A fusion protein for preventing pneumococcal infection and its application

Abstract

The present invention relates to a fusion protein, an immunogenic composition, a recombinant vaccine, and a molecular architecture design and application for preventing pneumococcal infection. The present invention starts from the tertiary structure of the protein molecules of ply and PhtD, and after creative screening, finally selects the C-terminal domain of the ply protein and the N-terminal domain of the phtD protein to construct a fusion protein, and then further adds elements such as the Fc domain and STABILON. The fusion protein molecule of the present invention can weaken tissue lesions caused by pneumococcal infection, has good immunogenicity, plays an effective preventive and immune protection role, and effectively prevents pneumococcal infection. The immunogenic composition, fusion protein, and recombinant vaccine for preventing pneumococcal infection of the present invention have broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, in particular to the field of immune medicine technology, and specifically relates to a pneumococcal humoral immune vaccine and its application. Background Art

[0002] Safe and effective vaccines are considered the best measures to prevent and control pneumococcal infections. The capsular polysaccharide (CPS) of Streptococcus pneumoniae is the core antigen in current vaccine development. CPS is a carbohydrate substance coated on the surface of bacteria. It is the main virulence factor of Streptococcus pneumoniae and a key component for inducing the production of protective antibodies. Currently, there are more than 90 serotypes of Streptococcus pneumoniae, and each serotype has a specific CPS structure. In the life cycle of Streptococcus pneumoniae, CPS plays a key role. It not only protects the bacteria from the attack of the host immune system, but also participates in the interaction between bacteria and host cells. It is an important part of the infection process.

[0003] The main types of pneumococcal vaccines on the market include polysaccharide vaccines (PPV) and conjugate vaccines (PCV). PPSV directly uses purified CPS as an antigen, while PCV combines CPS with a protein carrier (such as tetanus toxoid) to enhance immunogenicity, especially in infants and young children. There are many commercialized pneumococcal vaccines at home and abroad, such as 13-valent pneumococcal conjugate vaccine (PCV13) and 23-valent pneumococcal polysaccharide vaccine (PPV23). These vaccines can effectively reduce the clinical symptoms caused by pneumococcal infection, but there are still some limitations. First, they can only cover limited serotypes and cannot completely prevent all pneumococcal infections. In addition, with the widespread use of vaccines, non-vaccine serotypes of pneumococci have gradually increased, causing the problem of serotype replacement.

[0004] In order to overcome the limitations of existing vaccines, researchers are actively developing new pneumococcal vaccines. For example, the prior art patent CN104208671A discloses a polyvalent pneumococcal conjugate vaccine and a preparation method thereof, the patent CN110179974B introduces a polyvalent pneumococcal polysaccharide-protein conjugate vaccine and a preparation method thereof, and the patent CN1635904A proposes a polyvalent pneumococcal vaccine based on conjugated protein and its application. These new vaccines are intended to expand serotype coverage, improve immunogenicity, or reduce production costs.

[0005] However, the above vaccines are still mainly based on traditional polysaccharide and conjugate vaccine technologies. Traditional polysaccharide vaccines have limited effects in infants and young children, and conjugate vaccines have high production costs. Both face the problem of serotype replacement. Therefore, it is imperative to develop a new pneumococcal vaccine that can provide broad-spectrum protection, has low production costs, and is suitable for all age groups.

[0006] With the development of genomics, proteomics and immunology, the development of a new generation of pneumococcal vaccines has become an important direction for vaccine development. Among them, new vaccines designed based on conservative antigenic proteins are very promising. The new vaccines are based on the conserved protein antigens of pneumococcus, such as pneumococcal surface protein A (PspA) and pneumococcal surface antigen A (PsaA). These proteins are highly conserved in different serotypes and are expected to provide broad-spectrum protection.

[0007] Compared with traditional polysaccharide and conjugate vaccines, vaccines designed based on conserved antigen proteins have several advantages: (1) they are not restricted by serotype and are expected to provide broad-spectrum protection; (2) the production process is relatively simple and the cost is low; and (3) they can be combined with antigens from other pathogens to develop multivalent vaccines.

[0008] In summary, the development of a new generation of pneumococcal vaccines is imminent, and it will be the key to solving various problems of current traditional pneumococcal vaccines. The new vaccine is not only expected to provide broader protection, but also may reduce production costs, making it easier to promote and use the vaccine worldwide. These studies are not only crucial to controlling pneumococcal infections, but also provide new ideas and methods for the development of other bacterial vaccines. Therefore, although the prior art discloses a variety of new pneumococcal vaccines, the art is still in urgent need of a fusion protein, immune composition, and vaccine that weakens tissue lesions caused by pneumococcal infection, has good immunogenicity, plays an effective preventive and immune protection role, and effectively prevents pneumococcal infection. Summary of the invention

[0009] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a new fusion protein, immunogenic composition, recombinant vaccine, and molecular architecture design and application for preventing pneumococcal infection. The present invention starts from the tertiary structure of the protein molecules of ply and PhtD, combines immune epitopes, designs a variety of different ply truncation types and PhtD truncation types and combines them. After creative screening, the C-terminal domain of the ply protein and the N-terminal domain of the phtD protein are finally selected to construct a fusion protein, and then further add Fc domains, STABILON and other elements. The present invention finds that the new fusion protein molecule can weaken the tissue lesions caused by pneumococcal infection, has good immunogenicity, plays an effective preventive and immune protection role, and effectively prevents pneumococcal infection. The present invention also provides corresponding recombinant nucleic acids, gene expression cassettes, vectors, host cells, pharmaceutical compositions, vaccines, uses, etc.

[0010] One aspect of the present invention provides a fusion protein, characterized in that, from the N-terminus to the C-terminus, it comprises the following elements:

[0011] (a) an N-terminal domain polypeptide fragment of the pneumococcal histidine triad protein PhtD; and

[0012] (b) C-terminal domain polypeptide fragment of pneumolysin (ply).

[0013] Furthermore, the polypeptide fragments of element (a) and element (b) are derived from Streptococcus pneumoniae.

