BE1031628B1 - APPLICATION OF CIRCHIFIA IN THE PRODUCTION OF NOVEL VACCINES AGAINST BACTERIAL INFECTIONS - Google Patents

Abstract

The present invention discloses an application of circHIF1α in the production of novel vaccines against bacterial infections and relates to the field of biomedical technology. The nucleotide sequence of circHIF1α is as shown in SEQ ID NO.1. In the present invention, Glaesserella parasuis (G. parasuis) is used as a representative of Gram-negative bacteria, Streptococcus suis 2 (SS2) and Staphylococcus aureus (S. aureus) are used as Gram-positive bacteria, and the important role of circHIF1α in the adhesion and invasion of the above-mentioned bacteria to and into host cells is investigated at the in vitro level and the inhibitory effect of circHIF1α on bacterial infection is investigated at the in vivo level, and the results confirm that circHIF1α can inhibit bacterial adhesion to and invasion into host cells, thereby reducing the harm of the bacteria to the organism.

Description

APPLICATION OF CIRCHIFIA IN THE PRODUCTION OF NOVEL

VACCINES AGAINST BACTERIAL INFECTIONS

TECHNICAL FIELD

The present invention relates to the field of biomedical technology and, in particular, to an application of circHIF1a in the production of novel vaccines against bacterial infections.

STATE OF THE ART

So far, many RNAs are known that are transcribed by eukaryotic genomes but cannot code for proteins. They are called non-coding RNAs (non-coding RNA,

NcRNAs), including rRNAs, tRNAs, snRNAs, snoRNAs and microRNAs.

Among them, circular RNA (circRNA) is a newly discovered class of circular

RNAs that are widely distributed in eukaryotic cells and mostly originate from protein-coding genes. The process of formation of circRNAs differs from the normal splicing of mRNAs, circRNAs are formed by reverse splicing, where the downstream 5' donor splice site is joined to the upstream 3' acceptor splice site by a phosphodiester bond to form a single-stranded covalent closed

to form a loop.

Over the past decade, circular RNAs (circRNAs) have emerged as an important class of non-coding RNA molecules, and many circRNAs play key roles in cancer development and pathogenesis through different mechanisms of action. CircRNAs typically exhibit tissue-restricted and cancer-specific

expression patterns, and data from previous studies have shown that these molecules have potential clinical applications as diagnostic, prognostic and predictive

Biomarkers. Due to their special circular structure, circRNAs are characterized by a high

stability and RNase resistance, and the interest in the development of circRNA synthesis techniques and in exploring the application of synthetic circRNAs in

Diseases has increased in recent years with the increasing understanding of the physiological

Functions of natural circRNAs have increased. Currently, circRNAs are not only used in biosensors and drugs, but also as alternative therapeutic proteins, peptides and

Vaccines. Previous studies have reported that a circRNA vaccine against neococcal

Coronavirus has better immune protection than mRNA, but so far no circRNA-based bacterial vaccine has been developed, so the applicability of circRNA-

vaccines in the treatment of bacterial infections needs to be further investigated.

CONTENT OF THE PRESENT INVENTION

The object of the present invention is to provide an application of circHIF1a in the production of novel vaccines against bacterial infections in order to

The present invention has found that circHIF1a can inhibit both the adhesion and invasion of bacteria to and into host cells, and thus reduce the damage caused by the bacteria to the organism, so that circHIF1a can be used in the production of novel vaccines against bacterial

infections.

To achieve the above objectives, the present invention provides the following solutions:

The present invention provides an application of circHIF1a in the production of novel vaccines against bacterial infections, wherein the nucleotide sequence of circHIF1a is as shown in SEQ ID NO. 1.

Furthermore, circHIF 1a acts as a protection against bacterial infections by inhibiting bacterial adhesion to and/or invasion into host cells.

Furthermore, the bacteria are gram-negative and/or gram-positive.

The gram-negative bacteria are G/aesserella parasuis, the gram-positive bacteria are Streptococcus suis 2 or Staphylococcus aureus.

The present invention also provides an application of a biomaterial overexpressing circHIF1a in the production of novel bacterial vaccines, wherein the

Nucleotide sequence of circHIF1a as shown in SEQ ID NO.1;

The circHIF 1a overexpressing biomaterial is an expression vector or a host cell that overexpresses circHIF 1a.

Furthermore, the biomaterial acts as protection against bacterial infection by overexpressing circHIF1a and inhibiting bacterial adhesion to and/or invasion into host cells.

