A vaccine composition comprising a recombinant g protein of henipavirus

Abstract

The application discloses a vaccine composition containing a recombinant G protein of Hendra virus. Specifically, a multimer and a monomer are constructed by using the recombinant G protein of Hendra virus, and a better in-vivo protection effect than DNA is obtained; and the construction of the tandem protein also effectively solves the problem of poor broad-spectrum property of the vaccine. Meanwhile, the in-vivo protection effect evaluation of the vaccine can be quickly and safely realized by detecting the fluorescence residue in the mouse in-vivo through a VSV pseudovirus live imaging model.

Description

Technical Field

[0001] This invention relates to the field of recombinant protein vaccines, and more specifically to vaccine compositions comprising recombinant Nipah virus protein polymers. Background Technology

[0002] Nipah virus (NiV) is a novel paramyxovirus with a mortality rate as high as 40%-75%. NiV is carried by fruit bats and can spread widely among livestock and humans through various routes, including contaminated materials and food, posing a significant challenge to public health.

[0003] The NiV gene encodes six structural proteins: N, P, M, F, G, and L. The G protein is a naturally occurring tetrameric structure, with its extracellular domain core including the stem, neck, and head regions. The G protein can be used for NiV vaccine design and construction. However, current vaccines against Nipah virus infection still fail to induce sufficient protective antibodies. Furthermore, NiV, as a highly lethal and virulent virus, is listed as a biosafety level 4 pathogen, limiting the evaluation of the in vivo protective efficacy of related vaccines or antibodies. Efficient diagnostic methods are also lacking during vaccine development. There is an urgent need to research new vaccine evaluation methods and NiV vaccines. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention designs monomeric, trimeric, and tetrameric recombinant proteins based on the G protein structure, and evaluates the immunogenicity, immunogenicity, and cross-protection against Hendra virus (HeV) strains of different recombinant G proteins and DNA vaccines. Furthermore, a VSV pseudovirus in vivo imaging system is constructed to evaluate the in vivo protective effects of different immunogens.

[0005] On the one hand, this application provides an immunogenic composition.

[0006] In some embodiments, the immunogenic composition comprises a tetramer, trimer, and / or monomer of a G protein. In some specific embodiments, the immunogenic composition comprises a tetramer of a G protein; in some specific embodiments, the immunogenic composition comprises a trimer of a G protein; in some specific embodiments, the immunogenic composition comprises a monomer of a G protein.

[0007] In some embodiments, the monomer includes at least two G protein domains in tandem. In some specific embodiments, the monomer includes the head domains of at least two G proteins in tandem.

[0008] In some embodiments, the G protein is derived from Hennipa virus. In some specific embodiments, the G protein is derived from Nipah virus; in some specific embodiments, the G protein is derived from Hendra virus; in some specific embodiments, the G protein is derived from both Nipah virus and Hendra virus.

[0009] In some embodiments, the two tandem G protein domains in the G protein monomer include: HeV G protein head domain-HeV G protein head domain, NiV G protein head domain-NiV G protein head domain, HeV G protein head domain-NiV G protein head domain, or NiV G protein head domain-HeV G protein head domain.

[0010] In some specific embodiments, the G protein monomer comprises: HeV G protein domain-NiV G protein domain; in some specific embodiments, the G protein monomer comprises: NiV G protein domain-HeV G protein domain.

[0011] In some specific embodiments, the G protein monomer includes: HeV G protein head domain - NiV G protein head domain; in some specific embodiments, the G protein monomer includes: NiV G protein head domain - HeV G protein head domain.

[0012] In some embodiments, the G protein tetramer includes all functional domains of the G protein; in some specific embodiments, the G protein tetramer includes the stem domain, neck domain, and / or head domain of the G protein.

[0013] In some embodiments, the G protein trimer includes all functional domains of the G protein; in some specific embodiments, the G protein trimer includes the stem domain, neck domain, and / or head domain of the G protein.

[0014] In some embodiments, the G protein trimer includes the head domain of the G protein.

