biological systems. Contrastingly, the trinitrophenyl residue (TNP-X) covalently bound to chitin gel, and was released by lysozyme degradation with an initial burst. If release of CBD-FGF-2 were simply dependent on lysozyme degradation of the chitin gel, the release behavior of CBD-FGF-2 would be similar to that of TNP-X, with an initial burst. Therefore, we propose that CBD-FGF-2 repeats the cycle of binding, release, and re-binding to the chitin gel during degradation. This system allows for a time-dependent, controlled release of CBD-FGF-2 without an initial burst.