[0014] Furthermore, the amino acid sequence of the N-terminal domain polypeptide fragment of PhtD is shown as SEQ ID NO.1; the amino acid sequence of the C-terminal domain polypeptide fragment of ply is shown as SEQ ID NO.2.

[0015] Furthermore, the fusion protein comprises, from N-terminus to C-terminus, an N-terminal domain polypeptide fragment of PhtD, a spacer sequence, and a C-terminal domain polypeptide fragment of ply; the amino acid sequence of the spacer sequence is as shown in SEQ ID NO: 7; and the amino acid sequence of the fusion protein is as shown in SEQ ID NO.3.

[0016] Furthermore, the N-terminus of the fusion protein also comprises a signal peptide and / or an Fc domain of human immunoglobulin heavy chain constant region γ1 protein (Immunoglobulin Heavy Constant Gamma 1, IGHG1).

[0017] Furthermore, the signal peptide is derived from human Azurocidin protein, and its amino acid sequence is shown in SEQ ID NO: 4; the amino acid sequence of the Fc domain of the human immunoglobulin heavy chain constant region γ1 protein is shown in SEQ ID NO: 5.

[0018] Furthermore, the C-terminus of the fusion protein also contains the C-terminal polypeptide fragment STABILON of the human S5a / PSMD4 proteasome subunit, and its amino acid sequence is shown in SEQ ID NO.6.

[0019] Furthermore, the various elements of the fusion protein may be optionally connected via a connecting peptide.

[0020] Furthermore, the connecting peptide is a spacer sequence, a GGS linker sequence or a GS linker sequence.

[0021] Furthermore, the amino acid sequence of the spacer sequence is shown in SEQ ID NO: 7, the amino acid sequence of the GGS linker sequence is shown in SEQ ID NO: 8, and the amino acid sequence of the GS linker sequence is shown in SEQ ID NO: 9.

[0022] Furthermore, the fusion protein comprises, from the N-terminus to the C-terminus, the Fc domain of IGHG1, the N-terminal domain polypeptide fragment of PhtD, the C-terminal domain polypeptide fragment of ply, and the STABILON.

[0023] Furthermore, the fusion protein comprises, from the N-terminus to the C-terminus, the signal peptide of the human Azurocidin protein, the Fc domain of IGHG1, the N-terminal domain polypeptide fragment of PhtD, the C-terminal domain polypeptide fragment of ply, and the STABILON.

[0024] Furthermore, the amino acid sequence of the signal peptide derived from human Azurocidin protein is shown as SEQ ID NO: 4, the amino acid sequence of the Fc domain of IGHG1 is shown as SEQ ID NO: 5, the amino acid sequence of the N-terminal domain polypeptide fragment of PhtD is shown as SEQ ID NO.1, the amino acid sequence of the C-terminal domain polypeptide fragment of ply is shown as SEQ ID NO.2, and the amino acid sequence of STABILON is shown as SEQ ID NO.6.

[0025] Furthermore, the Fc domain of IGHG1 and the N-terminal domain polypeptide fragment of PhtD are connected through a GGSlinker sequence, the N-terminal domain polypeptide fragment of PhtD and the C-terminal domain polypeptide fragment of ply are connected through a spacer sequence, and the C-terminal domain polypeptide fragment of ply and the STABILON are connected through a GS linker sequence; wherein the amino acid sequence of the spacer sequence is shown in SEQ ID NO: 7, the amino acid sequence of the GGS linker sequence is shown in SEQ ID NO: 8, and the amino acid sequence of the GS linker sequence is shown in SEQ ID NO: 9.

[0026] Furthermore, the fusion protein also contains other Streptococcus pneumoniae antigen proteins.

[0027] Furthermore, the other pneumococcal antigen proteins are selected from one or more of the following: pneumococcal surface protein A (Pneumococcal surface protein A, PspA), pneumococcal surface antigen A (Pneumococcal surface adhesion A, PsaA), and pneumococcal cholesterol binding protein A (Pneumococcal choline-binding protein A, PcpA).

[0028] Another aspect of the present invention provides a recombinant nucleic acid molecule, characterized in that it encodes the fusion protein of the present invention.

[0029] Another aspect of the present invention provides a recombinant gene expression cassette, characterized in that it comprises the recombinant nucleic acid molecule of the present invention.

[0030] Another aspect of the present invention provides a recombinant vector, characterized in that it comprises the recombinant nucleic acid molecule of the present invention or the recombinant gene expression cassette of the present invention.

[0031] Another aspect of the present invention provides a recombinant host cell, characterized in that it comprises the recombinant nucleic acid molecule of the present invention, or the recombinant gene expression cassette of the present invention, or the recombinant vector of the present invention.

[0032] Another aspect of the present invention provides an immunogenic composition or pharmaceutical composition, characterized in that it comprises one or more fusion proteins described in any one of the present invention, and / or one or more recombinant nucleic acid molecules, and / or one or more recombinant gene expression cassettes described in the present invention, and / or one or more recombinant vectors described in the present invention, and / or one or more recombinant host cells described in the present invention.

[0033] Furthermore, the immunogenic composition or pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0034] Another aspect of the present invention provides a recombinant vaccine, characterized in that it comprises one or more fusion proteins described in any one of the present invention, and / or one or more recombinant nucleic acid molecules, and / or one or more recombinant gene expression cassettes described in the present invention, and / or one or more recombinant vectors described in the present invention, and / or one or more recombinant host cells described in the present invention, and / or one or more immunogenic compositions or pharmaceutical compositions described in the present invention.

[0035] Furthermore, the immunogenic composition, the pharmaceutical composition or the recombinant vaccine of the present invention further comprises an antiviral agent.

[0036] Furthermore, the antiviral agent is an antiviral agent for diseases caused by Streptococcus pneumoniae.