[0015] In some embodiments, the stem domain of the G protein includes an amino acid sequence as shown in SEQ ID NO: 4 or having at least 90% identity with and the same function as it;

[0016] In some embodiments, the neck domain of the G protein includes an amino acid sequence as shown in SEQ ID NO: 5 or having at least 90% identity with and the same function as it;

[0017] In some embodiments, the head domain of the G protein includes an amino acid sequence as shown in SEQ ID NO: 6 or 7, or having at least 90% identity with and functioning the same amino acid sequence.

[0018] In some embodiments, the G protein tetramer forms a tetramer through post-translational self-assembly; in some specific embodiments, the G protein tetramer forms a stable tetramer structure through post-translational self-assembly.

[0019] In some embodiments, the G protein trimer forms a trimer through post-translational self-assembly; in some specific embodiments, the G protein trimer forms a stable trimer structure through post-translational self-assembly.

[0020] In some embodiments, the structure of the G protein tetramer is designed as follows: the signal peptide of the original G protein sequence is replaced with the tPA signal peptide, and a His tag and a GS linker are added after tPA;

[0021] In some implementations, the G protein trimer is designed as follows: the entire stem and neck regions of the original sequence are deleted, the signal peptide of the original G protein sequence is replaced with the tPA signal peptide, and a His tag and GSG linker are added after tPA. The G protein head domain is connected through the T4 heterotrimeric motif and the GS linker.

[0022] In some implementations, the G protein monomer is designed as follows: the entire stem and neck regions of the original sequence are deleted, the signal peptide of the original G protein sequence is replaced with the tPA signal peptide, and a His tag and a GSG linker are added after tPA, with the two G protein head domains connected by the GS linker.

[0023] In some embodiments, the G protein tetramer comprises a first recombinant G protein as shown in SEQ ID NO: 1;

[0024] In some embodiments, the G protein trimer includes a second recombinant G protein as shown in SEQ ID NO: 2;

[0025] In some embodiments, the G protein monomer comprises a third recombinant G protein as shown in any one of SEQ ID NO: 3, 8 or 9; in some specific embodiments, the G protein monomer comprises a third recombinant G protein as shown in SEQ ID NO: 3; in some specific embodiments, the G protein monomer comprises a third recombinant G protein as shown in SEQ ID NO: 8; in some specific embodiments, the G protein monomer comprises a third recombinant G protein as shown in SEQ ID NO: 9.

[0026] On the one hand, the present invention provides a nucleic acid molecule.

[0027] In some embodiments, the nucleic acid molecule encodes an immunogenic composition as described in any of the above embodiments.

[0028] On the one hand, the present invention provides a recombinant expression vector.

[0029] In some implementations, the recombinant expression vector comprises a nucleic acid molecule as described in any of the above embodiments.

[0030] On the one hand, the present invention provides a host cell.

[0031] In some embodiments, the host cell expresses the immunogenic composition as described in any of the preceding claims; or contains the nucleic acid molecule as described in claim 5; or contains the recombinant expression vector as described in any of the preceding claims.

[0032] On the one hand, the present invention provides a method for preparing an immunogenic composition.

[0033] In some implementations, the method includes:

[0034] Culture medium is obtained by culturing host cells as described in any of the above; and the expressed recombinant protein is isolated from said culture medium to prepare an immunogenic composition.

[0035] On the one hand, the present invention provides a vaccine composition.

[0036] In some embodiments, the vaccine composition comprises an immunogenic composition as described in any of the above, a nucleic acid molecule as described in any of the above, or a recombinant expression vector as described in any of the above;

[0037] And a vaccine-acceptable vector;

[0038] In some implementations, the vaccinologically acceptable carriers include aluminum hydroxide, aluminum phosphate, aluminum hydroxide / CpG, aluminum phosphate / CpG, MF59, AS01, AS03, etc.

[0039] In some implementations, the vaccinologically acceptable carrier includes aluminum hydroxide / CpG (i.e., aluminum hydroxide and CpG). Attached Figure Description

[0040] Figure 1This diagram illustrates the construction of recombinant G protein tetramers, trimers, and monomers. In this diagram, NiV GM represents the extracellular domain of the NiV Malaysian strain G protein; G-Tetramer represents the sequence design of the recombinant G protein tetramer; G-Trimer represents the sequence design of the recombinant G protein trimer; G-Dimer represents the sequence design of the recombinant G protein monomer; SP represents the signal peptide; Stalk represents the G protein stem domain; Neck represents the G protein neck domain; Head represents the G protein head domain; tPA represents the tissue plasminogen activator signal peptide; Linker represents the linker; T4 represents the heterotrimeric motif; and 8×His represents the histidine tag.

