Glycoconjugate vaccine

Abstract

The invention relates to a divisional application of 201980029219.5, in particular to a method for preparing a The invention relates to application of streptococcus pneumoniae protein antigens NanA, PiuA and Sp0148 as carriers of immunogenic streptococcus pneumoniae capsular polysaccharide. For example, the invention can provide assistance for glycoconjugate vaccines to produce protective immune responses against a variety of capsular serotypes. The present invention provides glycoconjugates, vaccine compositions, and methods of making and using the same.

Description

[0001] This application is a divisional application of the Chinese application with application number 201980029219.5, application date May 2, 2019, and invention name “Sugar Conjugate Vaccine”. Technical Field

[0002] The present invention relates to a glycoconjugate vaccine and a method for preparing the same. Background Art

[0003] Streptococcus pneumoniae (pneumococcus) is an obligate human pathogen and one of the most common causes of pneumonia, sepsis, and meningitis, thus causing considerable morbidity and mortality worldwide. Pneumococcal meningitis is of particular concern due to its high mortality, especially in children and the elderly, and the resulting neurological sequelae that reduce quality of life.

[0004] The introduction of pneumococcal conjugate vaccines (PCVs) has been highly effective in reducing pneumococcal infections, including meningitis caused by vaccine serotypes (1-8). For example, children vaccinated with PCVs can have a significantly reduced incidence of pneumococcal infections caused by vaccine serotypes in the unvaccinated adult population due to herd immunity (1, 2, 4, 13). However, prior art PCVs have three major disadvantages. First, the major serotypes (STs) causing disease vary geographically and in age, but the formulation of existing PCVs is fixed and not easily changed. Therefore, existing PCVs have different effects on the incidence of pneumococci in different populations and / or different diseases (9). For example, pneumococcal meningitis continues to cause a large amount of morbidity and mortality worldwide, particularly in sub-Saharan Africa, where pneumococcal meningitis affects 98 children per 100,000 children each year (38). Second, because there are more than 90 pneumococcal serotypes and PCVs prevent colonization of the nasopharynx (the bacterium’s usual ecological niche) with only 13 of these, the introduction of PCVs has been associated with a large expansion of nonvaccine serotypes, including in cases of meningitis (5, 7, 8). The natural reproductive capacity of the bacterium suggests that the number of pneumococcal serotypes is likely to increase further, with an increase in disease incidence as nonvaccine serotypes are present (10–13). Third, the high cost of PCVs has limited their use in low- and middle-income countries (LMICs), where the burden of disease is greatest, and has hampered their cost-effectiveness in adults (14, 15). The need for a low-cost pneumococcal vaccine that can prevent disease in both children and adults, has the flexibility to adapt to changes in pneumococcal ecology, and provides some form of cross-serotype protection remains a global priority.

[0005] Current PCV manufacturing is expensive and time-consuming, requiring a multistep process of purification of individual capsular polysaccharides and chemical coupling to a protein carrier. Wren’s group has pioneered an alternative approach, called protein-glycoprotein coupling technology (PGCT), which uses the Campylobacter oligosaccharyltransferase CjPglB to produce recombinant protein / glycan constructs in Escherichia coli (16, 17). PGCT requires expression of the target glycan gene in E. coli, a gene encoding a carrier protein antigen with an additional “sugar tag” sequence to ensure protein / glycan conjugation, and the coupling enzyme CjPglB (18). Vaccine production is achieved by a one-step Ni 2+ Produced from batch cultures of E. coli by affinity chromatography, this approach can be easily scaled up for production. In addition, PGCTs could allow for greater flexibility in the serotypes included in PCVs, allowing vaccines to be tailored to the dominant serotypes in different target populations and geographic locations and to be rapidly reformulated in response to changes in S. pneumoniae ecology.

[0006] Another advantage of PGCTs is that different protein antigens can be easily conjugated to the capsular antigens. Since the first commercialization of a chemical conjugate vaccine for Haemophilus influenzae type b in 1990, the basic science of chemical conjugate vaccines has barely evolved. To date, only four major carrier proteins have been licensed for glycoconjugate vaccine formulations: inactivated toxin of Clostridium tetani, Corynebacterium diphtheriae (CRM197), a surface-expressed protein of Haemophilus influenzae (protein D), and Neisseria meningitidis serogroup B (19, 20). The immunogenic potential of the various proteins has not been tested, and the efficiency of antibody responses to the glycan component of the glycoconjugates varies between different peptide chains (21, 22). Identifying other proteins that can stimulate good antibody responses when fused to glycan antigens will aid in the development of glycoconjugate vaccines. Using antibodies to S. pneumoniae proteins as carrier proteins may offer the additional advantage of inducing an adaptive response to the protein. A number of pneumococcal protein antigens have been described above and, when used as vaccines, have been shown to induce protective immunity in animal models through antibody-mediated opsonophagocytosis (23), inhibition of bacterial protein function (24,25), and stimulation of Th17 cell responses (26-28), and some of these antigens have been tested in phase I trials in humans. Protective pneumococcal protein antigens could be used as carrier proteins in the PGCT approach to prepare PCVs that elicit serotype-independent immunity mediated by adaptive immunity to the protein components. Such vaccines may also have a theoretical advantage in preventing meningitis because antibodies against selected surface protein antigens could prevent penetration of the blood-brain barrier (29-31).

[0007] PCGT has been used to produce an effective prototype vaccine against Francisella tularensis (32), and a PGCT vaccine against Shigella dysenteriae has recently completed phase I trials (33). More recently, PGCT has been shown to be useful for preparing glycoconjugates with four different types of S. pneumoniae capsular serotypes, including serotype 4 (17). However, it remains to be determined whether PCVs made with PGCT can induce protective anti-capsular responses and whether they would have efficacy similar to that of existing commercial PCVs. Furthermore, there are no data on whether S. pneumoniae protein antigens can serve as effective carrier proteins for capsular antigens and still stimulate a protective response. Summarize

[0009] The inventors have recognized that pneumococcal protein antigens can be used as carriers of immunogenic pneumococcal capsular polysaccharides in saccharide conjugate vaccines, which can generate protective immune responses against multiple capsular serotypes. The present invention adopts the following technical solutions:

[0010] The first aspect of the present invention provides a glycoprotein comprising a Streptococcus pneumoniae protein antigen glycosylated with a Streptococcus pneumoniae capsular polysaccharide.

[0011] Preferred protein antigens of Streptococcus pneumoniae include NanA, PiuA and Sp0148, with PiuA being most preferred.

[0012] The second aspect of the present invention provides a vaccine composition comprising one or more glycoproteins of the first aspect.

[0013] The third aspect of the present invention provides a method for treating pneumococcal infection, comprising: administering the glycoprotein of the first aspect or the vaccine composition of the second aspect to an individual in need thereof.

[0014] A fourth aspect of the invention provides the glycoprotein of the first aspect or the vaccine composition of the second aspect for use in a method of treating a Streptococcus pneumoniae infection in a subject.

[0015] Treatment of pneumococcal infection according to the third and fourth levels may include prophylactic or protective treatment.

[0016] Other aspects and embodiments of the present invention are described in detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Vaccination with recombinant glycoproteins is shown to generate antibodies against pneumococcal capsule and carrier proteins, as measured by ELISA in antisera obtained from mice (n=8) vaccinated with recombinant glycoproteins (filled circles) or homologous unglycosylated antigens (open squares). Figure 1AE show anti-capsular polysaccharide antibodies measured using plates coated with purified type 4 capsular polysaccharide, with controls of antisera from Prevnar-13 (filled circles) and sham-vaccinated (open squares) animals, respectively. Figure 1 FH shows antibodies against carrier protein measured by sandwich ELISA from mice vaccinated with the combination vaccine (Combo) (Sp4) (filled circles) or the combination vaccine (open squares) using monoclonal anti-His capture antibody and recombinant unglycosylated carrier protein. Data are shown as mean ± SEM values ​​of duplicate experiments.

[0019] Figure 2 Vaccination with recombinant glycoproteins was shown to generate antibodies that recognized both homologous and heterologous pneumococcal isolates. Anti-ST4 ( Figure 2 A) and anti-ST2 ( Figure 2 B) Antibodies. Data are mean ± SEM from three different cultures. *P < 0.05 vs PBS, #P < 0.05 Protein and glycoprotein detection was performed using one-way ANOVA with Bonferroni post-test. Figure 2 C shows immunoblot analysis of concentrated lysates from overnight cultures of ST4 and ST2 using mixed antisera from glycosylated and unglycosylated vaccine groups. Molecular weight markers are given in kilodaltons.