[0037] Another aspect of the present invention provides the use of one or more fusion proteins described in any one of the present invention, and / or one or more recombinant nucleic acid molecules, and / or one or more recombinant gene expression cassettes described in the present invention, and / or one or more recombinant vectors described in the present invention, and / or one or more recombinant host cells described in the present invention, and / or one or more immunogenic compositions or pharmaceutical compositions described in the present invention, and / or one or more recombinant vaccines described in the present invention in the preparation of vaccines or biological immunization drugs for prevention and / or vaccination or in the preparation of drugs for preventing diseases caused by Streptococcus pneumoniae.

[0038] Furthermore, the drug is used to prevent tissue infection caused by Streptococcus pneumoniae.

[0039] Furthermore, the tissue infection is lung tissue infection.

[0040] Furthermore, the diseases caused by Streptococcus pneumoniae include lung tissue infection, pneumonia, meningitis, otitis media, bacteremia, sinusitis, endocarditis, arthritis, peritonitis, and endophthalmitis.

[0041] The fusion protein, immunogenic composition, recombinant vaccine, etc. of the present invention have the following beneficial technical effects:

[0042] 1. The present invention finds that the new fusion molecule has good immunogenicity and can provide immune protection, and can be used in the development of nucleic acid vaccines or subunit vaccines. The immunogenic composition of the present invention can provide effective immune protection against infection with Streptococcus pneumoniae;

[0043] 2. The present invention can be used as a universal antigen framework for nucleic acid vaccines or subunit vaccines, has strong immunogenicity, and compared with traditional polysaccharide vaccines, is not restricted by serotype and can induce broad-spectrum immune protection;

[0044] 3. The present invention starts from the tertiary structure of ply and PhtD protein molecules, combines immune epitopes, designs a variety of different ply truncated types and PhtD truncated types and combines them. After creative screening, the C-terminal domain of ply protein and the N-terminal domain of phtD protein are finally selected to construct a fusion protein. The truncated polypeptide fragment has a complete and stable 3D structure;

[0045] 4. Example 5 of the present invention shows that the fusion molecule based on the present invention can effectively promote the high-abundance expression and secretion of bacterial proteins in eukaryotic cells, and the expressed protein structure is correct and stable, which is conducive to the presentation of immune epitopes, thereby further improving the immune protection effect, and providing new technology for the field of immune drug design of multivalent pneumococcal vaccines. In particular, vaccine C (comprising the PhtD protein N-terminal domain-Ply protein C-terminal domain fusion protein) has the highest expression level in the supernatant, proving that compared with the separately expressed truncated protein Ply protein C-terminal domain and PhtD protein N-terminal domain (vaccine A, vaccine B), the fusion protein has the highest secretion efficiency and is more conducive to antigen presentation and activation of humoral immunity;

[0046] 5. Example 6 of the present invention shows that in the mouse nasal infection model, the comparison results of the bacterial load (number of plate colonies) in the lung tissue of mice after immune challenge showed that compared with the blank control group mice (PBS immunization), the pneumococcal load of the experimental group mice (vaccine C immunization) was significantly reduced, and the bacterial load of the blank control group mice was about 450 times that of the experimental group mice. The vaccine based on the present invention can provide a good immune prevention effect, and in 4 / 5 individuals, it can almost completely resist pneumococcal infection (lung tissue bacterial load ≤ 5). This result proves that the recombinant nucleic acid vaccine C based on the present invention can provide effective immune protection in the mouse model, and the vaccinated mice can produce protective immunity to prevent pneumococcal infection;

[0047] 6. Example 6 of the present invention also shows that, after challenge, the alveolar structure of mice in the vaccine C immunization group was relatively intact, with only a small amount of inflammatory cell infiltration and no obvious thickening of the alveolar wall. This result proves that the immune protection induced by the recombinant nucleic acid vaccine C of the present invention in the mouse model can weaken the tissue lesions caused by pneumococcal infection and play a good preventive role. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 It is a schematic diagram of the 3D structure of the full-length PhtD protein, the N-terminal domain polypeptide of the PhtD protein, the full-length ply protein, and the C-terminal domain polypeptide of the ply protein of the present invention.

[0049] Figure 2Schematic diagram of the molecular structure of the vaccine used in the comparative experiment in the example.

[0050] Figure 3 Schematic diagram of the non-limiting molecular structure of the fusion protein expressed by the vaccine of the present invention.

[0051] Figure 4 Schematic diagram of the vaccine template plasmid comprising the present invention.

[0052] Figure 5A-5C The peak diagram and purity test results of the vaccine of the present invention are shown in FIG. Figure 5A This is the quality control peak diagram and purity test results of vaccine A. Figure 5B This is the quality control peak diagram and purity test results of vaccine B. Figure 5C This is the quality control peak diagram and purity test results of vaccine C.

[0053] Figure 6 This is the expression effect of the vaccine of the present invention after transfection of HEK293T cells in vitro.

[0054] Figure 7 The present invention is an immunization and sampling process for the vaccine of the present invention in a mouse nasal drip infection model.

[0055] Figure 8A-B The preventive and protective effect of the vaccine of the present invention in the mouse nasal drip infection model after immunization; wherein Fig. 8A Comparison of bacterial load (number of colonies on the plate) in the lung tissue of mice after immunization and challenge. Figure 8B The results are statistical results of bacterial load.

[0056] Fig. 9 Comparison of HE staining results of lung tissue sections after immunization of mice. DETAILED DESCRIPTION

[0057] The term "Streptococcus pneumoniae" refers to Streptococcus pneumoniae , also known as Pneumococcus ( Diplococcus pneumonia ) or Pneumococcus, is a spherical, Gram-positive, alpha-hemolytic bacterium belonging to the genus Streptococcus.

[0058] The term "pneumococcal infection" refers to a variety of diseases caused by Streptococcus pneumoniae, including pneumonia, acute sinusitis, otitis media, meningitis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, cellulitis, brain abscess, etc.

[0059] The term "PhtD" refers to a member of the pneumococcal histidine triad (Pht) protein family of Streptococcus pneumoniae. The family includes four member proteins: PhtA, PhtB, PhtD and PhtE.