[0041] Figure 2 This section presents the expression, purification, and identification results of recombinant G protein tetramers, trimers, and monomers. Specifically, 2A: SDS-PAGE, Western blot, and high-performance liquid chromatography (HPLC) molecular weight determination results of recombinant G protein tetramers; 2B: SDS-PAGE, Western blot, and HPLC molecular weight determination results of recombinant G protein trimers; and 2C: SDS-PAGE, Western blot, and HPLC molecular weight determination results of recombinant G protein monomers.

[0042] Figure 3 This indicates the results of the detection of neutralizing antibody levels induced by recombinant G protein and DNA vaccines. Specifically, 3A: Detection of neutralizing NiV Malaysian strain antibody levels induced by recombinant G protein and DNA vaccines (3 weeks after the second immunization); 3B: Detection of neutralizing NiV Malaysian strain antibody levels induced by recombinant G protein and DNA vaccines (12 weeks after the second immunization); 3C: Differences in antibody levels induced by recombinant G protein against 5 different NiV strains and 5 different HeV strains.

[0043] Figure 4 This indicates the in vivo protective efficacy test results of recombinant G protein and DNA vaccines. Specifically, 4A: in vivo imaging fluorescence detection in mice after immunization with the recombinant G protein and DNA vaccines; 4B: quantitative fluorescence residue in mice after immunization with the recombinant G protein and DNA vaccines; and 4C: linear correlation between the level of neutralizing antibodies induced in immunized mice and the in vivo imaging fluorescence residue signal value.

[0044] Figure 5 This represents the SDS-PAGE results of three different G protein monomers.

[0045] Figure 6 This indicates the detection of neutralizing antibodies induced by sequential immunization with three different G protein monomers. Specifically, 6A represents the detection of neutralizing antibodies against the NiV strain induced by sequential immunization with three different G protein monomers, and 6B represents the detection of neutralizing antibodies against the HeV strain induced by sequential immunization with three different G protein monomers.

[0046] Figure 7 This indicates the detection of cellular immunity levels induced by sequential immunization with three different G protein monomers. Specifically, 7A: IFN-γ level detection induced by sequential immunization with three different G protein monomers; 7B: IL2 level detection induced by sequential immunization with three different G protein monomers; and 7C: IL4 level detection induced by sequential immunization with three different G protein monomers. Detailed Implementation

[0047] The following description of specific embodiments further illustrates this application, but it is not intended to limit the scope of this disclosure. Those skilled in the art can make various modifications or improvements based on the teachings of this application without departing from its basic ideas and scope. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0048] Example 1: Sequence design, modification and preparation of G protein from NiV Malaysian strain.

[0049] 1.1 Experimental Methods

[0050] G protein tetramer amino acid sequence design: The original signal peptide was replaced with the tPA signal peptide, and a His tag (HHHHHHHH; SEQ ID NO:10) and a linker (GSGGGGS; SEQ ID NO:11) were added after tPA. Figure 1 );

[0051] G protein trimer amino acid sequence design: The entire stem and neck regions of the original sequence were deleted. The original signal peptide was replaced with the tPA signal peptide, and a His tag (HHHHHHHH) and a short linker (GSG) were added after tPA. The G protein head domain was then linked via a T4 heterotrimeric motif and a long linker (GSGGGGS). Figure 1 );

[0052] G protein monomer amino acid sequence design: The entire stem and neck regions of the original sequence were deleted. The original signal peptide was replaced with the tPA signal peptide, and a His tag (HHHHHHHH) and a short linker (GSG) were added after tPA. The two G protein head domains were linked by the linker (GSGGGGS). Figure 1 ).

[0053] The recombinant G protein tetramer, trimer, and monomer sequences were all constructed into the pcDNA3.1 vector.

[0054] Recombinant G protein tetramers, trimers, and monomeric plasmids were transformed into DH5α competent cells and plated. Single clones were picked and activated. 1 mL of bacterial culture was placed in 200 mL of LB medium and cultured at 37°C and 200 rpm / min for 12 h. Plasmids were then extracted using an endotoxin-free plasmid extraction kit and used for subsequent recombinant protein expression and DNA immunization.

