Recombinant protein and preparation method thereof, vaccine protein adjuvant containing recombinant protein and use of recombinant protein for preventing respiratory diseases

Abstract

The invention provides a recombinant protein and a preparation method thereof, a vaccine protein adjuvant containing the recombinant protein and application of the recombinant protein to prevention of respiratory diseases. The recombinant protein disclosed by the invention achieves the effect of preventing respiratory diseases through various efficacy experiments.

Description

Technical Field

[0001] This invention relates to a recombinant protein and its preparation method, a vaccine protein adjuvant containing the recombinant protein for use in vaccines, and the use of the recombinant protein for the prevention of respiratory diseases. Background Technology

[0002] The respiratory mucosal immune system appears to be the primary target of most human pathogens. Therefore, a unique and effective pre-existing immune response within the respiratory mucosal compartments can prevent infection. However, a major limitation in mucosal vaccine development is that antigens applied to the mucosa typically elicit a relatively weak immune response, a need to avoid inducing severe reactions to a large number of harmless antigens in the environment. To overcome this barrier, mucosal adjuvants must be integrated into the vaccine composition to enhance the immune response. Adding appropriate adjuvants to vaccine formulations will contribute to a stronger induction of protective immunity.

[0003] However, traditional vaccine adjuvants suffer from insufficient stability and cytotoxicity. To address these issues, those skilled in the art urgently need to develop novel, more effective, and safer pharmaceutical products for the prevention of respiratory immune diseases to benefit the vast population in need. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide a recombinant protein comprising a B5 subunit (hereinafter referred to as LTB) variant of E. coli type II heat labile enterotoxin, wherein the LTB variant comprises at least one amino acid sequence via circular permutation (CP). (The object is to provide a method for modifying proteins using circular permutation and its application in vaccine adjuvant development.)

[0005] Another object of the present invention is to provide a method for producing the recombinant protein as described above, comprising cyclic recombination of the B5 subunit (LTB) of wild-type Escherichia coli type II heat labile enterotoxin.

[0006] In an embodiment of the invention, the cyclic recombination is performed by breaking a specific loop of the B5 subunit (LTB) of wild-type Escherichia coli type II heat labile enterotoxin and connecting the original end via a GSGS linker.

[0007] In an embodiment of the invention, residues 13-14, 31-32, or 52-53 of the LTB variant are broken to generate new N-terminals and C-terminals.

[0008] In an embodiment of the present invention, residues 13-14 are Thr13-Thr14(T13-T14,TT).

[0009] In an embodiment of the present invention, residues 31-32 are Asn31-Asn32 (N31-N32,NN).

[0010] In an embodiment of the present invention, residues 52-53 are Ala52-Lys53 (A52-K53, AK).

[0011] In embodiments of the present invention, at least the amino acid sequence via cyclic recombination (CP) is selected from the group consisting of: sequence identification number 1, sequence identification number 2, sequence identification number 3, and combinations thereof.

[0012] In embodiments of the present invention, the recombinant protein may further comprise a 6xHis-tag sequence, wherein the 6xHis-tag sequence is conjugated to the C-terminus of the LTB variant.

[0013] Another object of the present invention is to provide a protein adjuvant for a vaccine comprising the recombinant protein as described above.

[0014] Another object of the present invention is to provide the use of the recombinant protein as described above for the preparation of pharmaceuticals for the prevention of respiratory diseases.

[0015] In embodiments of the present invention, the respiratory disease is selected from the group consisting of: influenza, severe acute respiratory syndrome (SARS), severe special infectious pneumonia (COVID-19), and other diseases transmitted through the respiratory tract.

[0016] In embodiments of the present invention, recombinant proteins containing the amino acid sequences indicated by sequence identification number 1 and sequence identification number 2 do not have or have reduced GD1a binding ability.

[0017] In embodiments of the present invention, the recombinant protein maintains its binding ability to TLR 2 / 1.

[0018] In embodiments of the present invention, the dosage form of the pharmaceutical product may be an intramuscular injection, an injection, nasal drops, a spray, a nasal spray administration, or an inhaler.

[0019] In summary, through the results illustrated in the following examples, this invention utilizes an LTB protein scaffold to invent a novel LTB circular recombinant protein, which can be used to develop novel respiratory mucosal protein adjuvants. Novel LTB adjuvants with good structural stability are designed using circular recombinant design. The ability to activate TLR2 / 1 receptors is maintained using circular recombinant design, resulting in novel LTB adjuvants with comparable or higher antibody activity. Furthermore, the design of novel LTB adjuvants using circular recombinant design can reduce GD1a ganglioside binding capacity, thereby reducing cytotoxicity. The use of novel LTB adjuvants designed with circular recombinant design induces better immune response stimulation.

[0020] This invention utilizes the concept of circular recombination to design three LTB circular recombination variants (CP) and evaluates their GD1a binding affinity and structural stability using nuclear magnetic resonance (NMR) spectroscopy. Among the novel LTB circular recombination variants, TT exhibits excellent structural stability and demonstrates superior protein adjuvant activity. NN, on the other hand, shows structural instability similar to WT and is more prone to aggregation. Both TT and NN lose their GD1a binding affinity through circular recombination but retain their ability to activate TLR 2 / 1. AK exhibits superior structural stability, retains GD1a binding affinity, but has lower TLR 2 / 1 activation ability. These circular recombination variants endow LTB with different protein adjuvant activities, allowing for use for various indications and potentially advancing the future development of mucosal vaccines, particularly for respiratory diseases such as influenza.