[0060] The term "ply" refers to pneumolysin, which is one of the main virulence factors of Streptococcus pneumoniae. It is a pore-forming toxin composed of 471 amino acids that has the ability to destroy host cells and interfere with the host immune response.

[0061] The term "N-terminal domain" refers to a functional or structural unit near the amino terminus (N-terminus) of a protein molecule, which usually has an independent three-dimensional structure, or contains a specific amino acid sequence or conserved region, thereby having a specific biological function. Further, the "N-terminal domain" of the present invention adopts the N-terminal domain of PhtD, and its amino acid sequence is shown in SEQ ID NO: 1.

[0062] The term "C-terminal domain" refers to a functional or structural unit near the carboxyl terminus (C-terminus) of a protein molecule, which is located at the other end of the protein opposite to the N-terminal domain, and usually has an independent three-dimensional structure, or contains a specific amino acid sequence or conserved region, thereby having a specific biological function. Further, the "C-terminal domain" of the present invention adopts the C-terminal domain of ply, and its amino acid sequence is shown in SEQ ID NO: 2.

[0063] The term "immune response" refers to a humoral response, a cellular response, or both a humoral and a cellular response in an organism. The immune response can be assayed, including but not limited to measuring the presence of antibodies that specifically recognize proteins or cell surface proteins, measuring T cell activation or proliferation, and / or measuring one or more cytokine activity modulation or expression modulation.

[0064] The term "administration" or "vaccination" refers to administration based on the nucleic acid vaccine or vaccine composition of the present invention preferably via intramuscular or subcutaneous routes, although other routes of administration can also be used, for example, oral, intranasal (e.g., aerosol or other non-injection administration), intralymph node, intradermal, intraperitoneal, rectal or vaginal administration, or by a combination of routes. Administration in the neck muscle of the animal is preferred. The administration regimen can be adjusted to provide optimal immunity using an accelerated regimen (boosting regimens).

[0065] The term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0066] The term "recombinant nucleic acid molecule" refers to a polynucleotide having sequences that are not linked together in nature. The recombinant polynucleotide can be included in a suitable vector, and the vector can be used to transform into a suitable host cell. The polynucleotide is then expressed in the recombinant host cell to produce, for example, a "recombinant polypeptide", "recombinant protein", "fusion protein", etc.

[0067] The term "recombinant expression vector" refers to a DNA structure used to express a polynucleotide encoding a desired polypeptide, for example. A recombinant expression vector may include, for example, a collection of genetic elements that regulate gene expression, such as promoters and enhancers; (2) a structural or coding sequence that is transcribed into mRNA and translated into protein; and (3) appropriate transcription and translation start and stop sequences. The recombinant expression vector is constructed in any suitable manner and any vector may be used, including plasmids, viruses, phages, and transposons. Possible vectors for use in the present disclosure include, but are not limited to, chromosomal, non-chromosomal, and synthetic DNA sequences, such as viral plasmids, bacterial plasmids, phage DNA, yeast plasmids, and vectors derived from a combination of plasmids and phage DNA, DNA from viruses such as lentivirus, retrovirus, vaccinia, adenovirus, fowlpox, baculovirus, SV40, and pseudorabies. Both self-replicating and non-self-replicating vectors are included.

[0068] The term "mRNA" refers to messenger RNA, which is translated into Chinese as "messenger ribonucleic acid". It is a type of single-stranded ribonucleic acid that is transcribed from a strand of DNA as a template, carries genetic information and can guide protein synthesis.

[0069] The term "5'-UTR", which refers to the "5' untranslated region" or "5'UTR", is a portion of a gene that is transcribed into a primary RNA transcript (pre-mRNA) and is located upstream of the coding sequence. The primary transcript is the initial RNA product, containing introns and exons, produced by transcription from DNA. Many primary transcripts must undergo RNA processing to form physiologically active RNA. The processing to form mature mRNA includes modifying the ends, removing introns, capping and / or shearing off individual rRNA molecules from the precursor RNA. Therefore, the 5'UTR of an mRNA is a portion of the mRNA that is not translated into protein and is located upstream of the coding sequence. In a genomic sequence, the 5'UTR is usually defined as the region located between the transcription start site and the start codon. The 5' untranslated region (5'UTR) of vertebrate mRNA can be tens to hundreds of bases in length.

[0070] The term "3'-UTR", meaning "3'-untranslated region" or "3'UTR", refers to a region located at the 3' end of a gene, downstream of the stop codon of the protein coding region, and which is transcribed but not translated into an amino acid sequence, or to the corresponding region in an RNA molecule. The 3'-untranslated region typically extends from the stop codon of the translation product to a poly(A) sequence that is typically attached after the transcription process. The 3'-untranslated region of mammalian mRNA typically has a homology region known as the AAUAAA hexanucleotide sequence. This sequence may be a poly(A) attachment signal and is often located 10 to 30 bases upstream of the poly(A) attachment site. The 3'-untranslated region may contain one or more inverted repeats and may fold to produce a stem-loop structure that acts as a barrier to exoribonucleases or interacts with proteins (e.g., RNA binding proteins) that are known to increase RNA stability.

[0071] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including progeny of such cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and progeny derived therefrom. Host cells are any type of cell system that can be used to produce recombinant vaccines based on the present invention, including eukaryotic cells, e.g., mammalian cells, insect cells, yeast cells; and prokaryotic cells, e.g., E. coli cells. Host cells include cultured cells.

[0072] The terms "individual", "patient" or "subject" include mammals. Mammals include, but are not limited to, domestic animals (e.g., pigs, cows, sheep, cats, dogs, and horses, etc.), primates (e.g., humans and non-human primates such as monkeys), and rodents (e.g., rabbits, mice, and rats, etc.).

[0073] The terms "transformation, transfection, and transduction" have the meanings generally understood by those skilled in the art, namely, the process of introducing exogenous DNA or RNA into a host.