[0055] Recombinant G protein expression: Recombinant protein was obtained by transient expression in HEK293 suspension cells. Cells were cultured to 3 × 10⁶ cells before transfection. 6 - 4×10 6 300 μg of plasmid and 900 μl of PEI were mixed at a ratio of 1:3 and added to 100 mL of cells. After 3 h of transfection, 100 mL of fresh culture medium was added and the cells were cultured continuously at 37 °C, 8% CO2, and 120 rpm / min for 7 days.

[0056] Purification of recombinant G protein: After expression, the cell supernatant was centrifuged at 2000×g for 10 min, then transferred to a clean centrifuge tube for a second centrifugation at 8000 rpm for 30 min. The supernatant was then transferred to a clean container for purification. The instrument was cleaned and a purification column was pre-packed before purification. After equilibrating the nickel column for 20 min, the protein was injected at a flow rate of 5 mL / min. Impurities were washed with equilibration buffer for 10 min, and finally, the target protein was eluted with 0.5 M imidazole. The solution was collected based on the peak value. After purification, the protein was concentrated using a 10 kD ultrafiltration tube, and the buffer system was replaced. The ultrafiltered protein was quantified using the BCA method.

[0057] 1.2 Experimental Results

[0058] SDS-PAGE, Western blot, and high-performance liquid chromatography molecular weight determination experiments of the purified target protein showed that the recombinant G protein tetramer had a molecular weight of approximately 282.7 kD. It is relatively unstable in its native state and readily depolymerizes into a monomeric conformation (molecular weight approximately 163.1 kD). Simultaneously, aggregates of some recombinant G protein tetramers (molecular weight approximately 610.9 kD) were detected. Figure 2 A). Recombinant G protein trimers exhibit monomeric, monomeric, and trimeric bands under electrophoretic conditions. In the native state, they primarily exhibit a trimeric conformation with a molecular weight of approximately 194.9 kD. Higher molecular weight aggregates (approximately 423.3 kD) are also present. Figure 2 B). The recombinant G protein monomer exhibited a single monomer band during both electrophoresis and high-performance liquid chromatography detection, with a molecular weight of approximately 118.9 kDa. Figure 2 C).

[0059] Example 2: Immunization Procedure of Recombinant G Protein and DNA Vaccine

[0060] Recombinant G protein was administered via intramuscular injection, with each mouse receiving 10 μg of protein, 250 μg of aluminum hydroxide, and 20 μg of CpG. For the DNA vaccine, each mouse was injected intramuscularly with 50 μg of plasmid, followed by electrical stimulation to enhance the effectiveness of the DNA vaccine. A second immunization was administered 3 weeks after the first, with blood samples collected from the orbital cavity at 3 and 12 weeks after the second immunization.

[0061] Example 3: Neutralizing Antibody Detection

[0062] 3.1 Experimental Methods

[0063] The serum to be tested was inactivated in a 56℃ water bath for 30 min. In a 96-well plate, the first column was set up as a cell control, the second as a virus control, and columns three through twelfth as the test sample area. 1.5 μL of serum was added to the first well of the test sample area, along with 148.5 μL of culture medium. 100 μL of culture medium was added to each of the remaining wells. After mixing thoroughly in the first row of the test sample area, 50 μL was transferred to the next row, and this was serially diluted 3-fold. After dilution, 50 μL of diluted pseudovirus was added to each well in columns two through twelfths. The plates were incubated at 37℃ for 1 h. 50,000 293T cells were added to each well, and the plates were incubated at 37℃ for 24 h. The 96-well plate was then removed, the supernatant was discarded, substrate was added, and the plates were analyzed using a microplate reader.

[0064] 3.2 Experimental Results

[0065] The levels of neutralizing antibodies induced in mice were measured at 3 and 12 weeks after the second immunization. The results showed that the neutralizing antibodies induced by recombinant G protein tetramer, trimer, and monomer against the Malaysian NiV strain were significantly higher than those induced by the DNA immunization strategy, with differences ranging from 12 to 137 times. Figure 3 (A and B). The recombinant G protein tetramer induced neutralizing antibodies at 12 weeks post-second immunization as well as at 3 weeks post-second immunization. Recombinant G protein trimers and dimers showed a decrease in neutralizing antibody levels, approximately 1.5 to 2 times. DNA immunohistochemistry induced neutralizing antibodies with the G protein trimer structure exhibited a longer-lasting and more potent effect.