[0020] Figure 3 Flow cytometric analysis of antibody deposition on Streptococcus species is shown. Figure 3 A shows examples of flow cytometric histograms of these antibodies deposited on a Streptococcus strain expressing the S. pneumoniae serotype 4 capsule, termed Staphylococcus aureus (Sp4), in 10% antisera from the glycosylated (red shading) and non-glycosylated (grey shading) vaccine groups, with mock-vaccinated sera (dashed line) as a control. Figure 3 B shows antibody deposition measured on S. pneumoniae (Sp4) in 10% mouse antisera (n=8) from glycosylated (filled circles) and non-glycosylated (open circles) vaccine groups using flow cytometry analysis. Red dots indicate responses at reduced antiserum concentrations (5% vs. 10%) in high titer samples. *p<0.05 tested using Kruskal-Wallis and Dunn post hoc tests (vs PBS). Figure 3 C shows examples of flow cytometry histograms of antibody deposition on the TIGR4 S. pneumoniae strain in 2% (red shading), 0.2% (grey shading) and 0.02% (dashed line) antisera of the glycosylated vaccine group. Figure 3D shows the deposition of antibodies on TIGR4 in pooled antisera from mice vaccinated with glycosylated or non-glycosylated vaccines. Deposition titers were determined using bacteria incubated with decreasing concentrations of Prevnar-13 antiserum to generate a standard curve. Results are shown as mean ± SEM values ​​of replicates.

[0021] Figure 4 Antibody deposition on non-serotype 4 pneumococci is shown. Figure 4 AD shows representative histograms and antibody deposition on homologous and heterologous pneumococcal isolates in 1% mixed antiserum in mice vaccinated with glycosylated NanA (grey shading), Sp0148 (red shading), or PiuA (blue shading) or normal mouse serum (dashed line). Black columns represent the percentage of PE+ bacteria and grey columns represent the gMFI of the positive population. Gates were set so that 5-10% of the events in the NMS reaction were PE+ to account for strain-specific differences in autofluorescence. Data are shown as mean ± SEM of technical replicates. Figure 4 E shows immunofluorescence staining of homologous and heterologous pneumococcal isolates using antisera from mice vaccinated with the combined vaccine (green channel) and pneumococcal pan-potent serum (red channel).

[0022] Figure 5 The effects of antiserum samples on the interaction of S. pneumoniae with human neutrophils are shown. Figure 5 A shows an example of flow cytometry histograms of fresh human neutrophils incubated with FAM-SE labeled TIGR4 after opsonization with glycosylated (red shading) or non-glycosylated (grey shading) vaccine groups and 5% rabbit complement in 20% antiserum. Sham-vaccinated serum (dashed line) served as a control. Figure 5 B shows the percent association of fresh human neutrophils with TIGR4 when opsonized in 20% antiserum (n=8) and 5% rabbit complement from the glycosylated (filled circles) or non-glycosylated (open) vaccine groups. Antiserum from animals vaccinated with Prevnar-13 (filled circles) and mock vaccinated (open squares) served as controls. *p<0.05 using Kruskal-Wallis and Dunn post hoc test (vs PBS). Figure 5 E shows the percent association of fresh human neutrophils with non-type 4 pneumococci after opsonization in 20% antiserum (n=6) and 5% rabbit complement from the glycosylated (closed circles) or non-glycosylated (open squares) vaccine groups. Antiserum from animals vaccinated with Prevnar-13 (closed circles) and mock vaccinated (open squares) served as controls.

[0023] Figure 6Vaccination with recombinant glycoconjugates provides homologous but not heterologous protection against pneumococcal pneumonia. Mice were vaccinated with a combination of glycosylated and non-glycosylated vaccines and treated with 1 × 10 7 CFU TIGR4 challenged intranasally 48 hours (7A-B) or with serotype 4 (7C-E). Bacterial burden determined by serial dilution and plating. *p<0.05 using Kruskal-Wallis and Dunn post hoc test (vs PBS). Specific description

[0025] The present invention relates to recombinant glycoproteins for use in vaccine compositions, in particular glycoproteins comprising pneumococcal protein antigens glycosylated with pneumococcal capsular polysaccharides. These glycoproteins generate protective immune responses against multiple pneumococcal serotypes and can be used to prevent or treat pneumococcal infections, such as meningitis.

[0026] The glycoprotein described herein may comprise a pneumococcal protein antigen and one or more N-linked pneumococcal capsular polysaccharides, i.e., the capsular polysaccharide may be covalently linked to the asparagine residues of the protein antigen (N-glycosylation). The capsular polysaccharide and the pneumococcal protein antigen may be heterologous, i.e., the pneumococcal protein antigen may not be glycosylated by the capsular polysaccharide in the natural system.

[0027] Bacterial pathogen cells, such as Streptococcus pneumoniae, are usually surrounded by capsular polysaccharides that form a protective outer layer around the cells. Capsular polysaccharides can interact directly with host B cells and induce antibody synthesis in the absence of T cells. A Streptococcus pneumoniae capsular polysaccharide may contain one or more B cell epitopes. The polysaccharides in the capsule are homopolymers or heteropolymers of monosaccharide subunits linked by glycosidic bonds. The configuration of monosaccharide subunits in the capsular polysaccharide of cells of different serotypes of bacterial pathogens varies from one another. For example, more than 90 different Streptococcus pneumoniae capsular serotypes have been identified to date.

[0028] The pneumococcal capsular polysaccharide of the glycoprotein described herein may include common pneumococcal serotypes, such as 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F. The structures of capsular polysaccharides from pneumococcal serotypes are well known in the art (Bentley and Spratt PLoS Genet. 2006 Mar; 2(3): e31. Epub 2006 Mar 10).

[0029] In some preferred embodiments, the capsular polysaccharide may be a serotype 4 capsular polysaccharide.

[0030] The glycoproteins described herein may comprise one or more capsular polysaccharide moieties, such as two, three, four, five or more capsular polysaccharide moieties. In some embodiments, the glycoprotein may comprise two capsular polysaccharide moieties. For example, the capsular polysaccharide moieties may be linked to glycosylation sequences within the glycoprotein, such as at the N and C termini of the protein antigen.

[0031] The multiple capsular polysaccharide moieties attached to the glycoprotein may be the same polysaccharide or different polysaccharides.

[0032] The pneumococcal protein antigen can be any protein expressed on pneumococcus capable of eliciting an immune response, preferably a T cell response. For example, the pneumococcal protein can contain one or more T cell epitopes. Preferred pneumococcal protein antigens can induce cross-protective immunity against a variety of pneumococcal strains and support a strong antibody response to the capsular polysaccharide attached thereto.

[0033] Suitable S. pneumoniae proteins are known in the art and include neuraminidase A (NanA; spr1536;

[0034] EC3.2.1.18), pneumococcal heme transport system lipoprotein PiuA (Sp1872) and Sp0148. Most preferably, PiuA (Sp1872) is selected from the group consisting of:

[0035] Suitable PiuA protein antigens may comprise the amino acid sequence of database accession numbers KGI34864.1, AOG58833.1, ANO37655.1, KGI33012.1 or SEQ ID NO: 3, or a fragment or variant of any of these.

[0036] Suitable NanA protein antigens may comprise the amino acid sequence of database accession number NP_359129.1 or SEQ ID NO: 6, or a fragment or variant of any of these. For example, the protein antigen may comprise the N-terminal lectin-like domain of NanA.

[0037] Suitable Sp0148 protein antigens may comprise the amino acid sequence of database accession number ABJ55394.1 or WP_000724951.1 or SEQ ID NO: 9, or a fragment or variant of any of these.

[0038] A variant of a reference polypeptide sequence, such as one of SEQ ID NOs: 3, 6 or 9, can comprise an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the reference polypeptide sequence.

[0039] Usually refer to the algorithm GAP (GCG Wisconsin PackageTM , Accelrys, San Diego CA) to define nucleotide and amino acid sequence identity. GAP uses the Needleman & Wunsch algorithm to align two complete sequences to maximize the number of matches and minimize the gaps. Typically, default parameters are used with a gap creation penalty of 12 and a gap extension penalty of 4. Use of GAP may be preferred, but other algorithms such as BLAST, BLASTP or BLASTN (using the method of Altschul et al., FASTA (using the method of Pearson and Lipman, or PSI-Search using the Smith-Waterman algorithm) may also be used, and default parameters are typically used [54-56, 75].

[0040] For example, a protein antigen may comprise an amino acid sequence that differs from a reference polypeptide sequence, such as one of SEQ ID NOs: 3, 6 or 9, by insertion, addition, substitution or deletion of 1, 2, 3, 4, 5-10, 10-20, 20-30, 30-40 or more amino acids.

[0041] The protein antigen may be a fragment of a reference polypeptide sequence, such as one of SEQ ID NO: 3, 6 or 9, or a variant of a reference polypeptide sequence. A fragment is a truncated polypeptide consisting of fewer amino acids than the full-length sequence, the fragment comprising at least one immunogenic determinant of the full-length sequence and retaining immunogenicity. Suitable fragments may comprise at least 100, at least 150, at least 200, at least 250 or at least 300 amino acids of the full-length sequence.

[0042] Fragments of full-length protein antigens can enhance the immune response that recognizes the full-length antigen and the serotype of Streptococcus pneumoniae or the strain from which the protein antigen is derived (adjuvants can be added appropriately if necessary). For example, suitable fragments can be identified by computer simulation of potential immunogenic sites, and then fragment groups are generated and tested in vivo in mammalian protection models.