[0074] The term "drug combination" or "pharmaceutical composition" refers to auxiliary materials widely used in the field of drug production. The main purpose of using a carrier is to provide a pharmaceutical composition that is safe to use, stable in nature and / or has specific functionality, and also to provide a method so that it can be effectively absorbed in the body of a subject. A pharmaceutically acceptable carrier can be an inert filler or an effective component that provides a certain function for the pharmaceutical combination (such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient in the composition). Non-limiting examples of pharmaceutically acceptable carriers include, but are not limited to, adhesives, suspending agents, emulsifiers, diluents (or fillers), granulating agents, adhesives, disintegrants, lubricants, anti-adhesives, glidants, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents and sweeteners.

[0075] The term "prevention" means that before a subject develops a disease, the symptoms after developing the disease are alleviated by exposing the subject to (e.g., administering) the recombinant vaccine, composition, etc. according to the present invention, compared to when the subject has not developed the disease. It does not necessarily mean that the disease must be completely suppressed.

[0076] Unless defined otherwise or clearly indicated by the context, all technical and scientific terms in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0077] The present invention discloses a method for preparing a novel pneumococcal vaccine and its application. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the same. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are deemed to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0078] In the fusion protein and encoding nucleic acid and elements thereof, preparation methods and applications provided by the present invention, the raw materials and reagents used can all be obtained commercially.

[0079] The present invention is further illustrated below in conjunction with the examples, wherein, as a preferred embodiment, a nucleic acid vaccine framework is selected for preparing a recombinant vaccine.

[0080] Example 1 Design of domain polypeptide truncation of ply protein and PhtD protein

[0081] Due to the cytotoxicity of ply protein and the large molecular weight of PhtD protein, both antigenic proteins are not suitable for full-length expression or construction of fusion proteins in full-length form for vaccine preparation. Therefore, it is necessary to truncate or modify ply protein and PhtD protein to ensure that the truncated or modified new molecules have a stable tertiary structure while retaining the immunogenicity of the proteins, so as to achieve beneficial effects such as easier secretion expression, strong immunogenicity, and no cytotoxicity.

[0082] According to the above ideas, the present invention starts from the tertiary structure of the protein molecules of ply and PhtD, combines the immune epitopes, and selects polypeptide fragments with stable structures. A variety of different ply truncation types and PhtD truncation types are designed and combined. After screening, the C-terminal domain of the ply protein and the N-terminal domain of the phtD protein are finally selected. Preferably, the amino acid sequence of the N-terminal domain of the PhtD protein is as shown in SEQ ID NO: 1, and the amino acid sequence of the C-terminal domain of the ply protein is as shown in SEQ ID NO: 2. Figure 1 As shown, the selected polypeptide fragment has a complete 3D structure. The selected polypeptide fragment is used as an antigen sequence for the vaccine design and comparative test of the subsequent examples of the present invention.

[0083] Example 2 Construction of the recombinant nucleic acid vaccine of the present invention

[0084] In order to prepare a recombinant nucleic acid vaccine comprising the antigen of the present invention and compare whether the vaccine based on the present invention has a good in vitro expression effect, the vaccine molecular architecture involved in the embodiment is as follows: Figure 2 , Figure 3 As shown. For example, Figure 2 Schematic diagram of the molecular structure of vaccine A and vaccine B used in the comparative experiment in the example. Figure 3 The schematic diagram of the protein molecule expressed by the vaccine C used in the embodiment of the present invention includes the following elements from the N-terminus to the C-terminus: signal peptide, Fc domain, target antigen region (N-terminal domain of PhtD protein and C-terminal domain of PhtD protein, which can be connected by a spacer sequence), STABILON, and each element can be directly connected or connected by different linkers, such as GGS linker or GS linker. Figure 2 , Figure 3 The differences between the three vaccines shown in the diagram, Vaccine A, Vaccine B, and Vaccine C, are that the target antigen regions are different. The target antigens are the C-terminal domain of the Ply protein, the N-terminal domain of the PhtD protein, the N-terminal domain of the PhtD protein, and the C-terminal domain of the PhtD protein (the two can be connected by a spacer sequence). Figure 2 , Figure 3The recombinant nucleic acid vaccine of the protein shown in the embodiment first constructs a gene expression cassette for expressing the antigen sequence described in the present invention. The expression cassette comprises, from the 5' end to the 3' end, 5'UTR, CDS region, 3'UTR, PolyA, wherein the CDS region comprises the fusion molecule architecture described in the present invention. Subsequently, the complete gene expression cassette sequence is optimized based on codon degeneracy, and the DNA sequence is directly obtained by gene synthesis (commissioned to GenScript Corporation for synthesis). Finally, the synthesized gene expression cassette DNA sequence is inserted into an expression vector that can be used for in vitro RNA transcription, such as Figure 4 As shown, a vector plasmid for preparing a recombinant nucleic acid vaccine is obtained.

[0085] According to the above method, the carrier used in the subsequent examples was prepared:

[0086] (1) Preparation of vector based on the recombinant nucleic acid vaccine A of the present invention

[0087] Step a: synthesizing a "signal peptide-Fc domain of human IGHG-C-terminal domain of ply-STABILON" fusion gene fragment, the Fc domain of human IGHG and the C-terminal domain of ply are connected by a GGS linker sequence whose amino acid sequence is shown in SEQ ID NO: 8, the C-terminal domain of ply and STABILON are connected by a GS linker sequence whose amino acid sequence is shown in SEQ ID NO: 9, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 4, the amino acid sequence of the Fc domain of human IGHG is shown in SEQ ID NO: 5, the amino acid sequence of the C-terminal domain of ply is shown in SEQ ID NO.2, and the amino acid sequence of STABILON is shown in SEQ ID NO: 6.

[0088] Step b: Construct a nucleic acid vaccine framework vector.

[0089] The nucleic acid vaccine framework vector comprises a 5'-UTR and a 3'-UTR, and can be a vector for producing any form of RNA vaccine or a vector for producing a DNA vaccine.

[0090] Step c: preparing recombinant plasmid.

[0091] The gene synthesized in step a is inserted into the vector structure of step b to obtain a vector for preparing the recombinant nucleic acid vaccine A based on the present invention.