[0066] Three weeks after the second immunization, the levels of neutralizing antibodies against different NiV and HeV strains of the recombinant G protein were measured to evaluate the cross-protective effect of the NiV recombinant G protein. The results showed that the tetramer, trimer, and monomer constructed based on the NiV G protein all exhibited higher neutralizing antibody activity against NiV strains than against HeV strains. Figure 3 C. Table 1).

[0067] Table 1. Differences in neutralizing antibodies against NiV and HeV strains from recombinant G protein.

[0068]

[0069] Note: All gene IDs are from the NCBI database.

[0070] Example 4: In vivo imaging fluorescence detection

[0071] 4.1 Experimental Methods

[0072] Twelve weeks after the second immunization, mice in the experimental group were infected with a pseudovirus via intraperitoneal injection at a dose of 4.58 × 10⁵ TCID₅₀ per mouse. In vivo fluorescence imaging was performed 6 hours post-infection. Each mouse was injected with 200 μL of fluorescein potassium salt 5–7 minutes before detection, immediately anesthetized, and neatly placed in the in vivo imaging system. An 8 × 8 Binging value was selected for bioluminescence imaging. All mice were adjusted to a uniform grayscale range, and areas of the same size were circled for quantification of fluorescence values.

[0073] 4.2 Experimental Results

[0074] In vivo imaging results showed that no or very little fluorescence residue was observed in the recombinant G protein tetramer, trimer, and monomer immunization groups. However, after DNA immunization, except for the monomeric DNA vaccine group which showed less fluorescence residue (a difference of 1.3 times), the other two groups of mice showed significant fluorescence signal values ​​(5.1 times and 3.3 times higher than the recombinant G protein immunization group, respectively). This indicates that the in vivo protective effect of recombinant G protein is significantly better than that of the DNA immunization strategy. Figure 4 A and B). Linear fitting was performed based on the neutralizing antibody levels in each immunization group and the fluorescence values ​​detected by in vivo imaging mice. The results showed that the fluorescence signal value after pseudovirus infection in mice was negatively correlated with the level of immune-induced neutralizing antibodies (R² = 0.8124). Figure 4 C).

[0075] Example 5: Enhancement of broad-spectrum immunity

[0076] To address the poor cross-neutralizing activity of the NiV recombinant G protein against HeV, this experiment further modified two tandem NiVG head domain monomeric proteins (NiV-NiV), replacing one of the head domains with HeV (HeV-NiV and NiV-HeV, respectively). Figure 5 The study compared the differences in neutralizing antibody induction using a sequential immunization strategy. Sequential immunization included three strategies: two DNA injections, one DNA injection plus one recombinant protein injection, and two recombinant protein injections. For DNA immunization, the dose was 50 μg / mouse, administered intramuscularly to the leg muscles followed by electrical stimulation to enhance transfection. For recombinant protein immunization, the dose was 5 μg / mouse, combined with aluminum hydroxide / CpG (50 μg / mouse and 20 μg / mouse, respectively), also administered via intramuscular injection to the leg. A booster immunization was performed 4 weeks after the first immunization, and blood samples were collected 3 weeks after the second immunization to measure neutralizing antibody levels.

[0077] Experimental results showed that the level of neutralizing antibodies induced by NiV recombinant G protein tetramer was superior to that of the three different monomers (approximately 2-fold) in terms of neutralizing activity against NiV strains. However, in the neutralizing antibody results against HeV strains, the levels of neutralizing antibodies induced by recombinant G protein tetramer and monomers containing two NiV G protein head domains in tandem were significantly lower than those induced by HeV-NiV or NiV-HeV. Figure 6 The sequential immunization strategy, namely DNA immunization + recombinant protein, did not significantly improve the level of neutralizing antibody induction. However, the NiV-HeV group showed a certain degree of improvement in the level of IL-4 induced by cellular immunity, which was statistically different from the blank control group (statistical method: Kruskal-Wallis test). Figure 7 ).