[0043] Suitable protein antigens for use in vaccine compositions may lack a signal peptide sequence.

[0044] The pneumococcal protein antigen may contain a glycosylation sequence. A glycosylation sequence is a short sequence of consecutive amino acids within a protein that forms the attachment site for the polysaccharide portion. The capsular polysaccharide may be covalently linked to an asparagine residue within the glycosylation sequence of the protein antigen.

[0045] The choice of glycosylation sequence may depend on the oligosaccharyl transferase used to produce the glycoprotein. A suitable glycosylation sequence recognized by Campylobacter jejuni PglB may comprise the sequence D / EYNXS / T, wherein Y and X are any amino acids except P. For example, the glycosylation sequence may comprise the sequence DQNAT. Other glycosylation sequences used with other oligosaccharyl transferases can be easily determined.

[0046] The glycosylation sequence may be heterologous, i.e., the sequence may not be naturally present in the sequence of the protein antigen and may be incorporated therein by recombinant techniques. Suitable methods for incorporating glycosylation sequences into protein antigen sequences are well known in the art. In some embodiments, the heterologous glycosylation sequence may be located on both sides of the linker residue, for example, one or more G residues, such as G or GG.

[0047] The protein antigen may comprise one glycosylation sequence or multiple glycosylation sequences. For example, the protein antigen may comprise two glycosylation sequences. Preferably, the glycosylation sequence may be located at the N-terminus of the protein antigen (or adjacent to the N-terminal leader sequence) or the C-terminus of the protein antigen (or adjacent to the C-terminal purification tag).

[0048] In some embodiments, the protein antigen can be coupled to a leader peptide to direct the secretion of the glycoprotein from the cell into the culture medium as a precursor. A range of suitable leader peptides are known in the art and include the DsbA leader sequence

[0049] MKKIWLALAGLVLAFSASAAQ or variants thereof. The leader peptide may be heterologous to the protein antigen, i.e. it may be a leader sequence that does not naturally bind to the protein antigen. The leader peptide may be located at the N-terminus of the precursor glycoprotein. After the precursor is expressed to produce the glycoprotein, the leader peptide is removed by post-translational processing.

[0050] The glycoprotein may further comprise a purification tag. A purification tag is one of the heterologous amino acid sequences that forms a specific binding pair. The other heterologous amino acid sequence of the specific binding pair can be bound to the polypeptide to detect, separate and / or purify the polypeptide containing the purification tag. For example, a purification tag can form an epitope that is bound by an antibody molecule.

[0051] Various suitable purification tags are known in the art, including, for example, MRGS(H)6, DYKDDDDK(FLAG TM ), T7-, S-(KETAAAKFERQHMDS), poly-Arg(R 5-6 )、poly-His(H 6-10 )、poly-Cys(C4)、poly-Phe(F 11 )、poly-Asp(D 5-16), Strept-tag II (WSHPQFEK), c-myc (EQKLISEEDL), HA influenza tag

[66] , Glu-Glu-Phe tag

[67] , Tag.100 (Qiagen; 12aa tag derived from mammalian MAP kinase 2), Cruz tag 09TM (MKAEFRRQESDR, Santa Cruz Biotechnology Inc.), and Cruz tag 22TM (MRDALDRLDRLA, Santa Cruz Biotechnology Inc.).

[0052] In some preferred embodiments, a poly-His purification tag can be used, such as a C-terminal (His) 10 sequence. After expression, the glycoprotein containing the poly-His purification tag can be separated by affinity chromatography using an affinity resin containing bound divalent nickel or cobalt ions. The use of affinity resins to purify polyhistidine-tagged glycoproteins is well known in the art. The poly-His tag can be removed by protease cleavage after purification to produce a protein antigen as needed.

[0053] Suitable pneumococcal protein antigens comprising a glycosylation sequence, a His tag and a leader peptide may comprise the amino acid sequence of any of SEQ NOs: 2, 5 or 7, or a fragment or variant of any of these. Also disclosed herein are SEQ NOs: 2, 5 or 7 without a leader peptide and optionally a His tag.

[0054] The above glycoprotein can be prepared by any suitable method. Preferably, the glycoprotein is prepared by protein-glycosylation coupling technology (PGCT) (see Examples 16-18, 41, 42). For example, the method for producing glycoprotein described herein may include:

[0055] Expression in microbial cells;

[0056] (i) oligosaccharyltransferase;

[0057] (ii) Streptococcus pneumoniae capsular polysaccharide; and

[0058] (iii) Streptococcus pneumoniae protein antigens;

[0059] Oligosaccharyltransferases glycosylate pathogen protein antigens with pathogen capsular polysaccharides to produce glycoproteins.

[0060] The microbial cell may be a prokaryotic cell, preferably an Escherichia coli cell.

[0061] The microbial cell for producing glycoprotein can comprise (i) heterologous nucleic acid encoding oligosaccharyl transferase, (ii) heterologous capsular polysaccharide biosynthesis locus, and (iii) heterologous nucleic acid encoding pathogen protein antigen. The heterologous nucleic acid and locus can be present on an extrachromosomal plasmid in the microbial cell, or integrated into the genome of the microbial cell.

[0062] Methods for producing microbial cells include transforming microbial cells with:

[0063] (i) a heterologous nucleic acid encoding an oligosaccharyltransferase;

[0064] (ii) a heterologous S. pneumoniae capsular polysaccharide biosynthesis site; and

[0065] (iii) a heterologous nucleic acid encoding a protein antigen of Streptococcus pneumoniae.

[0066] Suitable methods for transforming microbial cells are well known in the art. Heterologous nucleic acids and loci can be included in one or more vectors such as plasmids. In some embodiments, heterologous nucleic acids and loci can all be included in different vectors. In other embodiments, two or more heterologous nucleic acids and heterologous loci can also be included in the same vector.

[0067] For expression of oligosaccharyltransferases, protein antibodies and capsular polysaccharides, microbial cells can be cultured under standard conditions in a cell culture vessel such as a bioreactor or fermentor.

[0068] Oligosaccharyltransferases (EC 2.4.1.119) catalyze the glycosylation of asparagine residues of the glycosylation sequence of a protein antigen to a capsular polysaccharide. Suitable oligosaccharyltransferases for attachment to any particular capsular polysaccharide substrate can be easily identified using standard techniques. In some preferred embodiments, the oligosaccharyltransferase may be PglB, preferably bacterial PglB, such as Helicobacter pylori PglB, Desulfovibrio PglB, Campylobacter lari PglB and Campylobacter jejuni PglB. Preferred PglB may be an amino acid sequence comprising database accession number ASI87642.1 or YP_002344519.1 or SEQ ID NO: 10 or variants and fragments thereof.

[0069] The pneumococcal protein antigens and pneumococcal capsular polysaccharides are described in detail above. The pneumococcal capsular polysaccharide for connection via PGCT can be connected to an undecenyl pyrophosphate anchor. In some embodiments, for example, when the oligosaccharyl transferase is Campylobacter jejuni PglB, the capsular polysaccharide can be included in a reducing end sugar containing an acetylamino group at the C2 position.

[0070] The pneumococcal capsular polysaccharide can be produced in microbial cells from heterologous capsular polysaccharide biosynthetic loci or operons. The polysaccharide biosynthetic locus is a region of the pneumococcal genome that contains genes required for the production of capsular polysaccharides in microbial cells. The polysaccharide biosynthetic locus is located between the AliA and DexB genes in the pneumococcal genome and has four conserved genes at its N-terminus. The polysaccharide biosynthetic loci for different pneumococcal serotypes have been reported in the art (see, for example, Bentley and Spratt PLoS Genet. 2006 Mar, 2 (3): e31. Epub 2006 Mar 10) and can be identified in any pneumococcal genome using standard genetic analysis techniques.

[0071] The biosynthetic loci of the pneumococcal capsular polysaccharide can be obtained from cells of pneumococcal serotypes and cloned into vectors expressed in microbial cells to produce the capsular polysaccharide. The technology of producing capsular polysaccharides from heterologous biosynthetic loci in cells has been established in the art (16-18, 41, 42).

[0072] After the glycoprotein is produced in the microbial cell, it can be separated and / or purified, which can be achieved using any convenient method known in the art. Techniques for purifying recombinant glycoproteins are well known in the art and include, for example, HPLC, FPLC or affinity chromatography. In some embodiments, as described above, an affinity tag on the glycoprotein can be used for purification.

[0073] The glycoprotein can be formulated with a pharmaceutically acceptable excipient as described herein and optionally an adjuvant and / or one or more different glycoproteins, for example to produce an immunogenic formulation or vaccine composition for therapeutic use. Vaccine compositions are described in detail above.

[0074] Both the capsular polysaccharide and the protein antigen are immunogenic, and can induce an adaptive immune response to the capsular polysaccharide and the protein antigen in an individual to whom the glycoprotein is administered. The immune response can include a T cell-dependent immune response and a T cell-independent immune response, such as the production of antibodies. In some preferred embodiments, the glycoprotein can simultaneously induce a T cell-dependent immune response and a T cell-independent immune response.