[0092] (2) Preparation of vector based on the recombinant nucleic acid vaccine B of the present invention

[0093] Step a: synthesizing a "signal peptide-Fc domain of human IGHG-N-terminal domain of PhtD-STABILON" fusion gene fragment, the Fc domain of human IGHG and the N-terminal domain of PhtD are connected by a GGS linker sequence whose amino acid sequence is shown in SEQ ID NO: 8, the N-terminal domain of PhtD and STABILON are connected by a GS linker sequence whose amino acid sequence is shown in SEQ ID NO: 9, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 4, the amino acid sequence of the Fc domain of human IGHG is shown in SEQ ID NO: 5, the amino acid sequence of the N-terminal domain of PhtD is shown in SEQ ID NO.1, and the amino acid sequence of STABILON is shown in SEQ ID NO: 6.

[0094] Step b: Construct a nucleic acid vaccine framework vector.

[0095] The nucleic acid vaccine framework vector comprises a 5'-UTR and a 3'-UTR, and can be a vector for producing any form of RNA vaccine or a vector for producing a DNA vaccine.

[0096] Step c: preparing recombinant plasmid.

[0097] The gene synthesized in step a is inserted into the vector structure of step b to obtain a vector for preparing the recombinant nucleic acid vaccine B based on the present invention.

[0098] (3) Preparation of vector based on the recombinant nucleic acid vaccine C of the present invention

[0099] Step a: synthesizing a fusion gene fragment of "signal peptide-Fc domain of human IGHG-N-terminal domain of PhtD-C-terminal domain of ply-STABILON", wherein the Fc domain of human IGHG and the N-terminal domain of PhtD are connected by a GGS linker sequence having an amino acid sequence as shown in SEQ ID NO: 8, the N-terminal domain of PhtD and the C-terminal domain of ply are connected by a spacer sequence having an amino acid sequence as shown in SEQ ID NO: 7, the C-terminal domain of ply and STABILON are connected by a GS linker sequence having an amino acid sequence as shown in SEQ ID NO: 9, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 4, the amino acids of the Fc domain of human IGHG are shown in SEQ ID NO: 5, the amino acids of the N-terminal domain of PhtD are shown in SEQ ID NO.1, the amino acids of the C-terminal domain of ply are shown in SEQ ID NO.2, and the amino acid sequence of STABILON is shown in SEQ ID NO: 6.

[0100] Step b: Construct a nucleic acid vaccine framework vector.

[0101] The nucleic acid vaccine framework vector comprises a 5'-UTR and a 3'-UTR, and can be a vector for producing any form of RNA vaccine or a vector for producing a DNA vaccine.

[0102] Step c: preparing recombinant plasmid.

[0103] The gene synthesized in step a is inserted into the vector structure of step b to obtain a vector for preparing the recombinant nucleic acid vaccine C based on the present invention.

[0104] Table 1 Amino acid sequences of proteins involved in the present invention

[0105] Amino acid sequence and sequence number PhtD N-terminal domain polypeptide fragment CSYELGRHQAGQVKKESNRVSYIDGDQAGQKAENLTPDEVSKREGINAEQIVIKITDQGYVTSHGDHYHYYNGKVPYDAIISEELLMKDPNYQLKDSDIVNEIKGGYVIK VDGKYYVYLKDAAHADNIRTKEEIKRQKQERSHNHNSRADNAVAAARAQGRYTTDDGYIFNASDIIEDTGDAYIVPHGDHYHYIPKSDLSASELAAAQAYWNGKQGS (SEQ ID NO: 1) Ply C-terminal domain polypeptide fragment EVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDRNGQDLTAHFFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND (SEQ ID NO: 2) PhtD N-terminal domain-Ply C-terminal domain CSYELGRHQAGQVKKESNRVSYIDGDQAGQKAENLTPDEVSKREGINAEQIVIKITDQGYVTSHGDHYHYYNGKVPYDAIISEELLMKDPNYQLKDSDIVNEIK GGYVIKVDGKYYVYLKDAAHADNIRTKEEIKRQKQERSHNHNSRADNAVAAARAQGRYTTDDGYIFNASDIIEDTGDAYIVPHGDHYHYIPKSDLSASELAAAQ AYWNGKQGSGGSGGGGSGGEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGA YVAQYYITWDELSYDHQGKEVLTPKAWDRNGQDLTAHFFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND (SEQ ID NO: 3) Signal peptide MTRLTVLALLAGLLASSRA (SEQ ID NO: 4) Fc domain of human IGHG EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 5) STABILON KDGKKDKKEEDKK (SEQ ID NO: 6) Spacer sequence GGSGGGGSGG (SEQ ID NO: 7) GGS linker GGSGGSGGSG (SEQ ID NO: 8) GS linker GSGSGSG (SEQ ID NO: 9)

[0106] Example 3 Preparation of the recombinant nucleic acid vaccine of the present invention

[0107] (1) Preparation of capped mRNA vaccines

[0108] Step a: The vector plasmid used to produce the capped mRNA vaccine in Example 2 is linearized by enzyme digestion to obtain a linearized plasmid for in vitro transcription.

[0109] Step b: The linearized plasmid is subjected to an in vitro co-transcription capping reaction to add a 7-methylated guanylate cap structure to the 5' end of the transcribed mRNA, and the template DNA is degraded.

[0110] (2) Preparation of non-capped mRNA vaccines

[0111] Step a: The vector plasmid used to produce the non-capped mRNA vaccine in Example 2 is linearized by enzyme digestion to obtain a linearized plasmid for in vitro transcription.

[0112] Step b: The linearized plasmid is subjected to an in vitro non-capped transcription reaction and the template DNA is degraded.

[0113] (3) DNA vaccine preparation

[0114] Step a: Amplify the vector plasmid used to produce the DNA vaccine in Example 2 to obtain a large amount of target plasmid for purification.

[0115] Step b: Use an endotoxin-free plasmid extraction and purification kit to extract and purify the target plasmid.