[0021] The following will further illustrate the embodiments of the present invention. The examples listed below are for illustrative purposes only and are not intended to limit the scope of the present invention. Any person skilled in the art can make some modifications and refinements without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the contents defined in the appended claims. Attached Figure Description

[0022] Figure 1 This image shows the GD1a binding site of the B5 subunit (LTB) of type II heat labile enterotoxin from E. coli. There are two possible GD1a binding sites on the LTB surface. Figure 1 This shows the key residues responsible for binding.

[0023] Figure 2This invention demonstrates a strategy using circular permutation (CP) to remove ligand binding. The invention breaks the ligand-binding-associated protein peptide chain and connects the original ends via linkers to design a GD1a-binding-deficient LTB while retaining the same structure as the original strain, thus maintaining its ability to activate TLR 2 / 1.

[0024] Figure 3 This paper presents a novel design for LTB ring-shaped recombination structures. Three CPs, named TT, NN, and AK, are designed by disconnecting the corresponding rings and connecting the original ends via GSGS linkers.

[0025] Figure 4A and Figure 4B The purification of LTB and CP constructs is shown, wherein... Figure 4A Showing the FPLC gel filtration curves of LTB and CPs. Dashed lines indicate the presence of stable pentamers in LTB WT, TT, NN, and AK (dashed lines). Incorrectly folded LTB in WT and NN forms aggregates, which elute before the pentamer peak, as indicated by asterisks. Figure 4B SDS-PAGE analysis of purified LTB and CPs pentamers in the presence and absence of (+) β-mercaptoethanol showed that the purified products achieved excellent purity.

[0026] Figure 5 The circular dichroism (CD) spectra at different temperatures show that LTB CPs have the same secondary structure characteristics as WT.

[0027] Figure 6 The NMR HSQC spectra show the structural stability of LTB CPs. LTB WT and NN show broadened resonance lines, indicating significant dynamics in the backbone and a tendency to form polymers in aqueous solution. LTB TT and AK show strong and dispersed resonances, indicating correct folding and good structural stability.

[0028] Figure 7The X-ray structures of LTB TT and AK are shown, and compared with those of LTB WT. (A) The comparison of the structure of LTB TT (green) with that of WT (orange) shows great structural similarity. The CP sites of LTB TT are indicated by arrows, with the representative major GD1a binding sites indicated by red circles. GD1a coordinates are docked onto the LTB TT structure, and the major GD1a binding sites are interrupted due to the break in the protein backbone. (B) The comparison of the structure of LTB AK (cyan) with that of WT (orange) shows great structural similarity. The CP sites of LTB AK are indicated by arrows, with the representative major GD1a binding sites indicated by red circles. GD1a coordinates are docked onto the LTB AK structure, and the GD1a binding sites are intact. LTB AK retains the major GD1a binding sites, but the minor binding sites are disrupted.

[0029] Figure 8 Display NMR 1D 1 H titration experiment. Comparison of five NMR 1D 1 H-spectroscopy, namely the spectra of free LTB TT, free GD1a, TT to GD1a 1:1, TT to GD1a 1:2, and TT to GD1a 1:3. In the presence of LTB TT, the GD1a signal remains unchanged, and in subsequent titrations, the GD1a signal intensity is consistent with the titration factor, indicating that LTB TT lacks the ability to bind GD1a.

[0030] Figure 9 Display NMR 1D 1 H titration experiment. Comparison of five NMR 1D 1 H-spectroscopy, namely the spectra of free LTB NN, free GD1a, NN to GD1a 1:1, NN to GD1a 1:2, and NN to GD1a 1:3. In the presence of LTB NN, the GD1a signal remains unchanged, and in subsequent titrations, the GD1a signal intensity is consistent with the titration factor, indicating that LTB NN lacks the ability to bind GD1a.

[0031] Figure 10A The NMR HSQC spectrum of LTB TT titrated with GD1a is shown. Containing... 15 NMR samples of N LTB TT were prepared in NMR buffer (50 mM Tris-HCl, pH 7.0, 150 mM NaCl, 10% D₂O). GD1a was titrated into the LTB solution to prepare samples with the desired molar ratios of 1:1 and 1:2. The respective HSQC spectra were obtained at 298 K. The presence of GD1a did not interfere with the LTB TT signal, indicating no binding between the two molecules.

[0032] Figure 10B The NMR HSQC spectrum of LTB AK titrated with GD1a is shown. Containing... 15 NMR samples of N LTB AK were prepared in NMR buffer (50 mM Tris-HCl, pH 7.0, 150 mM NaCl, 10% D2O). GD1a was titrated into the LTB solution to prepare samples with the desired molar ratios of 1:1 and 1:2. The respective HSQC spectra were obtained at 298 K. The presence of GD1a caused broadening and subsequent disappearance of the LTB AK signal, indicating significant binding between the two molecules.

[0033] Figure 11 This study demonstrates the activity of LTB CPs in induced human TLR 2 / 1. Human embryonic kidney cell line HEK 293A cells, after gene transfection, exhibit hTLR 2 / 1 heterodimer and NF-κB-driven luciferase. After cell treatment with LTB and culture at 37°C for 5 hours, the binding of LTB to hTLR 2 / 1 triggers intracellular signaling pathways, activating the NF-κB promoter and leading to the expression of downstream luciferase genes. Luciferase activity was measured by adding luciferase receptors, and the results represent the activity of LTB CPs in activating TLR 2 / 1. TT and NN showed activities similar to WT, while AK, although exhibiting weaker activity, still demonstrated the ability to activate hTLR 2 / 1 heterodimer and downstream NF-κB. Pam3CSK4, a synthetic lipopeptide that activates TLR2 / 1, was used as a positive control.