[0075] Glycoproteins can provide a protective immune response against Streptococcus pneumoniae. For example, when a glycoprotein individual is exposed to Streptococcus pneumoniae, the glycoprotein may stimulate, promote or enhance a protective immune response. Glycoproteins can stimulate a protective immune response against a serotype of Streptococcus pneumoniae or a serotype of Streptococcus pneumoniae from which the capsular polysaccharide and / or protein antigens are derived, rather than just a serotype of Streptococcus pneumoniae from which the capsular polysaccharide and / or protein antigens are derived. For example, a glycoprotein can stimulate a protective immune response against multiple serotypes of Streptococcus pneumoniae. In some embodiments, a protective immune response may correspond to a single serotype.

[0076] Vaccine compositions may be formulated using one or more glycoproteins as described above.

[0077] A vaccine composition is an immunogenic preparation comprising one or more immunogenic components that is capable of generating a protective immune response in an individual to the one or more immunogenic components. The immunogenic components of the vaccine compositions described herein may be comprised of Streptococcus pneumoniae protein antigens of one or more glycoproteins and Streptococcus pneumoniae capsular polysaccharides.

[0078] The vaccine compositions described herein include protein and polysaccharide antigens from Streptococcus pneumoniae that are capable of eliciting an immune response against Streptococcus pneumoniae in an individual. For example, the protein and polysaccharide antigens may be immunoreactive to serum exposed to Streptococcus pneumoniae, such as human serum exposed to Streptococcus pneumoniae.

[0079] The vaccine composition may comprise one glycoprotein or multiple glycoproteins as described above, preferably multiple glycoproteins, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different glycoproteins. The multiple glycoproteins in the composition may comprise multiple different protein antigens and / or multiple different capsular polysaccharides. For example, the vaccine composition may comprise glycoproteins having multiple different pneumococcal capsular polysaccharides and the same pneumococcal protein antigens; multiple different pneumococcal protein antigens and the same pneumococcal capsular polysaccharides; or multiple different pneumococcal protein antigens and multiple different pneumococcal capsular polysaccharides.

[0080] The vaccine composition may further comprise an adjuvant. An adjuvant is a non-immunogenic agent that increases or enhances the immune response to an antigen in an individual (for review see for example and Vaccine Adjuvants: adjuvants: preparation methods and research protocols Ed O'Hagan (2000) Springer).

[0081] Suitable adjuvants include aluminum and aluminum salts, such as aluminum hydroxide and / or aluminum phosphate; oil emulsion compositions (or oil-in-water compositions), including squalene aqueous emulsions, such as emulsigen TM (MVP Technologies Inc), Addavax TM and MF59 TM (See, e.g., WO 90 / 14837); saponin preparations, such as QS21 and immunostimulatory complexes (ISCOMS) (See, e.g., US Pat. No. 5,057,540; WO 90 / 03184, WO 96 / 11711, WO 2004 / 004762, WO 2005 / 002620); Toll-like receptor (TLR) agonists, such as TLR7 agonists (See, e.g., WO 2012 / 117377), optionally in combination with an aluminum salt; a bacterial or microbial derivative, such as monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL), flagellin, an oligonucleotide containing a CpG-motif, rhamnose dipeptide (MDP) and / or trehalose dycorynemycolate (TDM), an ADP-ribosylated bacterial toxin, such as Escherichia coli heat-labile enterotoxin LT and cholera toxin CT and its non-enzyme binding subunits, a poly I:C anionic polymer, including an acrylic acid polymer (such as polymethyl methacrylate), acrylic acid cross-linked with allyl sucrose (such as CarbopolTM, Lubrizol and polyethyleneimine (PEI)), a cytokine selected from: GMCSF, interferon γ, interferon α, interferon β, interleukin 12, interleukin 23, interleukin 17, interleukin 2, interleukin 1, TGF, TNFα and TNFβ.

[0082] The method of preparing a vaccine composition may comprise:

[0083] One or more glycoproteins as described above are mixed with a pharmaceutically or veterinarily acceptable excipient and optionally an adjuvant.

[0084] The term "pharmaceutically acceptable" or "veterinarily acceptable" as used herein refers to compounds, materials, compositions and / or dosage forms that are suitable for use in contact with animal tissues (e.g., humans or other mammals) without excessive toxicity, irritation, allergic response or other problems or complications, and with a reasonable benefit / risk ratio, within the scope of sound veterinary or medical judgment. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

[0085] Suitable excipients and carriers include, but are not limited to, water, saline, buffered saline, phosphate buffer, alcohol / water solution, emulsion or suspension, and other conventionally used diluents, adjuvants and excipients may be added according to conventional techniques. Such carriers may include ethanol, polyols and suitable mixtures thereof, vegetable oils and injectable organic esters, and buffers and pH regulators may also be used, including but not limited to salts prepared from organic acids or bases. Representative buffers include, but are not limited to, organic acid salts, such as salts of citric acid (e.g., citrate), ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, phthalic acid, Tris, trimethylamine hydrochloride or phosphate buffer. The carrier of the injection drug may include sodium chloride solution, Ringer's dextrose, dextrose, trehalose, sucrose, lactated Ringer's solution or fixed oil. The carrier of intravenous injection may include liquid and nutritional supplements, electrolyte supplements, such as those based on Ringer's dextrose, etc. Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents (e.g., EGTA, EDTA), inert gases, etc., may also be provided in the pharmaceutical carrier. The vaccine compositions described herein are not limited by the choice of carrier. Within the technical scope of the art, the above-mentioned pharmaceutically acceptable components have appropriate pH, isotonicity, stability and other conventional properties.

[0086] Suitable carriers, excipients, etc. can be found in standard pharmaceutical textbooks, for example, Remington's Pharmaceutical Sciences

[71] and The Handbook of Pharmaceutical Excipients, 4 th edit., eds. R.C. Rowe et al., APhAPublications, 2003.

[0087] Vaccine compositions can be conveniently presented in unit dosage form and can be prepared by any method known in the pharmaceutical art. Such methods include the step of associating one or more isolated immunogenic polypeptides with the above-mentioned carriers or excipients which may constitute one or more auxiliary components. Typically, the formulation is prepared by uniformly and intimately combining the active compound with a liquid carrier, a finely divided solid carrier, or both.

[0088] Depending on the route of administration, the vaccine compositions described herein can be produced in various forms. For example, the vaccine compositions can be prepared in the form of a sterile aqueous solution or dispersion, suitable for injection, or prepared in a lyophilized form using a freeze-drying technique. Lyophilized vaccine compositions are typically kept at about 4° C. and can be reconstituted in a stable solution such as saline or HEPES with or without an adjuvant. The vaccine composition can also be in the form of a suspension or emulsion.

[0089] These vaccine compositions may include additives suitable for administration by any conventional route of administration. The vaccine composition may be prepared to be administered to a subject in the form of, for example, a liquid, powder, aerosol, tablet, capsule, enteric-coated tablet or capsule, or suppository. Therefore, the vaccine composition may also be in the form of, but not limited to, a suspension, a solution, an emulsion in an oily or aqueous vehicle, a paste, and an implantable sustained release or biodegradable preparation. Compositions for sustained release or implantation may include pharmaceutically acceptable polymers or hydrophobic materials, such as emulsions, ion exchange resins, slightly soluble polymers, or slightly soluble salts.

[0090] Preparations suitable for parenteral administration (e.g., by injection, including intramuscular injection) include aqueous and non-aqueous isotonic, pyrogen-free, sterile injection solutions, which may contain antioxidants, buffers, preservatives, stabilizers, bacteriostats, and solutes that are isotonic with the blood of the intended recipient; aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. Examples of isotonic excipients suitable for such preparations include sodium chloride injection, Ringer's solution or lactated Ringer's injection. Typically, the concentration of the active compound in the solution is from about 1 μg / ml to about 100 mg / ml, for example, from about 10 μg / ml to about 50 mg / ml. In some preparations for parenteral administration, the active ingredient can be provided in a dry (i.e., powder or granular) form so that it can be reconstituted with a suitable vehicle (e.g., sterile pyrogen-free water) before the parenteral administration of the reconstituted composition. Other useful parenteral preparations include those containing active ingredients in microcrystalline form, liposomal preparations, or preparations as components of a biodegradable polymer system.

[0091] The vaccine composition may be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition and require only the addition of a sterile liquid carrier, such as water for injection, immediately prior to use.

[0092] The vaccine composition can be administered to the subject by any convenient route of administration. In some embodiments, it is administered by a parenteral route, such as intramuscular, intranasal, transdermal or subcutaneous. For example, the vaccine composition can be administered by injection, preferably intramuscular injection.