[0116] Example 4 Quality Control of In Vitro Transcription of Recombinant Nucleic Acid and Vaccine Preparation of the Present Invention

[0117] Vaccine A (based on the recombinant nucleic acid vaccine A of the present invention), vaccine B (based on the recombinant nucleic acid vaccine B of the present invention), and vaccine C (based on the recombinant nucleic acid vaccine C of the present invention) were prepared by the method for preparing the capped mRNA vaccine in Example 3. The purity of the produced recombinant nucleic acid was tested, and the purity of the recombinant nucleic acid used in the experiment was greater than 90%. The quality control peak diagram of the recombinant nucleic acid of the present invention is as follows: Figure 5A , Figure 5B , Figure 5C The specific description is as follows: (1) The recombinant nucleic acid vaccine A based on the present invention has a purity of 93%, and the quality control peak diagram and purity test results are as shown in Figure 5A ; (2) The recombinant nucleic acid vaccine B based on the present invention has a purity of 94.6%. The quality control peak diagram and purity test results are as follows Figure 5B ; (3) The recombinant nucleic acid vaccine C based on the present invention has a purity of 90.1%. The quality control peak diagram and purity test results are as follows Figure 5C The above purity meets the quality requirements for cell transfection experiments and vaccine production.

[0118] Example 5 In vitro expression effect of the recombinant nucleic acid of the present invention

[0119] The vaccine in Example 4 was transfected into HEK293T cells using a cell transfection reagent. After 48 hours of in vitro culture, the protein was collected and tested by Western blot. The protein molecular weights of vaccine A, vaccine B, and vaccine C were calculated, as shown in Table 2.

[0120] Table 2 Protein molecular weight of vaccine A, vaccine B, and vaccine C

[0121] vaccine Protein molecular weight (kDa) A 52.6 B 55.9 C 77.6

[0122] Figure 6 The results of in vitro expression WB (Western blot) of HEK293 cells transfected with vaccines A, B, and C are shown. The antigens expressed by the three are all humoral immune antigens, and theoretically significant expression can be detected in the supernatant.

[0123] like Figure 6 As shown, the target protein signals were detected in the supernatant of vaccines A, B, and C, and the molecular weights were all in line with expectations. This proves that the truncated proteins and fusion proteins based on the present invention can not only be smoothly translated and correctly folded in eukaryotic cells, but also have stable structures and can be efficiently secreted outside the cells.

[0124] Among them, vaccine C had the highest expression level in the supernatant, proving that compared with the truncated protein expressed alone, the fusion protein had the highest secretion efficiency and was more conducive to antigen presentation and activation of humoral immunity. Therefore, vaccine C was subsequently used for animal testing to verify the immune protection effect.

[0125] Example 6 Preventive effect of the recombinant nucleic acid vaccine of the present invention in a mouse nasal drip infection model

[0126] In order to verify whether the nucleic acid vaccine based on the present invention has an immune protection effect, this example conducted an immunization and virus attack comparison experiment on vaccine-immunized mice (experimental group, i.e., vaccine C) and non-immunized mice (blank control group).

[0127] The experiment selected 12 Balb / c mice aged 6-8 weeks, weighing 18-25 g, all of which were kept in independent cages with constant temperature and humidity, and adapted to the environment for 3-7 days in advance. The temperature of the breeding room was 20-26°C, the humidity was 40-70%, and the light and dark alternated from 8 am to 8 pm, and the darkness from 8 pm to 8 am the next day; sufficient feed was continuously supplied, and the mice were free to take in unlimited amounts, and sterile water was drunk. The drinking water bottle was continuously supplied with water, and they were free to take in. After the mice were adaptively raised, they were randomly divided into 4 groups, 6 in each group, and each mouse was marked with an ear tag. As shown in Table 3, the dosage in this table and below refers to the amount of active ingredient.

[0128] Table 3 Grouping and immunization process of mouse immunization experiment in Example 5

[0129] Group vaccine Number of animals Immunization dose Immunization Procedure 1 Based on the recombinant nucleic acid vaccine C of the present invention 6 5μg (100μL) / piece / time Day 0, Day 21: Subcutaneous inoculation in the left groin 2 PBS (blank control) 6 100μL / piece / time Day 0, Day 21: Subcutaneous inoculation in the left groin

[0130] Each group of mice was immunized twice according to the immunization process in Table 3. On Day 35, the mice were challenged with isoflurane gas. After the mice were completely anesthetized, 40 μL of Streptococcus pneumoniae solution (5×10 6 CFU / mL) were dripped into the nose. The mice were raised normally after the dripping, and the clinical manifestations of the mice were observed and recorded every day. The mice were killed on Day 38, and samples were collected to detect the bacterial load of lung tissue and lung tissue pathological sections to evaluate the immune protection effect of the vaccine. The immunization, virus attack and sampling process is as follows Figure 7 shown.

[0131] Take 10 mg of lung tissue from each mouse, grind it and dilute it 1000 times, spread it on LB solid culture dishes, and detect the bacterial load in the lung tissue. Fig. 8A and Figure 8B shown. Fig. 8A This is a comparison of the bacterial load (number of colonies on the plate) in the lung tissue of mice after immunization with the virus. The results showed that compared with the blank control group mice (immunized with PBS), the pneumococcal load of the experimental group mice (immunized with vaccine C) was significantly decreased. Figure 8BThe results of bacterial load statistics show that the bacterial load of mice in the blank control group is about 450 times that of mice in the experimental group. The vaccine based on the present invention can provide a good immune prevention effect, and in 4 / 5 individuals, it can almost completely resist pneumococcal infection (lung tissue bacterial load ≤ 5). This result proves that the recombinant nucleic acid vaccine C based on the present invention can provide effective immune protection in the mouse model, and the vaccinated mice can produce protective immunity to prevent pneumococcal infection.