[0034] Figures 12A-12E The adjuvant efficacy of LTB TT and NN was demonstrated. (12A) Intranasal immunization procedure in a mouse model: Mouse experiments were conducted, and mouse serum and pulmonary bronchoalveolar fluid (BALF) were collected. Neuraminidase (NA)-specific IgG and IgA were detected using ELISA. BALB / c mice (n=5 per group) were immunized intranasally with recombinant NA protein of influenza strain N1 containing two mutant sites (N329T and K331T), adjuvanted with LTB WT, TT, or NN. Polyinosinic acid-polycytidylic acid (poly(I:C), a double-stranded RNA analog) was used as a positive control for adjuvants because it could trigger an antiviral immune response. ELISAs were used to measure the (12B) N1NA-specific IgG titer and (12C) N1NA-specific IgA titer in serum, as well as the (12D) N1NA-specific IgA titer and (12E) total IgA titer in BALF.

[0035] Figures 13A-13CThe activity of serum and BALF against influenza A / California / 04 / 2009 (H1N1) virus was shown. Enzyme-ligand agglutinin assay was used to measure the titer of the NA inhibitory antibody, i.e., the reduction in NA enzyme activity of the P1N1 virus (Pandemic Influenza A Virus). NAI activity against A / California / 04 / 2009 (H1N1) virus in serum (13A) and BALF (13B) was also shown. The corresponding half-maximal inhibitory concentration (IC50) was also presented for (13C). 50 (This refers to the half-inhibitory concentration of the antagonist being measured), with a titer of 50% reduction in viral N1NA enzyme activity. Results showed that, compared to N1NA protein without adjuvant, groups using poly(I:C), LTB WT, and its variants TT and NN as adjuvants induced significant N1NA inhibitory titers in serum and BALF. Among these, the use of TT adjuvant was found to result in higher N1NA titers in both serum and BALF.

[0036] Figure 14 This study demonstrated the protective effect against H1N1 virus after inoculation with recombinant N1NA protein, aided by the addition of LTB TT or LTB WT adjuvants. Three weeks after the final immunization, immunized mice (n=6 per group) were intranasally infected with 5×LD50 A / PR8 (H1N1) virus. Survival rate and body weight were monitored daily for 14 days post-infection. Mice were considered to have no effective protection and were deemed surviving if they experienced a weight loss exceeding 25%. Detailed Implementation

[0037] definition

[0038] The values ​​used in this paper are approximate, and all experimental data are expressed in the range of ±20%, preferably in the range of ±10%, and optimally in the range of ±5%.

[0039] Unless otherwise stated herein, the terms “a,” “the,” and similar terms used in this specification (especially in the claims below) shall be understood to include both singular and plural forms.

[0040] As used in this article, the terms "preventing" or "prevention" refer to the prevention or delaying of the onset of disease symptoms when a drug is used on an individual who is at high risk of developing the disease but does not have symptoms of the disease onset.

[0041] According to the present invention, E. coli type II heat labile enterotoxin has an AB5 structure, consisting of a catalytically active A-subunit and a pentamer B-subunit responsible for binding to the host glycocomplex. The A polypeptide is a catalytic G... sα Enzymes that regulate ADP-ribosylation of proteins can upregulate adenylate cyclase. B-pentamers are involved in the binding of gangliosides, a family of glycolipids found on the surface of mammalian cells. Type II heat-sensitive enterotoxins are potent mucosal adjuvants. The non-catalytic B-subunit of type II enterotoxin, LTB, interacts with toll-like receptors (TLRs). LTB has been shown to activate human monocytes or mouse macrophages in the TLR2 signaling pathway. TLR1, which forms a heterodimer with TLR2, has also been found to be stimulated by LTB. In the absence of the A-subunit, LTB initially binds to lipid raft-associated GD1a gangliosides, thereby recruiting the TLR2 / TLR1 signaling complex to the lipid raft. GD1a binding promotes the interaction between LTB and the TLR2 / TLR1 signaling complex. This interaction activates the inflammatory transcription factor NF-κB through the downstream intracellular transferor proteins MYD88 and TRIF. NF-κB activation results in the production of inflammatory cytokines such as TNF-α, IL-1β, IL-6, and IL-8, potentially leading to dose-limiting inflammation. This mechanism suggests an important link to the innate immune system, as the GD1a co-receptor promotes LTB signaling. LTB has been shown to be a potential immunomodulatory adjuvant.

[0042] According to this invention, the applicant believes that the main cause of the side effects of LT toxin is excessive binding to GD1a gangliosides. Modification of GD1a binding may alter the induced immunoreactivity. Based on this hypothesis, the applicant designed the B5 subunit (LTB) of type II heat labile enterotoxin using the concept of circular permutation (CP) to effectively modulate potential adjuvant activity. Prior art discloses a crystal complex structure of LTB and Neu5Ac-nLT, wherein four ganglioside ligands are bound in grooves between amino acid positions 12-16 of the monomer and amino acid positions 29-36 of the adjacent monomer. Figure 1 ). Figure 2This invention demonstrates a strategy for removing ligand-binding proteins using cyclic recombination. The invention involves breaking the ligand-binding-related protein peptide chain and linking the original ends via linkers, novelly designing a GD1a-binding defective LTB while retaining the same structure as the native strain and maintaining its ability to activate TLR 2 / 1. The applicant discovered that the potential binding sites for both LTB and GD1a are located on the loop; therefore, cyclic recombination is used to disrupt these potential GD1a binding sites, altering the binding affinity of LTB to GD1a.