[0093] The appropriate dosage of the vaccine composition varies from individual to individual or from group to group depending on the actual situation. Determining the optimal dosage will generally involve a therapeutic benefit level that balances the risks or harmful side effects of any administration. The selected dosage level depends on a variety of factors, including but not limited to the route of administration, the time of administration, and the secretion rate of the vaccine composition, other drugs, compounds and / or materials used in combination, as well as the species, type, maturity, sex, weight, condition and general health of the individual. The amount of the vaccine composition and the route of administration will ultimately be determined by a veterinarian or physician, although generally the dosage should achieve a serum concentration of the vaccine composition sufficient to produce a beneficial effect without causing substantial harmful or toxic side effects.

[0094] Treatment may include administering to an individual a therapeutically effective amount of a vaccine composition. A "therapeutically effective amount" refers to an amount of a vaccine composition that produces a desired therapeutic effect, commensurate with a reasonable benefit / risk ratio. For example, a suitable amount of a vaccine composition for administration to an individual may be an amount that produces a protective immune response against each polysaccharide or protein antigen present in the individual of the composition.

[0095] When an individual to whom the vaccine composition described herein is administered is exposed to Streptococcus pneumoniae, the individual to whom the vaccine composition is administered may exhibit acquired and / or adaptive immune responses to Streptococcus pneumoniae. These responses may provide protection against morbidity or mortality caused by Streptococcus pneumoniae infection. For example, the vaccine composition may reduce the likelihood of infection with Streptococcus pneumoniae, reduce the severity or duration of clinical symptoms of infection in an individual, prevent or delay the onset of clinical symptoms of infection, and prevent or reduce the risk of death in an individual after infection with Streptococcus pneumoniae.

[0096] In vivo administration of a dose can be carried out continuously or intermittently (for example, in batches at appropriate intervals). In some embodiments, the vaccine composition can be repeatedly administered to the same individual at sufficient time intervals to obtain a booster effect, for example, at least 1 week, 2 weeks, 3 weeks or 4 weeks, preferably 2 weeks, between two administrations. A booster dose can be initially administered to an individual after a certain dose, for example, during administration, an initial dose can be given at 1-4 weeks, and a booster dose can be given at 3-6 weeks old. The method for determining the most effective mode of administration and dosage is well known to those skilled in the art, and will vary with the preparation and the subject being treated. For example, in some embodiments, when a booster dose is administered after a period of time (for example, after 2-4 weeks), if the level of maternally derived antibodies in the mother is reduced, the initial dose of the vaccine composition can be administered after two weeks. Single or multiple administrations can be performed by selecting dosage levels and methods by a veterinarian or doctor.

[0097] The glycoprotein or vaccine composition described herein can be used in a method for treating an animal or human. The present invention provides a glycoprotein or vaccine composition described herein for use in treating a Streptococcus pneumoniae infection in an individual or a group of individuals; the present invention provides the use of the glycoprotein or vaccine composition described herein in the preparation of a medicament for treating a Streptococcus pneumoniae infection in an individual or an individual; and provides a method for treating a Streptococcus pneumoniae infection, comprising administering the glycoprotein or vaccine composition described herein to an individual or a group of individuals in need thereof.

[0098] Pneumococcal infection includes infection with any serotype or strain of Pneumococcus, such as Pneumococcal serotype 4, and clinical signs of Pneumococcal infection, and diseases associated with Pneumococcal infection, including bacterial meningitis, septicemia, pneumonia, otitis media, cardiac infection, pericarditis, septic arthritis, and spontaneous peritonitis.

[0099] S. pneumoniae infection can be identified or diagnosed using standard diagnostic criteria.

[0100] The treatments described herein can elicit an immune response in an individual or population upon exposure to S. pneumoniae. This can achieve a desired therapeutic effect, for example, increased protection or resistance against morbidity or mortality caused by S. pneumoniae infection.

[0101] More preferably, the treatment described herein may be a prophylactic or preventive treatment, i.e., the individual or population may not have a pneumococcal infection and / or may not show clinical signs of a pneumococcal infection at the time of treatment. In some embodiments, the individual or population may be susceptible to or at risk of infection with pneumococcal infection.

[0102] For example, glycoproteins or vaccine compositions can be used for vaccination or immunization of individuals or populations against Streptococcus pneumoniae. The treatment of Streptococcus pneumoniae infection described herein can prevent future Streptococcus pneumoniae infection in individuals or populations or mitigate its effects. Preventive or prophylactic treatments may reduce the susceptibility of individuals or populations to Streptococcus pneumoniae infection, prevent or inhibit Streptococcus pneumoniae colonization in the nasopharynx, reduce the risk or likelihood of infection with Streptococcus pneumoniae, delay or reduce the severity or duration of lesions or other clinical symptoms of Streptococcus pneumoniae infection, or prevent or delay the onset of clinical symptoms of Streptococcus pneumoniae in individuals or populations, and / or reduce or prevent the morbidity or mortality caused by Streptococcus pneumoniae infection.

[0103] Individuals or populations suitable for treatment with the glycoprotein or vaccine compositions described herein can be humans or non-human mammals, such as non-human mammals susceptible to S. pneumoniae infection, including equine mammals, such as horses.

[0104] The glycoprotein or vaccine composition described herein can reduce the incidence of morbidity or mortality associated with S. pneumoniae in a population relative to an unimmunized population. For example, a vaccinated population relative to an unimmunized population can reduce the incidence of lesions or other clinical symptoms of S. pneumoniae infection after exposure to S. pneumoniae. In some embodiments, vaccination of a population with a vaccine composition described herein can reduce the number of individuals in the population showing clinical symptoms of S. pneumoniae infection by at least 50%, more preferably at least 60%, at least 65%, at least 70%, or at least 75%, and most preferably at least 80% compared to an untreated population.

[0105] Other aspects and embodiments of the present invention provide the above aspects and embodiments, wherein the term "comprising" is replaced with the term "including", and in the above aspects and embodiments, the term "comprising" is replaced with the term "consisting essentially of".

[0106] It should be understood that, unless the context otherwise requires, this application discloses all combinations of any one of the above aspects and embodiments. Similarly, unless the context otherwise requires, this application discloses all combinations of preferred and / or optional features, either alone or in combination with any other aspect.

[0107] Modifications of the above embodiments, other embodiments and modifications thereof will be apparent to those skilled in the art from reading this disclosure and are within the scope of the present invention.

[0108] All documents and sequence database entries mentioned in this specification are herein incorporated by reference in their entirety for all purposes.

[0109] As used herein, "and / or" should be considered as a specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" will be considered as a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, just as if each were listed separately herein.

[0110] experiment

[0111] The following experiments used PGC to conjugate the brain endothelial cell infiltrate NanA (29), the Th17 stimulatory antigen Sp0148 (24), and the N-terminal fragment of the ABC transporter PiuA (34) to the cystic 4 serum antigen, and the efficacy of these glycoconjugates was tested using a mouse model of vaccination and infection with Streptococcus pneumoniae.

[0112] 1. Materials and Methods

[0113] 1.1 Bacterial strains and growth conditions

[0114] Table 1 lists the bacterial strains used in this study. Escherichia coli isolates were routinely cultured in modified superoptimal broth (SSOB) or agar at 28°C. Where appropriate, the cultures were supplemented with 100 μg / ml ampicillin, 20 μg / ml tetracycline and / or 80 μg / ml spectinomycin. S. pneumoniae was cultured on Columbia horse blood agar plates (E&O Laboratories) at 37°C + 5% CO2 or in liquid media in brain heart infusion (BHI). Where appropriate, the cultures were supplemented with 5 μg / ml gentamicin or 75 μg / ml streptomycin. S. mitis was cultured in Todd-Hewitt liquid media supplemented with 0.5% yeast extract at 37°C + 5% CO2. S. pneumoniae and S. mitis were cultured to an OD of approximately 0.4-0.8. 600 , and stored in 1 ml aliquots in 20% glycerol at -80°C. Stocking density was determined by serial dilution and plating.

[0115] 1.2 Synthesis and gene modification of carrier protein genes

[0116] DNA sequences, codons optimized for expression in E. coli, encoding neuraminidase A (NanA, Spd1504) and the ABC transporters PiuA (Sp1872) and Sp0148 were commercially synthesized in pUC57 and subcloned into pEXT21. The size of full-length NanA made it difficult to recombinantly produce in high yields in early experiments, necessitating the subcloning of the protein's N-terminal lectin-like domain, which is critical for protein function, from pEXT21 (nanA) as described in the Supplementary Methods above (29). To promote PglB glycosylation, the synthetic carrier protein sequence was modified with the sequence DQNAT, which has two spacer glycine residues at both ends and located at the N and C termini of the mature protein. Periplasmic targeting was promoted by exchanging the native signal peptide for the DsbA leader sequence MKKIWLALAGLVLAFSASAAQ. The C-terminal ten-His sequence can be used to facilitate purification of Ni-NTA. In NanA, internal EcoRI and XbaI sites were replaced by synonymous mutations. Commercial plasmid constructs were cloned into efficient library DH5α cells (Life Technologies) according to the manufacturer's instructions and stored in 20% glycerol at -80° C. The synthetic vector protein sequence was subcloned into the expression vector pEXT21 using restriction enzymes EcoRI and XbaI (New England Biolabs).