[0132] Part of the lung tissue of each mouse was removed, fixed with paraformaldehyde, embedded in paraffin, and sectioned. The sections were stained with hematoxylin-eosin dye, and the pathological changes of the lung tissue were observed under a microscope. Fig. 9 As shown, the blank control group mice (PBS immunization) had a large number of inflammatory cell infiltrations in the alveoli, local hemorrhages, obvious thickening of the alveolar walls, and obvious shrinkage of the alveoli; the experimental group mice (vaccine C immunization) had relatively intact alveolar structures, a small amount of inflammatory cell infiltration, and no obvious thickening of the alveolar walls. This result proves that the immune protection induced by the recombinant nucleic acid vaccine C of the present invention in the mouse model can weaken the tissue lesions caused by Streptococcus pneumoniae infection and play a good preventive role.

[0133] In summary, the immunogenic composition provided by the present invention can induce effective protective immunity in model animals and mice, and has a good preventive effect against pneumococcal infection. Therefore, the present invention can be applied to the production and development of immune drugs, filling the gap in the current research and development of broad-spectrum pneumococcal vaccines, and has extremely high commercial value and broad application prospects.

[0134] The above embodiments of the present disclosure are merely examples for clearly illustrating the present disclosure, and are not intended to limit the implementation methods of the present disclosure. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all implementation methods here. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present disclosure shall be included in the scope of protection of the claims of the present disclosure.

Claims

1. A fusion protein, characterized in that: From N-terminus to C-terminus, it contains the following elements: Element a, an N-terminal domain polypeptide fragment of the histidine triad protein PhtD; and Element b, a polypeptide fragment of the C-terminal domain of hemolysin, The polypeptide fragments of element a and element b are derived from Streptococcus pneumoniae; the amino acid sequence of the N-terminal domain polypeptide fragment of PhtD is shown in SEQ ID NO.1; the amino acid sequence of the C-terminal domain polypeptide fragment of hemolysin is shown in SEQ ID NO.

2.

2. The fusion protein according to claim 1, characterized in that The N-terminus of the fusion protein further comprises a signal peptide and / or the Fc domain of human immunoglobulin heavy chain constant region γ1 protein (IGHG1); or, the C-terminus of the fusion protein further comprises the C-terminal polypeptide fragment STABILON of the human S5a / PSMD4 proteasome subunit.

3. The fusion protein according to claim 2, characterized in that The signal peptide is derived from human blue pyrimidine protein, and its amino acid sequence is shown in SEQ ID NO: 4; the amino acid sequence of the Fc domain of IGHG1 is shown in SEQ ID NO: 5; and the amino acid sequence of STABILON is shown in SEQ ID NO.

6.

4. The fusion protein according to claim 2, characterized in that The fusion protein comprises, from N-terminus to C-terminus, the signal peptide derived from human blue pyrimidine protein, the Fc domain of the immunoglobulin heavy chain constant region γ1 protein, the N-terminal domain polypeptide fragment of PhtD, the C-terminal domain polypeptide fragment of the hemolysin, and the STABILON; wherein the amino acid sequence of the signal peptide derived from human blue pyrimidine protein is as shown in SEQ ID NO: 4, the amino acid sequence of the Fc domain of the immunoglobulin heavy chain constant region γ1 protein is as shown in SEQ ID NO: 5, the amino acid sequence of the N-terminal domain polypeptide fragment of PhtD is as shown in SEQ ID NO.1, the amino acid sequence of the C-terminal domain polypeptide fragment of the hemolysin is as shown in SEQ ID NO.2, and the amino acid sequence of the STABILON is as shown in SEQ ID NO.

6.

5. The fusion protein according to claim 4, characterized in that The Fc domain of the immunoglobulin heavy chain constant region γ1 protein and the N-terminal domain polypeptide fragment of PhtD are connected through a GGS linker sequence, the N-terminal domain polypeptide fragment of PhtD and the C-terminal domain polypeptide fragment of the hemolysin are connected through a spacer sequence, and the C-terminal domain polypeptide fragment of the hemolysin and the STABILON are connected through a GS linker sequence; the amino acid sequence of the spacer sequence is shown in SEQ ID NO: 7, the amino acid sequence of the GGS linker sequence is shown in SEQ ID NO: 8, and the amino acid sequence of the GS linker sequence is shown in SEQ ID NO:

9.

6. A recombinant nucleic acid molecule, characterized in that Encodes the fusion protein according to any one of claims 1 to 5.

7. A recombinant gene expression cassette, characterized in that: Comprising the recombinant nucleic acid molecule of claim 6.

8. A recombinant vector, characterized in that: Comprising the recombinant nucleic acid molecule according to claim 6, or the recombinant gene expression cassette according to claim 7.

9. A recombinant host cell, characterized in that Comprising the recombinant nucleic acid molecule according to claim 6, or the recombinant gene expression cassette according to claim 7, or the recombinant vector according to claim 8.

10. An immunogenic composition or pharmaceutical composition, characterized in that: Comprising a fusion protein according to any one of claims 1 to 5, and / or a recombinant nucleic acid molecule according to claim 6, and / or a recombinant gene expression cassette according to claim 7, and / or a recombinant vector according to claim 8, and / or a recombinant host cell according to claim 9.

11. A recombinant vaccine, characterized in that: Comprising a fusion protein according to any one of claims 1 to 5, and / or a recombinant nucleic acid molecule according to a claim, and / or a recombinant gene expression cassette according to a claim, and / or a recombinant vector according to claim 8, and / or a recombinant host cell according to claim 9, and / or an immunogenic composition or pharmaceutical composition according to claim 10.

12. Use of the fusion protein of any one of claims 1 to 5, and / or the recombinant nucleic acid molecule of claim 6, and / or the recombinant gene expression cassette of claim 7, and / or the recombinant vector of claim 8, and / or the recombinant host cell of claim 9, and / or the immunogenic composition or pharmaceutical composition of claim 10, and / or the recombinant vaccine of claim 11 in the preparation of a medicament for preventing diseases caused by Streptococcus pneumoniae.

13. The use according to claim 12, characterized in that The diseases caused by Streptococcus pneumoniae include lung tissue infection, pneumonia, meningitis, otitis media, bacteremia, sinusitis, endocarditis, arthritis, peritonitis, and endophthalmitis.

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