[0043] Thr13 and Thr14 in LTB are key residues for GD1a binding. Mutations in Thr13 and Thr14 in LTB result in reduced binding affinity for GD1a. The deoxygenated carbon at position 2 of the pyranose ring forms a hydrophobic contact with the methyl group of Thr13, and the carboxylic acid group of Neu5Ac acquires hydrogen bonds from the side-chain hydroxyl group of Thr14 and the main chain amine. Figure 1 ).

[0044] Furthermore, Neu5Ac-nLT forms hydrogen bonds with the side chain of Asn31 and the main chain amine group of Asn32. Figure 1 ).

[0045] Figure 3 This paper presents a novel design for LTB ring-shaped recombination structures. Three CPs, named TT, NN, and AK, are designed by disconnecting the corresponding rings and connecting the original ends via GSGS linkers.

[0046] Figure 1 This shows the GD1a binding site of LTB. There are two possible GD1a binding sites on the LTB surface. Figure 1 The key residues responsible for binding are shown. The applicant believes that LTB may have two binding sites with GD1a: a major binding pocket composed of amino acids 12-14, 30-33, and 92, and a minor binding pocket composed of amino acids 50-55. This is similar to the results obtained by NMR analysis in the following examples.

[0047] The applicant believes that the main GD1a binding site consists of the following amino acids: Arg12, Thr13, Thr14, Ile30, Asn31, and Asn32. Figure 1 ).

[0048] Furthermore, another ligand molecule was found to interact with residues Arg51, Lys53, Asp54, and Tyr55 at a minor site. This interaction may provide weaker GD1a binding, as the existing interactions are fewer than those at previous sites; therefore, positions 51–55 are considered minor GD1a binding sites.

[0049] In this invention, a cyclic recombination complex (CP) of LTB was designed, in which the original N-terminus and C-terminus were linked, and residues 13–14, 31–32, and 52–53 were broken to generate new N-termini and C-termini. A four-amino acid residue (GSGS) linker (sequence identification number: 4) was added between the original N-terminus and C-terminus. By disrupting the backbone linker at the disruption site, the GD1a binding site was disrupted, thereby reducing GD1a binding capacity. The structural properties of the candidates were evaluated by circular dichroism spectroscopy, NMR, and X-ray crystallography. These three CPs exhibited structural folding similar to that of natural proteins, demonstrating that these designs can create natural LTB variants. However, these three CPs yielded different structural properties, representing different possibilities for development into novel protein adjuvants.

[0050] Among these three CPs, the LTB variant was identified as having reduced binding to GD1a, better structural stability, and higher immunostimulatory activity compared to the native protein.

[0051] This invention demonstrates the feasibility of designing novel LTB adjuvant proteins using cyclic recombination. The newly designed LTB CP may contribute to the development of mucosal vaccines for influenza and other respiratory diseases. This invention addresses the urgent need for novel, effective, and safe mucosal adjuvants.

[0052] According to the present invention, the amino acid sequence of LTB TT is as shown in sequence identification number 1.

[0053] According to the present invention, the amino acid sequence of LTB NN is as shown in sequence identification number 2.

[0054] According to the present invention, the amino acid sequence of LTB AK is as shown in sequence identification number 3.

[0055] According to the present invention, a 6xHis-tag sequence can be conjugated to the C-terminus of the LTB to assist in purification.

[0056] The present invention is further illustrated by the following embodiments. These embodiments are provided for illustrative purposes only and are not intended to limit the scope of protection of the present invention. The scope of protection of the present invention is as shown in the appended claims.

[0057] The design process for the LTB circular recombination construct is as follows. To directly reduce the binding affinity of GD1a, this invention designs an LTB circular recombination that breaks the lineage bonds between residues 13–14, 31–32, and 52–53. A four-residue (GSGS) linker is added to connect the original N- and C-termini. The LTB CPs gene has been synthesized into the pET22b(+) vector plasmid. If necessary, a C-terminal His tag is added to aid purification.

[0058] The procedure for the expression and purification of LTB CPs is as follows. Protein expression was performed using *E. coli* BL21(DE3) (Invitrogen). Transfection was performed using a heat shock method, followed by development onto agar plates. After overnight incubation at 3°C, single colonies were picked and cultured in a small amount of Luria Bertani (LB) medium with shaking at 37°C overnight. Cells were then transferred to a large amount of LB medium and cultured with vigorous shaking at 37°C. When the OD600 reached 0.6, cells were induced with 1 mM IPTG at 16°C for 20 hours. Cells were harvested by centrifugation. The cell pellet was resuspended in lysis buffer (50 mM Tris buffer, pH 7.0 and 150 mM NaCl) and then lysed using a high-pressure homogenizer. Insoluble fractions were removed from the lysate by centrifugation at 15000g for 30 minutes, and the supernatant was loaded into a column filled with Ni-containing IMAC resin (GE Healthcare). Non-specifically bound proteins were removed using wash buffer (50 mM Tris buffer, pH 7.0, 150 mM NaCl, and 40 mM imidazole), and His-tagged LTB proteins were eluted using elution buffer (50 mM Tris buffer, pH 7.0, 150 mM NaCl, and 400 mM imidazole). After protein concentration at 4°C using a 10,000 Da Amicon membrane ultrafiltration (Millipore) filter, gel filtration was performed to purify the pentamer form of LTB. A HiLoad 16 / 60 Superdex-75 size exclusion column (GE Healthcare Life Sciences) was equilibrated using running buffer (50 mM Tris buffer, pH 7.0, and 150 mM NaCl). Under the control of the system controller, the flow rate is set to 1 mL / min.