[0117] 1.3 Preparation of expression strains

[0118] Overnight cultures of E. coli W3110 and W311B pB4-4 were inoculated at 1:100 and the OD 600 The culture was cooled on ice, precipitated at 4000 × g for 10 minutes, and washed sequentially with 0.5 volume and then 0.25 volume of ice-cold 10% glycerol. The competent cells were resuspended in 1 / 250 volume of ice-cold 10% glycerol and transformed in 50 μl aliquots in 0.2 cm test tubes with pEXT21 (nanA), pEXT21 (piuA) or pEXT21 (sp0148) at 2.5 kV, 200 Ω and 25 μF. In order to study the role of lipid-linked Sp4 in anti-capsule immunity, W3110 pB4-4 cells were transformed with pEXT21 (piuA). The isolates confirmed to be expressed were stored in 20% glycerol at -80°C.

[0119] 1.4 Recombinant protein preparation

[0120] W3110 pEXT21 (NanA / PiuA / Sp0148) and W311B pB4-4 pEXT20 pEXT21 (NanA / PiuA / Sp0148) isolates were grown overnight at 28°C and then subcultured into SSOB at a ratio of 1:100. Bacteria were grown for 2-3 h and then induced overnight at 28°C with 1 mM IPTG and 4 mM MnCl2. Cells were pelleted at 14,000 × g and lysed using a pressure cell homogenizer (Stanstead). Lysates were treated with 25 U / ml Benzonase nuclease (Sigma-Aldrich) for 20 min at room temperature and filtered at 0.2 μm using a Millex-GP syringe filter (Millipore). In the initial studies, recombinant (glyco)proteins were purified using a Ni-NTA purification system (Thermo Fisher Scientific) according to the manufacturer's instructions. For vaccination studies, the recombinant (sugar) protein was separated using a His-trap FF column from GE Healthcare and an AKTA purifier with a linear imidazole gradient of 25 to 250 mM. The fractions containing only the recombinant (sugar) protein were identified by SDS-PAGE and Coomassie Brilliant Blue staining, merged, and the buffer was exchanged into PBS using a Vivaspin 20 centrifugal concentrator (10000 MWCO). Protein concentration was determined using a Pierce Coomassie Plus (Bradford) assay kit (Thermo Fisher). The recombinant (sugar) protein was sterilized by filtration, standardized to 200 μg / ml in PBS, and stored at -80 ° C. Sample purity was confirmed by SDS-PAGE.

[0121] 1.5 SDS-PAGE and immunoblotting

[0122] Recombinant protein (10 μl) was mixed with 4 μl 4x LDS sample buffer (thermos fisher) and 2 μl 0.5M dithiothreitol (Sigma-Aldrich) and heated at 70°C for 10 minutes. Samples were separated on NuPAGE 12% bis-tris protein gels in MOPS buffer and transferred to nitrocellulose membranes using the iblot2 transfer system according to the manufacturer's instructions. The membrane was blocked with PBST supplemented with 2.5% skim milk powder (Marvel) and 0.1% normal goat serum (Thermo Fisher). Serotype 4 rabbit anti-capsule antibody (Statens Serum Institut, Denmark) and monoclonal mouse anti-His IgG (Abcam) were used as primary antibodies, diluted at 1:1000 and 1:5000 dilutions, respectively. After 1 h incubation, the membrane was washed three times with PBST and incubated with secondary goat anti-rabbit IgG (IRDye800) and goat anti-mouse IgG (IRDye680) conjugated antibodies at a dilution of 1:10000 for 45 min. The membrane was washed three more times in PBST and detected using the LI-COR Odyssey Fluorescence Imaging System (LI-COR Biosciences UK Ltd).

[0123] TIGR4 and D39 lysates were prepared by pressure lysis of overnight cultures. Lysates were filtered at 0.2 μm and concentrated 10-fold using a Vivaspin 20 centrifugal concentrator (10,000 MWCO). Aliquots of 10 μl of concentrated lysates were analyzed by immunoblotting using a 1:1000 dilution of mouse antiserum and a 1:10,000 dilution of goat anti-mouse IgG (IRDye800) as described above. Densitometry was performed using the LI-COR Odyssey Fluorescence Imaging System.

[0124] 1.6 Vaccination studies

[0125] Female CD1 mice, 5-6 weeks old, were purchased from Charles River and housed in 4 cages. For antiserum generation and pneumonia challenge studies, mice were vaccinated intraperitoneally on day 0 with 10 μg of recombinant (glyco)protein emulsified 1:1 in Sigma adjuvant (Sigma-Aldrich). Boosting immunizations were given subcutaneously on days 21 and 35. The positive control group was vaccinated and boosted with 20 μl of 1:5 PBS diluted Prevnar-13 (Wyeth). The negative control group was sham-vaccinated with PBS and Sigma adjuvant only. Colonization studies were performed using mice vaccinated intraperitoneally as described above and boosted intranasally with 2 μg of recombinant protein (without adjuvant) or 20 μl of diluted Prevnar-13 or PBS under light isoflurane anesthesia. For pneumonia studies, mice were given 1×10 7 Mice were infected with CFU TIGR4. Mice were culled 24 h after infection and the bacterial burden in the lungs and blood was assessed.

[0126] 1.7 Anti-capsule and anti-protein ELISA

[0127] To measure the anti-capsule reaction, Nunc Maxisorp 96-well plates were coated with 0.5 μg / well purified type 4 pneumococcal polysaccharide (Statens Serum Institut, Denmark) in PBS at 4°C overnight. The wells were blocked with PBST supplemented with 2.5% skim milk powder and 0.1% normal goat serum and incubated with a 10-fold dilution of mouse antiserum in PBS supplemented with 0.1% BSA and 0.01% sodium azide for 1 hour at 28°C. The wells were washed three times with PBST and incubated with a 1:20,000 dilution of HRP-conjugated goat anti-mouse IgG (Abcam) for 1 hour at 28°C. After washing three more times, bound antibodies were detected with 50 μl of tetramethylbenzidine. The reaction was stopped with 50 μl of 1 M H2SO4, and the OD was measured using an EL800 microplate reader (BioTekInstruments) and a reference wavelength of A630. 450 Appropriate anti-capsule antibody titers were obtained by comparison with a standard curve generated using an arbitrary titer of 1:10,000 Prevnar antiserum.

[0128] Anti-protein reactions were performed by sandwich ELISA using plates coated with 0.1 μg monoclonal rabbit anti-histidine IgG (Abcam) in PBS overnight at 4°C. Wells were washed three times with PBST and incubated with 1 μg / well of recombinant protein in PBS for 2 hours at 28°C. After washing three more times, the plates were blocked and incubated with 10-fold diluted mouse antiserum and HRP-conjugated goat anti-mouse IgG as described above.

[0129] For whole cell ELISA, grow TIGR4 and D39 cultures to an OD of approximately 0.4-0.8 600 , washed and resuspended to an OD of 0.4 in 10% glycerol 600 The plates were incubated with 100 μl / well of standardized bacteria at room temperature for 2 h, and the bacteria were fixed with 4% formaldehyde for 20 min. The washed plates were incubated at 28 °C for 2 h with 1:1000 dilution of mouse antiserum and HRP-conjugated goat anti-mouse IgG as described above.

[0130] 1.8 Antibody deposition assay

[0131] Antibody deposition assays were performed using frozen stocks of S. pneumoniae and S. microbiota as described previously. Briefly, 2 × 10 6 CFU / well bacteria were incubated with diluted mouse antiserum in a final volume of 50 μl at 37°C with stirring at 225 rpm for 30 minutes. Bacteria were pelleted at 3000 rpm, washed twice with PBS, and incubated with 1:100 diluted PE-conjugated goat anti-mouse IgG (25 μl / well) for 30 minutes. After two more washes, bacteria were coagulated with 200 μl / well of 4% paraformaldehyde, and the fluorescence intensity of the bacterial population was measured by flow cytometry using a FACSVerse system. The titer of TIGR4 was determined by comparison with a standard curve generated using anti-Prevnar antiserum at an arbitrary titer of 1:10000.

[0132] 1.9 Phagocytic uptake assay

[0133] Phagocytic uptake assays were performed using neutrophils purified from healthy human donors using the MACSexpress magnetic purification system according to the manufacturer's instructions. Frozen stocks of S. pneumoniae were resuspended in 1 ml of 0.1 M sodium bicarbonate and incubated with FAM-SE for 30 min at 37 °C. Bacteria were washed three times with PBS and then 2 × 10 6 CFU / well of bacteria were incubated with diluted mouse antiserum in a final volume of 50 μl at 37°C with agitation at 225 rpm for 30 min. 2×10 4 Neutrophils were added to 50 μl PBS and the reaction was incubated for another 30 minutes. The reaction was coagulated with 100 μl / well of 4% paraformaldehyde and the fluorescence intensity of neutrophils was measured by flow cytometry using the FACSVerse system.

[0134] 1.10 Statistical analysis

[0135] Statistical analysis was performed using GraphPad Prism with appropriate group size and number of comparisons. Flow cytometric data were analyzed using FlowJo software.