Furthermore, the bacteria are gram-negative and/or gram-positive.

Furthermore, the gram-negative bacteria are G/aesserella parasuis and the gram-positive bacteria are Streptococcus suis 2 or Staphylococcus aureus.

The present invention also provides a vaccine against bacterial infections, wherein the composition comprises biological material overexpressing circHIF1«œ, wherein the

Nucleotide sequence of circHIF1a as shown in SEQ ID NO.1;

The circHIF1a overexpressing biomaterial is an expression vector or a host cell that overexpresses circHIF 1a.

The vaccine also contains pharmaceutically acceptable excipients.

The present invention discloses the following technical effects:

In the present invention, G/aesserella parasuis (G. parasuis) as representative of the Gram-negative bacteria, Streptococcus suis 2 (SS2) and Staphylococcus aureus (S. aureus) as

Gram-positive bacteria, and the important role of circHIF 1a in adhesion and

Invasion of the above bacteria to host cells is investigated at the in vitro level and the inhibitory effect of circHIF 1a on bacterial infection is investigated at the in vivo level, and the results confirmed that circHIF1a inhibits bacterial adhesion to and invasion into

host cells, thereby reducing the damage the bacteria cause to the organism.

DESCRIPTION OF THE FIGURES

In order to describe the embodiments of the present invention or the technical solutions in

In order to illustrate the state of the art more clearly, the accompanying drawings to be used in the embodiments are briefly described below, and it is obvious that the drawings accompanying the following descriptions are only some

Embodiments of the present invention, and those skilled in the art may further

Drawings according to these drawings without any inventive effort being involved.

Fig. 1 is a volcano diagram of differentially expressed circRNA in exosomes;

Fig. 2 is a heatmap of differentially expressed circRNA in exosomes;

Fig. 3 is the significant downregulation of circHIF 10 determined by qRT-PCR;

Fig. 4 shows the changes in circHIF1a expression in exosomes(A) and in the

Cell sedimentation (B) secreted by PIEC cells infected with SS2;

Fig. 5 shows the changes in circHIF1a expression in exosomes(A) and in the

Cell sedimentation (B) secreted by PIEC cells infected with S. aureus;

Fig. 6 is a schematic representation of the structural analysis of circHIF 10;

Figure 7 is a schematic representation of the pLC5-ciR vector;

Fig. 8 is the G. parasuis adhesion-invasion immunofluorescence diagram (A) and the

Analysis results of adhesion (B) and invasion (C); all scale bars in A are 25 µm;

Fig. 9 is the SS2 adhesion-invasion immunofluorescence diagram (A) and the

Analysis results of adhesion (B) and invasion (C); all scale bars in A are 25 µm;

Fig. 10 is the S. aureus adhesion-invasion immunofluorescence diagram (A) and the

Analysis results of adhesion (B) and invasion (C); all scale bars in A are 25 µm;

Figure 11 shows the effect of circHIF1a on the survival of mice after various bacterial infections; where A-C represent infections with G. parasuis, SS2 and S. aureus, respectively;

Figure 12 is the effect of circHIF1a on bacterial content in the organs of mice infected with different bacteria, where A-C represent infections with G. parasuis, SS2 and S. aureus, respectively;

Fig. 13 is a pathological histological cross-section.

DETAILED DESCRIPTION

Various embodiments of the present invention will now be described in detail, which are not to be considered as limitations on the present invention, but are to be understood as more detailed descriptions of certain aspects, features and embodiments of the present invention.

It is to be understood that the terms described in the present invention are merely for

serve to describe specific embodiments and are not to be used to limit the present invention. In addition, the ranges of values given in the present invention are to be understood as the specific disclosure of each intermediate value between the upper and lower limits of that range. Any smaller range between any given value or range of statements and any other given value or intermediate value within the range is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. While the present invention describes only preferred methods and materials, any method and material similar or equivalent to those described herein may also be used in making or testing the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe methods and/or materials related to the cited documents. In the event of a conflict with any incorporated documents, the contents of this specification shall control.

Various modifications and variations may be made to the specific embodiments of the present invention without departing from the scope or spirit of the present disclosure, as will be apparent to those skilled in the art. Other embodiments apparent from the description of the present

The invention will also be obvious to those skilled in the art. The descriptions and

Embodiments of the present invention are exemplary only.

As used herein, “including,” “comprising,” “having,” “containing,” and the like are all open-ended terms meaning “including,” but not “limited to.”