[0059] The immunization and sampling procedures for mice are as follows. Female C57BL / 6 and BALB / c mice aged 6 to 8 weeks were purchased from the Laboratory Animal Center in Taiwan, China. These mice were intranasally immunized with different doses of recombinant influenza virus N1 neuraminidase (NA) (immunogen) and recombinant LTB protein (10 μg) (adjuvant) or PBS as a control group. N1NA contained two mutations, N329T and K331T, to enhance cross-reactivity between different viral strains. Mice were anesthetized before intranasal administration and then given a 30 μl mixture containing N1NA (with or without LTB) or the control group. All groups of mice were immunized three times at weeks 0, 3, and 6, and serum was collected at week 8. These mice were sacrificed at week 9 to collect lung flushing fluid (BALF).

[0060] The procedure for N1NA-specific antibody titer analysis is as follows: Antibodies in serum and BALF samples from immunized mice were quantified using ELISA. 100 μl (2 μg / ml recombinant N1NA protein) was coated onto 96-well ELISA pans and fixed at 4°C for 16–18 hours. Subsequently, the pans were blocked with blocking buffer (1% BSA in PBS) at 37°C for 2 hours. Serial dilutions of serum or BALF samples were added to each pan and incubated at room temperature for 1 hour. Then, HRP-conjugated goat anti-mouse IgG antibody (1:30,000) or HRP-conjugated goat anti-mouse IgA antibody (1:50,000) was added to the wells and incubated for another 1 hour at 37°C. Color development was performed using TMB chromogenic solution (BioLegend) and incubated at 37°C for 15 minutes. The reaction was then terminated with 2N H2SO4 and the sample was read from an ELISA reader (OD). 450 ) to perform the measurement.

[0061] The procedure for the neuraminidase inhibition assay is as follows: An ELISA plate coated with 100 μl (50 μg / ml) fetuin (Sigma) was incubated overnight at 4°C. The plate was then washed three times with PBST buffer and blocked with PBST buffer for 2 hours. The OD value measured by ELISA was then... 450 A certain amount of virus (H1N1 [A / Puerto Rico / 08 / 1934]) with a value of 2 was co-cultured with an equal volume of serially diluted serum at 37°C for 1 hour, then transferred to an ELISA plate coated with fetal globulin and incubated at 37°C for 1 hour. After washing three times with PBST, 100 μl (2.5 μg / ml) of lectin (Sigma) was added. After incubation at room temperature for 1 hour and washing three times with PBST buffer, TMB chromogenic solution was added to the plate and incubated in the dark for 15 minutes. The reaction was terminated with 2NH2SO4. OD was detected using an ELISA reader. 450 nm signal. A serum dilution that inhibits 50% of NA enzyme activity is defined as IC50. 50 value.

[0062] The procedure for viral challenge is as follows: 6–8 week old female BALB / c mice were administered three doses of subunit vaccine or a PBS-simulated control group. After the third dose, the immunized mice were given 5 times the mouse median lethal dose (5x LD50). 50 Mice were exposed to the H1N1 virus through their nasal cavity (A / Puerto Rico / 08 / 1934). Survival and body weight were monitored daily for two consecutive weeks; individuals with a body weight loss exceeding 25% were considered dead.

[0063] Example 1. A strategy for designing a circular recombination (CP) variant of the B5 subunit (LTB) of Escherichia coli type II heat-sensitive enterotoxin, which exhibits good structural stability.

[0064] Using LTB protein scaffolds, this invention is a strategy for designing novel protein adjuvants through circular recombination. This invention provides three CPs that introduce novel backbone breaks at the GD1a binding sites of Thr13-Thr14 (T13-T14), Asn32-Asn33 (N32-N33), and Ala52-Lys53 (A52-K53). The design will eliminate / reduce the GD1a binding capacity of the LTB.

[0065] Figure 4A and Figure 4B The purification of LTB and CP constructs is shown, in which Figure 4A The FPLC gel filtration curves of LTB and CPs are shown. The dashed lines indicate the presence of stable pentamers in LTB WT, TT, NN, and AK (dashed lines). Some LTB in WT and NN forms aggregates, which elute before the pentamer peak, as indicated by the asterisks. Figure 4B SDS-PAGE analysis of purified LTB and CPs pentamers in the presence and absence of (+) β-mercaptoethanol showed that the purified products achieved excellent purity.

[0066] All three CPs were well purified, and FPLC gel filtration curves demonstrated that they formed using the same pentamer. Figure 4A and Figure 4B ).

[0067] Figure 5 The circular dichroism (CD) spectra at different temperatures show that LTB CPs have the same secondary structure characteristics as WT.

[0068] Figure 6 The NMR HSQC spectra show the structural stability of LTB CPs. LTB WT and NN show broadened resonance lines, indicating that the protein readily aggregates at high concentrations. LTB TT and AK show strong and dispersed resonances, indicating correct folding and good structural stability.

[0069] CD and NMR confirmed that these three CPs have the same protein folding ( Figure 5 and Figure 6 ).