[0136] 1.11 Recombinant protein production

[0137] The N-terminal lectin-like domain of NanA was subcloned from pEXT21 (nanA) using the primers listed in Table S2. Two fragments of the commercially synthesized NanA construct were amplified from pEXT21 (nanA) using Q5 High Fidelity 2X premix (New England Biolabs) and primer pairs pEXT-F / nanA-R and pEXT-R / nanA-F at 98°C for 10s, 60°C for 30s, 72°C for 30s for 30 cycles. The fragments were PCR purified (QIAquick PCR purification kit, Qiagen) and absorbed with AvrII (New England Biolabs). The absorbed fragments were PCR purified and ligated using T3 DNA ligase (New England Biolabs). The reconnected reaction mixture was used as a template for NanA (N-terminal) amplification using the pEXT-F / pEXT-R primer pair as described above. The resulting insert was PCR purified and cloned into pEXT21 using restriction enzymes EcoRI and XbaI. Plasmid pEXT21(NanA), containing the lectin-like domain of NanA and the genetic components required for PglB glycosylation and Ni-NTA purification, was transformed into DH5α cells and stored in 20% glycerol at -80°C.

[0138] 2. Results

[0139] 2.1 Vaccination with recombinant glycoconjugates can generate antibodies against capsule and carrier proteins

[0140] NanA (Sp4), PiuA (Sp4), and Sp0148 (Sp4) were prepared from the isolates as described above and groups of eight female CD1 mice were vaccinated with either single vaccines (10 μg / mouse / injection) or in combination (4 μg / mouse / vaccination of each protein). Homologous non-glycosylated antigen, Prevnar-13, and PBS (Sham) vaccine groups were included as controls for anti-protein and anti-capsular responses. In sera recovered from mice 7 days after the previous vaccination, circulating anti-capsular antibodies exceeded the detection limit in all glycosylated antigen groups, while no reactivity was detected in the group vaccinated with protein only ( Figure 1AE). The levels of anti-capsular antibodies varied significantly between the groups but did not seem to correlate with the relative amounts of glycoproteins in the different preparations. Although the NanA sample had the highest degree of glycosylation, the PiuA glycoconjugate stimulated the most robust anti-capsular immune response ( Figure 1 A). Combination vaccination with all three glycoconjugates produced strong anti-capsular responses similar to those produced by Prevnar-13. Anti-carrier protein responses were assessed by sandwich ELISA and confirmed that all vaccine groups were seroconverted to the selected carrier protein ( Figure 1 FH). Regardless of whether they carried serotype 4 glycans or not, they all produced a strong humoral immune response to the protein antigen, indicating that glycosylation had no significant effect on protein antigenicity.

[0141] 2.2 Antibody recognition of Streptococcus pneumoniae in the sera of mice vaccinated with recombinant glycoconjugates

[0142] Antibody recognition of S. pneumoniae in sera of vaccinated mice was assessed using whole-cell ELISA and plates coated with the homologous type 4 S. pneumoniae TIGR4 as well as the heterologous type 2 S. pneumoniae D39 strain ( Figure 2 AB). Compared with the sera of mice vaccinated with Prevnar 13, the sera of mice vaccinated with the Sp0148 group of glycoconjugates had similar ELISA titers for TIGR4 recognition, while the sera of mice vaccinated with PiuA or the combination of the three protein glycoconjugates had higher titers ( Figure 2 A), mice vaccinated with the NanA glycoconjugate generated no significant increase in anti-TIGR4 titers. In mice vaccinated with the combination of unglycosylated PiuA and all three proteins, significant antibody titers against TIGR4 were also generated in the sera. As expected, there was no difference in the titers of D39 whole cell ELISA in sera recovered from mice vaccinated with the glycoconjugates or the corresponding proteins, while mice vaccinated with Sp0148 or all three proteins had a significant increase in antibody titers ( Figure 2 B). Recognition of native pneumococcal antigens was confirmed by immunoblotting of S. pneumoniae lysates with sera from mice vaccinated with different PGCT glycoconjugates and their homologous proteins ( Figure 2 C). Recognition of the three proteins selected for study was confirmed in TIGR4 lysates, as well as recognition of the type 4 pneumococcal capsule in samples probed with the glycoconjugate antiserum. Consistent with the results of the anti-capsule ELISA ( Figure 2A), the different proteoglycan conjugates showed different levels of reactivity with serotype 4 capsule, with the strongest signals coming from sera from mice vaccinated with PiuA (Sp4) and the glycoconjugate combination vaccine (Combo) (Sp4). Consistent with the results of the whole-cell ELISA, when D39 lysates were probed with sera from vaccinated mice, lipoprotein Sp0148 was also identified as well as PiuA, although the PiuA signal was close to the detection limit. Taken together, these data suggest that antibodies generated by vaccination with glycoconjugate molecules can recognize both anti-capsular and anti-protein antigens, and therefore can recognize both homologous and heterologous S. pneumoniae isolates.

[0143] 2.3 Vaccination with recombinant glycoconjugates stimulates the production of antibodies capable of promoting opsonization of live S. pneumoniae

[0144] A flow cytometric assay was used to assess whether vaccination with PGCT glycoconjugates could result in antibody recognition of whole live S. pneumoniae. To specifically investigate anti-capsular recognition, sera from the vaccinated groups were used to assess IgG binding to a Streptococcus mutant expressing Streptococcus pneumoniae type 4 (S. mitis (SpT4)) (36). Although no recognition of wild-type Streptococci was detected, individual sera from all groups used for the study recognized Streptococcus (SpT4) ( Figure 3 The extent of IgG binding varied between vaccine groups, with only sera from four and six mice in the Sp0148 and NanA (Sp4) vaccination groups recognizing S. pneumoniae (SpT4), whereas sera from all eight mice vaccinated with PiuA glycoconjugate or all three protein glycoconjugate vaccines elicited significant immunoglobulin binding to Streptococcus pneumoniae (SpT4) ( Figure 3 B). The levels of antibody binding in both groups were comparable to those achieved by the Prevnar-13 vaccinated group. No antibody deposition on Streptococcus (SpT4) strains was observed in sera from mice vaccinated with any of the unglycosylated protein antigens, confirming the absence of cross-reactivity between the pneumococcal carrier protein and the surface of S. pneumoniae cells. These data support the ELISA results that while each of the novel glycoconjugates selected for study stimulated an anti-Sp4 capsular immune response, the PiuA glycoconjugate stimulated a more robust response than the NanA and Sp0148 glycoconjugates.

[0145] The flow cytometric IgG binding assay was repeated using the TIGR4 strain. TIGR4 was incubated with decreasing dilutions of the pooled mouse antisera from the glycoconjugate-vaccinated mouse groups and showed a dose-dependent IgG binding ( Figure 3C). Comparison of gMFI readings with a standard curve generated using antisera from mice vaccinated with Prevnar-13 revealed higher surface antibody titers in reactions incubated with sera from mice vaccinated with the four glycoconjugates ( Figure 3 D). In addition, detectable levels of surface IgG binding were observed in sera from mice vaccinated with unglycosylated SpO148 or a combination of all three proteins ( Figure 3 D). IgG surface deposition was indistinguishable from negative controls in sera obtained from unglycosylated antigens of either NanA or PiuA. To assess recognition of individual protein antigens independent of capsular antibody recognition, IgG binding to S. pneumoniae of other capsular serotypes was assessed using a fixed concentration of mixed antisera from mice vaccinated with each glycoconjugate. IgG recognition of non-serotype 4 strains in sera from vaccinated mice was generally weaker than that of TIGR4 strains and varied from strain to strain ( Figure 4 AD), although high levels of IgG binding were observed to serotype 23F and reasonable levels to serotype 2F, very little binding was observed to 6B. The ability of different antigens to promote IgG binding varied between strains, with NanA induced to produce the weakest response to serotype 23F and Sp0148 induced to produce the weakest response to TIGR4, which may reflect differences in expression levels and surface accessibility of the individual protein antigens.

[0146] Visualization of antibodies binding to S. pneumoniae in the sera of mice vaccinated with PGCT glycoconjugates

[0147] Recognition of homologous and heterologous pneumococcal isolates was further investigated by studying antisera from the combination vaccine group and fluorescently labeled anti-mouse secondary antibodies using fluorescence microscopy ( Figure 4E). Incubation of the TIGR4 strain in sera from mice vaccinated with all three PGCT glycoconjugates produced bright, uniform surface staining on bacterial cells, similar to that observed in sera from Prevnar-13-vaccinated mice, confirming high levels of IgG binding to the homologous serotype. In contrast, fluorescent staining of non-serotype strains in sera from mice vaccinated with the PGCT glycoconjugate vaccine was weaker and less uniformly distributed, and more variable from strain to strain, according to flow cytometric data. However, when mice were vaccinated with a mixture of the three protein antigens, either glycosylated or unglycosylated, stained cocci were seen in all serotypes, including serotype 2 strains that were not recognized by sera from Prevnar-13-vaccinated mice. Together, the ELISA, flow cytometry, and immunofluorescence data demonstrate that PGCT glycoconjugates can induce anti-capsular IgG responses that vary in intensity with the carrier protein but (e.g., PiuA glycoconjugate) were similar in intensity to those following vaccination with Prevnar-13. Additionally, PGCT glycoconjugates stimulated significant antibody responses to protein antigens that could opsonize heterologous S. pneumoniae serotypes and potentially provide protection against multiple S. pneumoniae strains.