In the present invention, G. parasuis (G. parasuis), Streptococcus suis 2 ($SS2) and Staphylococcus aureus (S. aureus) are used as representatives of the gram-negative and gram-positive

Bacteria, and the important role of circHIF1a in the adhesion and invasion of the above-mentioned bacteria to and into host cells is demonstrated at the in vitro level and the inhibitory

Effect of circHIF1a on bacterial infection is investigated at the in vivo level to identify bacterial therapeutic targets to improve the efficacy of therapies against gram-negative and gram-positive bacterial infections. 5 Example 1 1. Screening and characterization of circHIF 10

In the present invention, exosomes from G. parasuis infected

Porcine iliac artery endothelial cells (n=3) or from non-infected porcine iliac artery

Endothelial cells (n=3) were extracted and subjected to circRNA sequencing to determine their

Expression profiles were determined, with a total of 80 upregulated circRNAs and 112 downregulated circRNAs being identified (Fig. 1-2). Significantly downregulated circHIF1a was identified by qRT-PCR (Fig. 3).

The present invention also relates to the expression of circHIF1a in the exosomes of

PIEC cells infected with SS2 or S. aureus as well as in the cell sediments to determine whether the significant downregulation of circHIF 14 in the exosomes of

G. parasuis infected porcine iliac artery endothelial cells (PIEC).

Results show that circHIF1a is also significantly downregulated in the exosomes of PIEC cells infected with SS2 or S. aureus, while circHIF1a expression in PIEC-

Cell sedimentation is upregulated (Fig. 4-5).

Ensemble software analysis and Sanger sequencing show that circHIF 10 is spliced from exons 2-6 of HIF1a from head to tail (Fig. 6). 2. Construction of the circHIF 1a overexpression vector

The 738 bp full-length sequence of circHIF1a is amplified by PCR and ligated to the plasmid pLC5-ciR (Fig. 7) via two enzymatic sites, EcoR | and BamH |, to obtain the circHIF10 overexpression vector.

Full-length sequence of circHIF1a (species: pig) (SEQ ID NO.1):

AATAAGTTCTGAACGTCGAAAAGAGAAGTCTAGAGATGCAGCCAGATCTCGACGAAGTAAA

GAGTCTGAAGTTTTTTATGAGCTTGCTCATCAGTTGCCACTTCCCCATAATGTGAGCTCACA

TCTTGATAAGGCTTCTGTTATGAGGCTTACCATCAGCTATTTTGCGTGTGAGGAAACTTCTAG

ATGCTGGGTGATTTGGATATTGAAGATGAAATGAAGGCACAGATGAATTGTTTTTATTTGAAA

GCCTTGGATGGTTTTGTTATGGTACTCACAGATGATGGTGACATGATTTATATTTCTGATAA

TGTGAACAAATACATGGGATTAACTCAGTTTGAACTAACTGGACACAGTGTGTTTGATTTTA

CTCATCCGTGCGACCATGAGGAAATGAGAGAAATGCTTACACACAGAAATGGCCTTGTGAA

AAAGGGTAAAGAGCAAAATACACAGCGGAGCTTTTTTCTCAGAATGAAGTGTACCTTAACTA

GCAGGGGGAGAACTATGAACATAAAGTCTGCAACATGGAAAGTACTTCACTGCACAGGTCA

TATTCATGTATATGATACCAACAATAACCAGTCTCAGTGTGGGTATAAGAAACCACCTATGA

CGTGCTTGGTGCTGATTTGTGAACCCATTCCTCATCCATCAAATATTGAAATTCCTTTAGAT

AGCAAGACATTTCTCAGTCGTCACAGTCTGGATATGAAATTTTCTTATTGTGATGAAAG.

The circHIF 10 full-length amplifications are as follows: circHIF1a-F:CGGAATTCTAATACTTTCAGAATAAGTTCTGAACGTCGAAA (SEQ ID NO.2); circHIF1a--R:CGGGATCCAGTTGTTCTTACCTTTCATCACAATAAGAAAAT (SEQ ID NO.3). 3. circHIF1a inhibits bacterial invasion and adhesion to PK-15 cells.