[0070] Figure 7The X-ray structures of LTB TT and AK are shown, and compared with those of LTB WT. (A) The structure of LTB TT (green) shows great structural similarity when compared with that of WT (orange). The CP sites of LTB TT are indicated by arrows, with representative GD1a major binding sites indicated by red circles. GD1a coordinates are docked onto the LTB TT structure, where the major GD1a binding site is interrupted due to the break in the protein backbone. (B) The structure of LTB AK (cyan) shows great structural similarity when compared with that of WT (orange). The CP sites of LTB AK are indicated by arrows, with representative GD1a major binding sites indicated by red circles. GD1a coordinates are docked onto the LTB AK structure, where the GD1a binding site is intact. LTB AK retains the major GD1a binding site, but the minor binding sites are disrupted.

[0071] TT exhibits a stable structure, and its strong NMR HSQC signal reflects this structural stability. The TT crystal structure in this embodiment shows the same structure as WT, except that the major GD1a binding sites are disrupted. Figure 7 ).

[0072] AK has a stable structure, and the strong NMR HSQC signal reflects its structural stability. The AK crystal structure in this embodiment shows the same structure as WT, but the main GD1a binding sites are still retained, while the secondary binding sites are destroyed. Figure 7 ).

[0073] NN exhibits structural elasticity and behaves very similarly to WT, but it is more prone to aggregation, thus making protein crystallization impossible. Compared to LTB WT, the three CPs designed using this method all demonstrated comparable or good structural stability.

[0074] Example 2. The present invention includes LTB CP variants that do not have GD1a ganglioside binding ability, such as TT and NN which do not have GD1a binding ability.

[0075] In this embodiment, NMR titration was used to observe the binding ability with GD1a. Figure 8 Display NMR 1D 1 H titration experiment. Comparison of five NMR 1D 1 H-spectroscopy, i.e., the spectra of free LTB TT, free GD1a, TT to GD1a 1:1, TT to GD1a 1:2, and TT to GD1a 1:3. In the presence of LTB TT, the GD1a signal remains unchanged, and in subsequent titrations, the GD1a signal intensity is consistent with the titration factor, indicating that LTB TT lacks the ability to bind GD1a.

[0076] This embodiment designs the LTB variant TT, which does not have GD1a binding ability. Figure 8 ).

[0077] Figure 9 Display NMR 1D 1 H titration experiment. Comparison of five NMR 1D 1 H-spectroscopy, namely the spectra of free LTB NN, free GD1a, NN to GD1a 1:1, NN to GD1a 1:2, and NN to GD1a 1:3. In the presence of LTB NN, the GD1a signal remains unchanged, and in subsequent titrations, the GD1a signal intensity is consistent with the titration factor, indicating that LTB NN lacks the ability to bind GD1a.

[0078] 1D 1H NMR spectroscopy showed no binding between GD1a and the LTB variant NN. Figure 9 ).

[0079] Figure 10A The NMR HSQC spectrum of LTB TT titrated with GD1a is shown. Containing... 15 NMR samples of N LTB TT were prepared in NMR buffer (50 mM Tris-HCl, pH 7.0, 150 mM NaCl, 10% D₂O). GD1a was titrated into the LTB solution to prepare samples with the desired molar ratios of 1:1 and 1:2. The respective HSQC spectra were obtained at 298 K. The presence of GD1a did not interfere with the LTB TT signal, indicating no binding between the two molecules.

[0080] The 2D NMR HSQC spectrum of TT showed that the presence of GD1a did not alter the TT signal. This further demonstrates that LTB TT does not bind to GD1a (Figure 10).

[0081] 2D NMR HSQC spectroscopy of NN cannot be performed because the HSQC signal of NN is poor.

[0082] Figure 10B The NMR HSQC spectrum of LTB AK titrated with GD1a is shown. Containing... 15 NMR samples of N LTB AK were prepared in NMR buffer (50 mM Tris-HCl, pH 7.0, 150 mM NaCl, 10% D2O). GD1a was titrated into LTB solutions to prepare samples with desired molar ratios of 1:1 and 1:2. The respective HSQC spectra were obtained at 298 K. The presence of GD1a affected the signal of LTB AK, broadening its spectral lines, indicating that GD1a promotes the polymerization of LTB AK, thus maintaining a bond between the two molecules.

[0083] Example 3. The new LTB variant is identical to LTB, possessing the ability to activate hTLR 2 / 1 and trigger intracellular signaling pathways to activate the NF-κB promoter.

[0084] Figure 11 This study demonstrates the activity of LTB CPs in induced human TLR 2 / 1. Human embryonic kidney cell line HEK 293A, after gene transfection, exhibits hTLR 2 / 1 heterodimer and NF-κB-driven luciferase. After cell treatment with LTB and culture at 37°C for 5 hours, the binding of LTB to hTLR 2 / 1 triggers intracellular signaling pathways, activating the NF-κB promoter and leading to the expression of downstream luciferase genes. Luciferase activity was measured by adding luciferase receptors, and the results represent the activity of LTB CPs in activating TLR 2 / 1. TT and NN showed activities similar to WT, while AK, although exhibiting weaker activity, still demonstrated the ability to activate hTLR 2 / 1 heterodimer and downstream NF-κB. Pam3CSK4, a synthetic lipopeptide that activates TLR2 / 1, was used as a positive control.

[0085] This embodiment designed LTB variants, TT, NN, and AK. Through activation of the TLR2 / 1 pathway, the three CPs exhibited comparable activities, such as expression of NF-κB-derived downstream luciferase genes. Figure 11 Therefore, LTB TT, NN, and AK have all been shown to have the same potential as LTB WT as protein adjuvants for enhancing immune responses.