[0078] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

[0079] The partial sequence of this invention is as follows:

[0080] First recombinant G protein

[0081]

[0082] (Note: Underlined parts are linkers, bold parts are His tags, italic parts are signal peptides, and the rest are extracellular domains of G proteins.)

[0083] Second recombinant G protein

[0084]

[0085] (Note: Underlined parts are linkers, wavy parts are heterotrimeric motifs, bold parts are His tags, italic parts are signal peptides, and the rest are G protein head domains.)

[0086] Third recombinant G protein

[0087]

[0088] (Note: Underlined parts are linkers, bold parts are His tags, italic parts are signal peptides, and the rest are G protein head domains.)

[0089] Stem structural domains

[0090]

[0091] Neck region

[0092]

[0093] NiV G protein head domain

[0094]

[0095] HeV G protein head domain

[0096]

[0097] >HeV-NiV monomeric protein

[0098]

[0099] (Note: Underlined parts are linkers, bold parts are His tags, italic parts are signal peptides, double underlined parts are the G protein head domains of NiV, and dashed underlined parts are the G protein head domains of HeV.)

[0100] NiV-HeV monomeric protein

[0101]

[0102] (Note: Underlined parts are linkers, bold parts are His tags, italic parts are signal peptides, double underlined parts are the G protein head domains of NiV, and dashed underlined parts are the G protein head domains of HeV.)

Claims

1. An immunogenic composition comprising a tetramer, trimer, and / or monomer of a G protein, said monomer comprising at least two G protein domains in tandem; said G protein being derived from Hennipa virus.

2. The immunogenic composition according to claim 1, wherein, The two tandem G protein domains in the G protein monomer include: HeV G protein head domain-HeV G protein head domain, NiV G protein head domain-NiV G protein head domain, HeV G protein head domain-NiV G protein head domain, or NiV G protein head domain-HeV G protein head domain.

3. The immunogenic composition according to claim 2, wherein, The structure of the G protein tetramer is designed as follows: the original G protein sequence signal peptide is replaced with tPA signal peptide, and a His tag and GS linker are added after tPA; The structural design of the G protein trimer is as follows: delete all stem and neck regions of the original sequence, replace the signal peptide of the original G protein sequence with tPA signal peptide, add His tag and GSG linker after tPA, and connect the G protein head domain through T4 heterotrimeric motif and GS linker. The structure of the G protein monomer is designed as follows: the entire stem and neck regions of the original sequence are deleted, the signal peptide of the original G protein sequence is replaced with the tPA signal peptide, and a His tag and a GSG linker are added after tPA. The two G protein head domains are connected by the GS linker.

4. The immunogenic composition according to claim 3, wherein, The G protein tetramer includes a first recombinant G protein as shown in SEQ ID NO:1, the G protein trimer includes a second recombinant G protein as shown in SEQ ID NO:2, and the G protein monomer includes a third recombinant G protein as shown in any one of SEQ ID NO:3, 8, or 9.

5. A nucleic acid molecule, characterized in that, Its code is the immunogenic composition as described in any one of claims 1-4.

6. A recombinant expression vector, characterized in that, It contains the nucleic acid molecule as described in claim 5.

7. A host cell, characterized in that, Its expression comprises the immunogenic composition as described in any one of claims 1-4; or comprises the nucleic acid molecule as described in claim 5; or comprises the recombinant expression vector as described in claim 6.

8. A method for preparing an immunogenic composition, characterized in that, The method includes: Culture medium is obtained by culturing the host cells as described in claim 7; and the expressed recombinant protein is isolated from the culture medium to prepare an immunogenic composition.

9. A vaccine composition, characterized in that, It includes the immunogenic composition as described in any one of claims 1-4, the nucleic acid molecule as described in claim 5, or the recombinant expression vector as described in claim 6; And a vaccine-acceptable vector; The vaccine-acceptable carriers include aluminum hydroxide, aluminum phosphate, aluminum hydroxide / CpG, aluminum phosphate / CpG, MF59, AS01, and AS03.

10. The vaccine composition according to claim 9, characterized in that, The vaccine-acceptable carriers include aluminum hydroxide / CpG.

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