Steps of adhesion invasion assay: 5x 105 PK-15 cells are cultured in a constant temperature incubator with 5% CO2 at 37°C overnight. circHIF1a overexpression vector and pLC5-ciR empty vector are transfected into PK-15 cells according to the instructions of lipo3000 reagent, and the

Incubation is continued at 37°C for 24 hours. They are then treated with various

Bacteria to allow bacterial adhesion. In this experiment, PK-15 cells are infected with S. aureus (MOI = 1) for 4 hours or with SS2 (MOI = 10) for 8 hours or with G. parasuis (MOI = 10) for 12 hours. Cells are washed thoroughly with PBS five times to remove nonspecific bacterial adhesions and then incubated with 100 μl of 0.25% trypsin for 10 minutes at 37°C. After incubation, 900 μl of pre-chilled PBS is added and cells are scraped from the culture plate by scratching the bottom. The scraped bacterial suspension is diluted and incubated on TSA medium with 0.1%

Nicotinamide adenine dinucleotide (NAD) and 5% serum. For the bacterial

Invasion assay: DMEM with 100 ug/ml gentamicin is added to the cultured PK-15 cells and washed twice with PBS. Cells are heated for 30 minutes to ensure that all extracellular bacteria are killed and then washed three times with PBS and intracellular bacteria are recovered as described above. All experiments are repeated three times.

Results:

Adhesion invasion assay confirms that after overexpression of circHIF1a in PK-15 cells,

Adhesion-invasion ability of G. parasuis and S. aureus is inhibited and the invasion ability of SS2 is significantly attenuated (Fig. 8-10). 4. circHIF 1a protects mice against bacterial infection

To determine the role of circHIF1a during bacterial infection of animals,

Balb/c mice infected with G. parasuis, SS2 or S. aureus: Three groups of Balb/c mice are infected with PK-15 cells overexpressing circHIF1a (5 x 106 cells/each) via the

tail vein, and the three control groups are injected in the same way and with the same dose of PBS. 24 hours later, 1

Cell injection group and 1 PBS group were selected to be infected with G. parasuis, SS2 and S. aureus, respectively.

The results show that the mortality rate of mice in the non-circHIF1a treated group is 250%, while the number of mice surviving after circHIF1a treatment is increased (Fig. 11). At the same time, in the circHIF1a treated group, the number of bacteria in various organs is significantly reduced (Fig. 12). The pathological sections show that the pathological changes in the organs in the circHIF1a treated group are also alleviated (Fig. 13).

In summary, the present invention demonstrates that circHIF 1a in the

Able to inhibit bacterial adhesion to and invasion of host cells, thereby reducing the damage caused by bacteria to the organism.

The above embodiments describe only with respect to the preferred mode of the present invention, and are not limited to the scope of the present invention, and various modifications and improvements made by those skilled in the art of the technical solutions of the present invention are intended to be incorporated in the

claims of the present invention without departing from the spirit of the present invention.

Claims (10)

1. An application of circHIF1a in the production of novel vaccines against bacterial infections, characterized in that the nucleotide sequence of circHIF1a is as shown in SEQ ID NO.1. 2. The use according to claim 1, characterized in that the circHIF1a acts as a protection against bacterial infections by inhibiting bacterial adhesion to and/or invasion into host cells. 3. The use according to claim 1, characterized in that the bacteria are gram-negative and/or gram-positive. 4. The use according to claim 1, characterized in that the gram-negative bacteria are Glaesserella parasuis; the gram-positive bacteria are Streptococcus suis 2 or Staphylococcus aureus. 5. An application of a biomaterial overexpressing circHIF1a in the production of novel bacterial vaccines, characterized in that the nucleotide sequence of circHIF1a is as shown in SEQ ID NO.1; the biomaterial overexpressing circHIF 14 is an expression vector or a host cell overexpressing circHIF 1a. 6. The use according to claim 5, characterized in that the biomaterial acts as a protection against bacterial infection by overexpressing circHIF 14 and inhibiting bacterial adhesion to and/or invasion into host cells. 7. The use according to claim 5, characterized in that the bacteria are gram-negative and/or gram-positive. 8. The use according to claim 5, characterized in that the gram-negative bacteria are Gracilaria parapsilosis; the gram-positive bacteria are Streptococcus suis 2 or Staphylococcus aureus. 9. A vaccine against bacterial infections, characterized in that the active ingredient comprises biological material overexpressing circHIF1a, wherein the nucleotide sequence of circHIF1a is as shown in SEQ ID NO.1; the biological material overexpressing circHIF1a is an expression vector or a host cell overexpressing circHIF 1a. 10. The vaccine according to claim 9, characterized in that the vaccine comprises pharmaceutically acceptable excipients.