[0086] The binding capacity of LTB protein to TLR 2 / 1 was determined using ELISA, with Pam3CSK4 as the positive control group. The results showed that TT and NN had almost the same binding capacity to TLR 2 / 1 as WT, indicating that TT and NN not only reduced their binding capacity to GD1a, but also maintained their binding capacity to TLR 2 / 1.

[0087] Compared to other mutations, AK has relatively low TLR1 / 2 activity, but it does not easily aggregate in aqueous solution, exhibits better structural stability, and retains a certain GD1a binding capacity.

[0088] Example 4. The new LTB variant has better potential than LTB as a protein adjuvant.

[0089] To confirm that LTB has antigen adjuvant activity, LTB was used as an adjuvant, and the N1NA protein (containing two mutation points, N329 and K331T) of the A / California / 04 / 2009 (H1N1) virus strain was used as a nasal spray vaccine to conduct immunization experiments on mice. Each group had five mice, and different LTB mutations, WT, TT, and NN were used as adjuvants for comparison. The mice were immunized with three doses of nasal spray at weeks 0, 3, and 6. Blood was collected at week 8 to obtain serum, and lung flushing fluid (BALF) was obtained by sacrificing the mice at week 9. Finally, ELISA was used to detect the IgG and IgA levels in the serum and BALF.

[0090] PBS and purified antigen were used as control groups, while polyinosinic acid-polycytidylic acid (poly(I:C), a double-stranded RNA analog) was used as a positive control. In both serum and BALF, the anti-N1 IgG and IgA titers in the adjuvanted group were significantly higher than those in the control group. In the control group, anti-N1 IgA was not even detectable in serum and BALF. Specifically, TT produced more anti-N1 IgG and IgA in serum and more anti-N1 IgA in BALF compared to WT and NN.

[0091] In BALF, the total IgA content was highest in NN, second highest in WT, and lowest in TT among the groups with adjuvants.

[0092] Figures 12A-12E The adjuvant efficacy of LTB TT and NN was demonstrated. (12A) Intranasal immunization procedure in mouse models: Mouse experiments were conducted, and mouse serum and BALF were collected. Neuraminidase (NA)-specific IgG and IgA were detected using ELISA. BALB / c mice (n=5 per group) were immunized intranasally with recombinant NA protein of influenza strain N1 containing two mutant sites (N329T and K331T), adjuvanted with LTB WT, TT, or NN, for three doses. Polyinosinic acid-polycytidylic acid (poly(I:C), a double-stranded RNA analog) was used as a positive control for adjuvants because it could trigger an antiviral immune response. ELISAs were used to measure the (12B) N1NA-specific IgG titer and (12C) N1NA-specific IgA titer in serum, as well as the (12D) N1NA-specific IgA titer and (12E) total IgA titer in BALF.

[0093] In a mouse model, after three doses of intranasal immunization, compared with the group using LTB WT, the combination of the influenza virus N1 neuraminidase (NA) immunogen with the protein adjuvants LTB TT and NN induced similar N1NA-specific IgG titers in serum. Figure 12B ).

[0094] In a mouse model, after three doses of intranasal immunization, the immunogen of influenza virus N1 neuraminidase (NA) binds to the protein adjuvant LTB TT, inducing significant N1NA-specific IgA titers in serum and bronchoalveolar lavage fluid (BALF). Figure 12C and 12D ).

[0095] Figures 13A-13C The activity of serum and BALF against influenza A / California / 04 / 2009 (H1N1) virus was shown. Enzyme-ligand agglutinin assay was used to measure the titer of the NA inhibitory antibody, i.e., the reduction in NA enzyme activity of the P1N1 virus (Pandemic Influenza A Virus). NAI activity against A / California / 04 / 2009 (H1N1) virus in serum (13A) and BALF (13B) was also shown. The corresponding half-maximal inhibitory concentration (IC50) was also presented for (13C). 50 (This refers to the half-inhibitory concentration of the antagonist being measured), with a titer of 50% reduction in viral N1NA enzyme activity. Results showed that, compared to N1NA protein without adjuvant, groups using poly(I:C), LTB WT, and their variants TT and NN as adjuvants induced significant N1NA inhibitory titers in serum and BALF. Specifically, the use of TT adjuvant was found to result in higher N1NA titers in both serum and BALF.

[0096] To test the neuraminidase inhibitory capacity of the antibodies, enzyme-linked lectin assay (ELLA) was used to detect neuraminidase inhibitory antibodies in each immunization group.

[0097] Experiments using H1N1 as the virus strain revealed that serum samples with adjuvants, even diluted 100-fold, still exhibited some antiviral activity, significantly higher than the control group. Furthermore, TT (transmissible thrombocytopenic precipitate) showed a relatively high IC50 value. 50 In terms of potency, the BALF group showed the same trend, with the adjuvanted group exhibiting significantly higher antiviral activity than the control group, and TT also had a relatively high IC50. 50 In terms of potency, this experiment demonstrated that among the three adjuvants, TT produced the most specific antibodies and induced the most neuraminidase inhibitory antibodies. The results indicate that TT, as an adjuvant, elicits a higher immune response to the antigen than WT and NN.

[0098] Serial dilutions of serum and diluted BALF were incorporated into the inhibition of binding between NA antibody and N1 protein. Results showed that immunization with N1 protein, along with LTB WT and its recombinant forms TT and NN, induced a significantly enhanced inhibitory titer upon binding to N1 protein without protein adjuvant. Figures 13A-13C ).