[0148] 2.5 Protective efficacy of mice vaccinated with recombinant glycoconjugates produced with PGCT

[0149] To determine whether the density of antibody deposition on TIGR4 was sufficient to promote opsonophagocytosis, a neutrophil uptake assay was performed using freshly isolated human neutrophils (37). Incubation in sera from mice vaccinated with either the PiuA(Sp4) glycoconjugate or the combination of the three proteins promoted neutrophil uptake of the TIGR4 pneumococcal strain, indicating that a functional anti-capsular humoral immune response was generated in response to recombinant type 4 capsular antigens prepared using PGCT ( Figure 5 A). However, sera from mice vaccinated with Sp0148(Sp4), NanA(Sp4), or the unglycosylated protein (alone or in combination) failed to promote neutrophil phagocytosis in this assay ( Figure 5 B). The combined conjugate also failed to promote neutrophil phagocytosis of non-serotype 4 strains tested ( Figure 5 CE).

[0150] The protective efficacy of glycoconjugates prepared using PGCT was evaluated using a mouse model of infection. Mice were vaccinated with Prevnar or a combination of all three PGCT glycoconjugates or a combination of unglycosylated proteins and then challenged by intranasal inoculation with the TIGR4 strain (pneumonia in a sepsis model) or serotype 4, which is known to cause meningitis. After 48 hours, mice were picked out to assess their infection levels by target organ CFU. In the TIGR4 challenge model, combined vaccination with the three PGCT glycoconjugates almost completely prevented sepsis and reduced lung CFU by approximately log 10 , which is very similar to the level of protection provided by the Prevnar-13 vaccine ( Figure 6 AB). Although not statistically significant, the median CFU / ml recovered from the blood was also reduced in mice vaccinated with the combination of unglycosylated proteins alone. In the meningitis model, vaccination with the three PGCT glycoconjugates completely prevented sepsis and meningitis and resulted in a 3 log reduction in lung CFU 10 ( Figure 6 CE), again, this was very similar to the level of protection provided by Prevnar-13 vaccination. For mice vaccinated with only the unglycosylated protein combination, the mean blood, lung and brain CFU were all lower than the negative control results, although only the blood data were statistically significant. These mouse model data suggest that protein antigens made using the S. pneumoniae capsule as well as PGCT can be highly protective against infection with the same S. pneumoniae serotype and provide levels of protection as good as existing commercially available PCV formulations. In addition, the protein component also generated a protective response that was independent of the capsular antigen, suggesting that the PGCT vaccine will be able to provide cross-serotype protection.

[0151] In the above experiments, proteoglycan conjugation technology was used to produce a low-cost multicomponent glycoconjugate vaccine containing three surface antigens glycosylated by recombinant serotype 4 capsules. The results of the study include: (a) S. pneumoniae glycoconjugates prepared using PGCT are as immunogenic as commercial PCV and provide similar levels of protection in mouse models of pneumonia and meningitis; (b) S. pneumoniae protein antigens can be used as carrier proteins for PCV vaccines instead of one of the established conjugate vaccine carrier proteins; (c) Glycoconjugates prepared using PGCT have significantly different anti-capsular immunogenicity depending on the carrier protein; (d) The carrier protein is immunogenic and can stimulate antibody responses that can provide a degree of protection against meningitis and sepsis that is independent of capsular antibodies. The data suggest that PGCT can provide an alternative method for making PCV that is less expensive than existing chemical conjugation methods and can produce similar levels of serotype-specific protection for the prevention of severe S. pneumoniae infections, including meningitis, in developing countries. In addition, PGCT can be formulated as a PCV containing a series of S. pneumoniae protein carrier proteins, thereby providing at least some degree of serotype-independent immunity.

[0152] A major finding that emerges from our data and has broad implications for vaccine development is the identification of other carrier proteins that can promote antibody responses to capsular antigens. Currently, only four proteins have been used to prepare glycoconjugates, but our data suggest that PiuA would be an alternative. The other two proteins used to generate glycoconjugates, Sp_0148 and NanA, were significantly less effective than PiuA in generating anti-glycan responses. Indeed, it is conceivable that most of the anti-capsular responses generated by the combination vaccine are due to the presence of glycosylated PiuA. The reason for such significant differences in the efficacy of different carrier proteins in promoting anti-capsular antibodies is not clear. Immunoblotting suggests that this is not simply due to the greater amount of PiuA glycoconjugates, as in fact the amount of NanA glycoconjugates appears to be the largest. The choice of carrier protein and glycosylation site is important for generating a strong anti-capsular IgG response, and recent data suggest that glycoconjugates generate antibody responses to glycans by directly presenting glycosylated antigen epitopes to T cell receptors via MHCII binding (21, 22). Such a mechanism would explain our data, as the strength of the anti-capsular antibody response would be influenced by the choice of carrier protein and by the site to which the capsular antigen is covalently attached. These data suggest that screening of other S. pneumoniae proteins should identify other carrier proteins for glycoconjugate vaccines that are able to generate strong antibody responses to capsule or other glycan antigens.

[0153] The major advantage of using pneumococcal protein antigens as carrier proteins for PCV prepared using PGCT is that these proteins can induce additional immune responses that may be protective. Immune responses to protein antigens may also include T cell-mediated immunity, including TH17 responses that could theoretically enhance mucosal immunity compared to pure antibody responses (24, 40). We investigated whether combinatorial glycoconjugates produced using PGCT that bind to the type 4 pneumococcal capsule conjugated to three conserved pneumococcal protein antigens, the endothelial cell infiltrating protein NanA (29), the Th17 stimulatory antigen Sp0148 (24), and the ABC transporter PiuA (34) could stimulate homologous and heterologous immunity against S. pneumoniae. Our data demonstrate that vaccination with carrier proteins generates robust antibody responses that recognize different live S. pneumoniae strains. Vaccination with these proteins alone resulted in a reduction in bacterial CFU in the blood and meninges following infection with S. pneumoniae, confirming the protective potential of antibodies to the carrier protein components. However, despite the selection of a known Th17-inducing antigen (SP_0148) for the vaccine, vaccination with the proteins alone failed to protect against lung infection, and the level of protection against systemic infection was weaker than that provided by the anti-capsular response. Furthermore, the antibody responses to the three proteins varied in their ability to recognize different S. pneumoniae strains.

[0154] In summary, the data presented here demonstrate that PGCT can be used to prepare S. pneumoniae glycoconjugates with efficacy comparable to existing commercial PCVs for preventing serotype-specific infection, while in addition stimulating protective immunity against heterologous serotypes.

[0155]

[0156] *For vaccine production

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Claims

1. A sugar conjugate, characterized in that include: Streptococcus pneumoniae protein antigens glycosylated with Streptococcus pneumoniae capsular polysaccharide, Wherein, the protein antigen is NanA and / or Sp0148.

2. The sugar conjugate according to claim 1, characterized in that: in, The NanA protein antigen comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:

6.

3. The sugar conjugate according to claim 1, characterized in that: in, The Sp0148 protein antigen comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:

9.

4. The sugar conjugate according to any one of claims 1 to 3, characterized in that: in, The protein antigen comprises a heterologous glycosylation sequence.

5. The sugar conjugate according to claim 4, characterized in that: in, The heterologous glycosylation sequence comprises the sequence D / EYNXS / T, wherein Y and X are any amino acids except P.

6. The sugar conjugate according to claim 4 or 5, characterized in that: in, The protein antigen comprises two or more of the heterologous glycosylation sequences.

7. The sugar conjugate according to any one of claims 4 to 6, characterized in that: in, The protein antigen comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NO:5 or SEQ ID NO:

8.

8. The sugar conjugate according to any one of claims 1 to 7, characterized in that: in, The capsular polysaccharide is a serotype 4 capsular polysaccharide.

9. A vaccine composition, characterized in that include: one or more glycoconjugates; as well as a pharmaceutically acceptable excipient, Wherein, the sugar conjugate is the sugar conjugate according to any one of claims 1 to 8.

10. The vaccine composition according to claim 9, characterized in that Also includes: Adjuvant.

11. A method for preparing a vaccine composition, characterized in that: include: mixing one or more glycoconjugates with a pharmaceutically acceptable excipient and optionally an adjuvant, Wherein, the sugar conjugate is the sugar conjugate according to any one of claims 1 to 8.

12. Use of a saccharide conjugate or a vaccine composition for the preparation of a medicament for treating a Streptococcus pneumoniae infection in an individual, characterized in that: in, The glycoconjugate is the glycoconjugate according to any one of claims 1 to 8, The vaccine composition is the vaccine composition according to claim 9 or 10.

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