[0099] Figure 14 This study demonstrated the protective effect against H1N1 virus after inoculation with recombinant N1NA protein, aided by the addition of LTB TT or LTB WT adjuvants. Three weeks after the final immunization, immunized mice (n=6 per group) were intranasally infected with 5×LD50 A / PR8 (H1N1) virus. Survival rate and body weight were monitored daily for 14 days post-infection. Mice were considered to have no effective protection and were deemed surviving if they experienced a weight loss exceeding 25%.

[0100] N1NA protein with LTB TT adjuvant was found to have the highest NA antibody inhibitory titer. In protective immunization mouse experiments, LTB TT showed better protective immunity than LTB WT during viral challenge. Figure 14 ).

[0101] In summary, based on the results illustrated in the above embodiments, this invention utilizes an LTB protein scaffold to invent a novel LTB circular recombinant protein, which can be used to develop novel respiratory mucosal protein adjuvants. Novel LTB adjuvants with good structural stability are designed using circular recombinant design. The ability to activate TLR2 / 1 receptors is maintained using circular recombinant design, resulting in novel LTB adjuvants with comparable or higher antibody activity. Furthermore, the design of novel LTB adjuvants using circular recombinant design can reduce GD1a ganglioside binding capacity, thereby reducing cytotoxicity. The use of novel LTB adjuvants designed with circular recombinant design induces better immune response stimulation.

[0102] This invention utilizes the concept of circular recombination to design three LTB circular recombination variants (CP) and evaluates their GD1a binding affinity and structural stability using nuclear magnetic resonance (NMR) spectroscopy. Among the novel LTB circular recombination variants, TT exhibits excellent structural stability and demonstrates superior protein adjuvant activity. NN shows structural instability similar to WT, exhibiting a higher tendency to aggregate. Both TT and NN lose their GD1a binding affinity through circular recombination but retain their ability to activate TLR 2 / 1. AK exhibits superior structural stability, is less prone to aggregation in aqueous solution, retains GD1a binding affinity, but has lower TLR 2 / 1 activation ability. These circular recombination variants endow LTB with different protein adjuvant activities, allowing for use for various indications and potentially advancing the future development of mucosal vaccines, particularly for respiratory diseases such as influenza.

[0103] The above description is illustrative only and not restrictive. Any equivalent modifications or alterations made without departing from the spirit and scope of this invention should be included in the appended claims.

Claims

1. A recombinant protein comprising a B5 subunit (LTB) variant of Escherichia coli (E. coli) type II heat-sensitive enterotoxin, wherein, The LTB variant contains at least one amino acid sequence via cyclic recombination.

2. The recombinant protein according to claim 1, wherein, The circular recombination is performed by breaking a specific loop of the B5 subunit (LTB) of wild-type Escherichia coli type 2 thermosensitive enterotoxin and connecting the original end via the GSGS linker.

3. The recombinant protein according to claim 2, wherein, The residues 13–14, 31–32, or 52–53 of the LTB variant are broken to generate new N-terminals and C-terminals.

4. The recombinant protein according to claim 3, wherein, The residues 13-14 are Thr13-Thr14.

5. The recombinant protein according to claim 3, wherein, The residues 31-32 are Asn31-Asn32.

6. The recombinant protein according to claim 3, wherein, The residues 52-53 are Ala52-Lys53.

7. The recombinant protein according to claim 1, wherein, The at least one amino acid sequence via cyclic recombination is selected from the group consisting of: sequence identification number 1, sequence identification number 2, sequence identification number 3, and combinations thereof.

8. The recombinant protein of claim 1, further comprising a 6xHis-tag sequence, wherein the 6xHis-tag sequence is conjugated to the C-terminus of the LTB variant.

9. A method for preparing the recombinant protein according to claim 1, comprising cyclic recombination of the B5 subunit (LTB) of wild-type Escherichia coli type II heat-sensitive enterotoxin.

10. The method according to claim 9, wherein, The circular recombination is performed by breaking the corresponding loop of the B5 subunit (LTB) of the wild-type Escherichia coli type 2 thermosensitive enterotoxin and connecting the original end via the GSGS linker.

11. The method according to claim 10, wherein, The residues 13–14, 31–32, or 52–53 of the LTB variant are broken to generate new N-terminals and C-terminals.

12. The method according to claim 11, wherein, The residues 13-14 are Thr13-Thr14.

13. The method according to claim 11, wherein, The residues 31-32 are Asn31-Asn32.

14. The method according to claim 11, wherein, The residues 52-53 are Ala52-Lys53.

15. A protein adjuvant for a vaccine comprising a recombinant protein according to any one of claims 1 to 8.

16. Use of a recombinant protein according to any one of claims 1 to 8 in the preparation of a pharmaceutical product for the prevention of respiratory diseases.

17. The use according to claim 16, wherein, The respiratory diseases mentioned are selected from the group consisting of: influenza, severe acute respiratory syndrome, severe special infectious pneumonia, and other diseases transmitted through the respiratory tract.

18. The use according to claim 16, wherein, Recombinant proteins containing the amino acid sequences shown by sequence identification numbers 1 and 2 have no or reduced GD1a binding ability.

19. The use according to claim 16, wherein, The recombinant protein maintains its binding ability to TLR 2 / 1.

20. The use according to claim 16, wherein, The dosage form of the pharmaceutical product is an intramuscular injection, injection, nasal drops, spray, nasal spray medication, or inhaler.

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